key: cord- -pw f asc authors: goyal, amit k.; rath, goutam; garg, tarun title: nanotechnological approaches for genetic immunization date: - - journal: dna and rna nanobiotechnologies in medicine: diagnosis and treatment of diseases doi: . / - - - - _ sha: doc_id: cord_uid: pw f asc genetic immunization is one of the important findings that provide multifaceted immunological response against infectious diseases. with the advent of r-dna technology, it is possible to construct vector with immunologically active genes against specific pathogens. nevertheless, site-specific delivery of constructed genetic material is an important contributory factor for eliciting specific cellular and humoral immune response. nanotechnology has demonstrated immense potential for the site-specific delivery of biomolecules. several polymeric and lipidic nanocarriers have been utilized for the delivery of genetic materials. these systems seem to have better compatibility, low toxicity, economical and capable to delivering biomolecules to intracellular site for the better expression of desired antigens. further, surface engineering of nanocarriers and targeting approaches have an ability to offer better presentation of antigenic material to immunological cells. this chapter gives an overview of existing and emerging nanotechnological approaches for the delivery of genetic materials. vaccine development offers an attractive and cost-effective preventive approach against deadly disease. new advances in immunology, molecular biology and biotechnology as low as for the development of unique, safe and effective against some dreadful diseases like hiv, cancer, hepatitis, tuberculosis, etc. (table ) . genetic immunization holds potential to discover new vaccines and may be an efficient vaccine delivery system. in the early s, dna vaccines burst into the scientific limelight. tang and johnston described the delivery of dna using a gene gun into the mice skin and felt that this could be a useful technique to generate antibody responses against specific transgene product (tang et al. ) . in , at the annual vaccine meeting at the cold spring harbor laboratory reported to drive both humoral and cellular immune responses against pathogens or tumor antigens in vivo by the use of dna vectors. merck pharmaceutical company reported that developed immune responses against influenza virus antigens in mice after injecting the naked plasmids intramuscularly (ulmer et al. ) . similarly, robinson proved the ability of dna plasmids against influenza virus antigens (fynan et al. ) . the capability of plasmids carrying hiv antigens or tumor antigens to generate immune responses and protection from tumor in mice has been described (wang et al. ). importantly, a dna vaccine affects humoral as well as cellular immunity. the use of the dna approach also promised to overcome the safety concerns associated with live vaccines-their reversion risks and their potential spread to unintended individuals, avoids the risks linked to the manufacture of killed vaccine (ruprecht ) . vaccines are generally composed of whole organism-either live and weakened or killed forms (first-generation vaccines). live, attenuated organisms such as smallpox and polio vaccines are able to induce killer t-cell (t c ) responses, helper t-cell (t h ) responses, and antibody immunity (fig. ) . first-generation vaccines providing maximum protection but associated with a risk that attenuated forms of a pathogen can revert to a dangerous form and may still be able to cause disease, especially in immune compromised vaccine recipients (e.g., aids patients). killed vaccines cannot generate specific killer t-cell responses and will be effective against limited diseases where cellular response is not essential (alarcon et al. ). these were the reasons which initiated the research for second-generation vaccines. second-generation vaccines were the subunit vaccines, consisting of defined protein antigens (such as tetanus or diphtheria toxoid), recombinant protein components (hepatitis b surface antigen), or surface proteins (influenza). these vaccines are able to generate th and antibody responses, but not killer t-cell responses. this reason again restricted the utility of these vaccines to limited number of diseases. today is the era of genetic immunization, which is nextgeneration vaccine (third generation), which seems to be highly effective till date. this strategy is based upon improved gene optimization, improved rna structural design, novel formulations and immune adjuvants, and more effective delivery approaches (alarcon et al. ; robinson and pertmer ) . at the cellular level, introduction of nanotechnology and the development of nanocarrier-based vaccines provide effective immunization through better targeting and by triggering antibody responses. in order to induce an effective protective immunity, these vaccines require boosting with agents called adjuvants. adjuvants and delivery vehicles have shown to protect antigens from degradation. the current trend toward many efforts to develop novel adjuvants and carrier have persistent on systems at the micro-and nanoscale. immunization by traditional vaccines requires the administration of live attenuated virus, killed organism, whereas dna vaccines can be constructed to encode specific antigenic determinants. dna vaccines are highly flexible, stable, easily stored, manufacture on large scale, encoding several types of genes including viral or bacterial antigens, and immunological and biological proteins (gengoux and leclerc ; kutzler and weiner ) . many potential advantages of dna vaccines are summarized in table . the gene of interest having the antigenic determinant is inserted into the recombinant vectors like multiple cloning region of plasmid by enzymatically, synthetically or by pcr and delivered to the inoculation site by one of several delivery methods like physical (gene gun, electroporation), viral (virosomes), or nonviral (liposomes, microspheres, nanospheres) to either skin (intradermally), subcutaneum, or muscle. the mechanisms by which dna vaccines produce antigen-specific immunity in vivo are under intense investigation, with an idealized model presented in fig. . figure represents the overview of the mechanisms of plasmid uptake and proteinaceous antigen expression by either somatic cells (e.g., myocytes, keratinocytes) at the site of injection or the resident antigen-presenting cells (apcs), the immature dendritic cells. the mechanisms include (a) direct targeting of dendritic cells (langerhans cells, i.e., skin dendritic cells) in gene gun administration of plasmid dna, which involves high-speed shooting of gold microbeads coated with plasmid dna into the upper layers of the skin or (b) "cross-priming," most likely in intramuscular injections or any parenteral injections where the somatic cells mentioned above primarily express the protein encoded by the the success of dna vaccines concerns improving their immunogenicity and safety. therefore, there is an urgent need for the development of potent and safe adjuvants and delivery systems that can be used with new generation of vaccines. as shown in fig. , there are several ways in which antigen expression and immunogenicity can be improved for the dna vaccine platform. there are lot of steps undertaken to modify immunogenicity and safety of dna vaccines (fig. ) . promoter is an important component of the plasmid that drives high levels of expression of the gene of interest. various promoters have been utilized to improve the expression of vaccine genes. the human cytomegalovirus (cmv) promoter has been extensively used for high levels of protein expression in mammalian cells (boshart et al. ) . however, there are some drawbacks associated with cmv promoters like chromatin condensation by histone deacetylase. recently, histone deacetylase inhibitors have been supplemented with cmv promoter-based plasmid that has shown increased expression of dna vaccine antigens (lai et al. immediate-early promoter one (ie ) have also demonstrated better gene expression in insect cells compared with cmv promoter (he et al. ). the porcine circovirus type capsid gene promoter has enhanced the antigen expression and immunogenicity in a hiv- plasmid vaccine (tanzer et al. ). regulation of transcriptional termination is a key element in control of gene expression within the framework of a single transcriptional promoter (barr et al. ) . so one of the most effective ways to increase protein production is through the use of codon optimization or by adopting species-specific codon changes (gustafsson et al. ) . plasmid backbone optimization has also been important contributory factor for dna vaccine. replacement of sv t polyadenylation and splicing signals of the paec plasmid vectors by synthetic intron and synthetic rabbit beta globin-based termination/polyadenylation sequences and cpg motif have enhanced the cell-mediated ifn-gamma-secreting activity. the rna polymerase ii dependent cytomegalovirus immediate early (cmv ie) enhancer/promoter and t promoter in psmcta and pshcta has been utilized to enhance the expression of antigenic substances (yu et al. ). viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. viral vector vaccines use live viruses to carry dna into human cells. it consists of a non-replicating virus that contains some defined genetic fig. factors affecting the immunogenicity of dna vaccines material from the pathogen to which immunity is desired. viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. viral vector vaccines carry dna into a host cell for production of antigenic proteins that can be tailored to stimulate a range of immune responses, including antibody, t helper cell (cd + t cell), and cytotoxic t lymphocyte (ctl, cd + t cell) mediated immunity (draper and heeney ) . retroviruses, parvoviruses, adenoviruses, lentiviruses, adeno-associated viruses, and the herpes simplex virus are being investigated for their ability to transfer dna. gene expression with high transfection efficiencies in tissues, such as kidney, heart, muscle, eye, and ovary, has been achieved by using viral vectors. advantages of viral-vectored vaccines include their ease of production, a good safety profile, ability to potentiate strong immune responses, infect a broad spectrum of cell types, triggering t-lymphocyte activation, potential for nasal or epicutaneous delivery and mucosal immunization (chamberlain ; galimi and verma ; lien and lai ; martin et al. ; mctaggart and al-rubeai ; wolf and jenkins ) . the recombinant retroviruses have the ability to integrate into the host genome in a stable fashion because it contains a reverse transcriptase that allows integration into the host genome. - kb is the typical maximum length of an allowable dna insert in a replication-defective viral vector. lentiviruses are a subclass of retroviruses. the unique feature of lentiviruses is to their ability to integrate into the genome of nondividing cells, whereas retroviruses can infect only dividing cells. when the virus enters the cell, the viral genome in the form of rna is reverse transcribed and produce dna, which is then inserted into the genome by the viral integrase (cattoglio et al. ). their primary applications are in gene therapy and vaccination but their limits use in basic research due to it does not integrate into the genome and is not replicated during cell division. respiratory, gastrointestinal and eye infections were commonly caused in humans after the contact with adenoviruses. adeno-associated virus (aav) is a small virus that infects both dividing and nondividing cells of humans and some other primate species and may incorporate its genome into that of the host cell with causes a very mild immune response. these features make aav a very attractive candidate for creating viral vectors for gene therapy (goff and berg ) . nowadays, incorporation of molecular adjuvants has been the main strategy for melioration of vaccines. co-injection of plasmids encoding cytokines, chemokines, or co-stimulatory molecules like death receptors, growth factors, adhesion molecules, toll-receptor ligands can be used individually or in combination to maximize substantial effect on the immune response in the clinic, in both prophylactic and therapeutic studies to plasmid-encoded antigen. for example, boost the humoral and cellular response when antigen co-administered with synthetic oligodeoxynucleotides containing unmethylated cpg motifs in mice (higgins et al. ) . recently, immunomodulation is based on targeting antigen-presenting cells (apc) "majorly macrophages" by using macrosialin promoter. the immune response of the constructed plasmids expressing jev envelope (e) protein under the control of aforesaid promoter and cytomegalovirus (cmv) immediate early promoter against jev have induces comparable immunity in comparison to ubiquitous promoter construct (ahsan and gore ) . nk group , member d (nkg d) is also reported as potent-activating receptor expressed by cells of the innate and adaptive immune systems. recombinant mouse cmv expressing the high-affinity nkg d ligand rae- γ has shown better expression and profound virus attenuation in vivo and could be a powerful to develop immunogenic hcmv vaccine (slavuljica et al. ) . in vivo dendritic cells (dc) targeting is an attractive approach with potential advantages in vaccine efficacy, cost, and availability. genetic targeting of the dc-specific cd c-driven active transcription factor xbp s to dc (xbp s/dc) has potentiated vaccine-induced prophylactic and therapeutic antitumor immunity in multiple tumor models (tian et al. ) . recently, heterodimeric antigen-presenting cells targeted multireceptor ligand approaches have been implemented to access the potential of more than one apc-specific targeting unit in the antigenic molecule. results revealed that heterodimeric barnase-barstar vaccine molecules were potent and provide a flexible platform for development of novel dna vaccines with increased potency (spang et al. ). some mechanisms of adjuvant action are discussed below: . vaccine adjuvants can increase the potency and immune response of small, antigenically weak synthetic or recombinant peptides in immunologically immature, immunosuppressed, or senescent individuals. . they can improve the immune response to stronger antigens in respect of speed, vigor, and persistence. for example, aluminum adjuvants adsorbed dtp elicit early and higher antibody response after primary immunization than do unadjuvanted preparations. . vaccine adjuvants can modulate antibody avidity, specificity, quantity, isotype, and subclass against epitopes on complex immunogens. . they target antigen to a cell-surface receptor on apcs by formation of multimolecular aggregates. . they can direct antigen presentations by direct peptide exchange on surface mhc molecules or by mhc class i or mhc class ii pathways by means of fusion or disruption of cell membranes (newman and powell ) . the most important characteristic of any adjuvanted vaccine is that it is more efficacious than the aqueous vaccine but unfortunately, the absolute safety of adjuvanted vaccines, or any vaccine, cannot be guaranteed. the real or theoretical risks of administering vaccine adjuvants are local acute or chronic inflammation, painful abscess, persistent nodules, ulcers, fever, hypersensitivity, anaphylaxis, chemical toxicity to tissues or organs, autoimmune arthritis, amyloidosis, anterior uveitis, glomerulonephritis or meningoencephalitis, immune suppression or oral tolerance, carcinogenesis, teratogenesis or abortogenesis and spread of a live vectored vaccine to the environment (edelman and tacket ; bussiere et al. ; goldenthal et al. ). . it must be safe, including freedom from side effects. . it should be affordable and stable. . it should be biodegradable or easily removed from the body after its adjuvant effect. . efficacy and immunogenicity should be achieved using fewer doses and/or lower concentrations of the antigen. . it should elicit a more vigorous protective or therapeutic immune response combined with the antigen than when the antigen is administered alone. . it must be defined chemically and biologically, so that there is no lot-to-lot variation in the manufactured product. freund's adjuvant is a solution of antigen emulsified in mineral oil and used as an immunopotentiator (booster). freund's complete adjuvant (fca) is composed of inactivated and dried mycobacteria whereas the incomplete form (fia) lacks the mycobacterial components. although, fca has been proved as a potent inducer of cell-mediated immunity and ability to boost the humoral immune response, but associated adverse side effects like sterile abscesses, granulomas, muscle indurations, plasma cell neoplasia, ascites and amyloidosis has limits its utility. a modified version of fca is known as freund's incomplete adjuvant (fia) in which antigen is administered in water-in-oil (w/o) emulsion but without mycobacterial components. it consists of a mixture of mineral oil (drakeol vr, bayol f, marcol ) ( % v/v) and emulsifier (mannide monooleate) ( % v/v) with an equal volume of aqueous solution of antigen. mechanism of the freund's adjuvants is allowing a gradual and continuous release of the antigen by establishment of a repository antigen-containing locus at the site of injection or interaction with mononuclear cells such as phagocytic cells, antigen presenting cells, etc. fia has been included in veterinary vaccines (rabies, hog cholera, canine hepatitis) (freund et al. ; fastier and hansen ; ott ) , as well as human vaccines (tetanus toxoid, influenza vaccines) (salk et al. ) . in general, both fia and fca are indeed very efficient in raising high antibody titers, induce cytotoxic t lymphocytes (ctl) and used in priming immunizations. morozova et al. investigated that development of inflammatory response in the rat myocardium after immunization rats with single subcutaneous injection of cardiac myosin ( ug/kg) with incomplete freund's adjuvant (ifa) (gjessing et al. ). there are very limited studies that have been conducted which signify their utility of freund's adjuvant for dna vaccination. it has been demonstrated that plasmid pv- cpg suspended in ifa has significantly enhanced both type of cellular and humoral immune responses to hbsag (luo et al. ). aluminum compounds [aluminum phosphate (alpo ), aluminum hydroxide (al (oh) ), and alum] precipitated vaccines are currently the most commonly used adjuvants with human and veterinary vaccines owing to their good track record of safety, low cost, and adjuvanticity with a variety of antigens (gupta et al. ; gupta and siber ) . however, aluminum adjuvants have certain limitations such as local reactions at the site of injection, ige antibody responses augmentation, ineffectiveness for some antigens, and inability to supplement cell-mediated immune responses . two methods are used to prepare vaccines and toxoids with aluminum compounds-in situ precipitation of aluminum compounds in the presence of antigen and adsorption of antigen onto preformed aluminum gel (aprile and wardlaw ; holt et al. ; hem and white ; gupta ) . the mechanism of adjuvanticity of aluminum compounds includes formation of a depot, efficient uptake by antigen-presenting cells, stimulation of immune competent cells of the body through induction of eosinophilia, and activation of macrophages and complement. recently, adjuvanticity of alum has been reported due to cell death and the subsequent release of host cell dna, which acts as a potent endogenous immunostimulatory signal-mediating alum adjuvant activity (marichal et al. ) . gupta et al. ( ) showed that diphtheria toxoid adsorbed aluminum phosphate induced significant antibody levels in rabbits. previously, manam et al. reported that aluminum phosphate adjuvant had shown no effect on the tissue distribution and integration frequency of delivery genetic materials (manam et al. ) . similarly, liang et al. ( ) showed the similar results indicated that there was not increase in hbsag expression when plasmid pcdna . -s mixed aluminum phosphate. however, they demonstrated the better antibody titer after intramuscular immunization of balb/c mice with pcdna . -s mixing aluminum phosphate adjuvant. this study revealed that aluminum phosphate has a potential for dna vaccination (liang et al. ) . recently, yu et al. have demonstrated the role of aluminum adjuvant for dna vaccines against botulinum neurotoxin (bonts) and shown induced protective humoral immune responses (yu et al. ) . combined use of il- with alum adjuvants for dna immunization have also demonstrated the significant change in the survival rates of the vaccinated animals against toxoplasma gondii (khosroshahi et al. ) . cytokines are a group of secreted low-molecular weight proteins by the cells of the innate and adaptive immunity that have a major role in cell-to-cell communication. cytokines play an important role in induction of immune responses during the processing and presentation of antigens. numerous cytokines including interleukin- (il- ), granulocyte-macrophage colony stimulating factor (gm-csf), and interleukin- (il- ) have been shown to significantly modulate the inflammatory process when given systemically. the local administration of il- increases local expression of major histocompatibility (mhc) class ii antigens and enhances skin antigen reactivity, but high bolus doses of il- cause hypotension, exacerbation of underlying autoimmune disease, and induce vascular leak syndrome. this studies revealed that exogenous il- could be a valuable adjunct in the treatment of immunodeficiency virus (hiv) infected human by decreases the frequency of apoptotic peripheral blood mononuclear cells (pmbcs), which may contribute to the increase in circulating cd + t cells. il- also induces b-cell activation and antibody synthesis in vitro (cordiali fei et al. ). among various improvement strategies, the incorporation of cytokine-expressing plasmids as molecular adjuvants has been widely studied in the past years, yet still without significant clinical application. this chapter reviews recent progress in the co-application of cytokine-encoding genes used for enhancement and direction of immunogenicity, as well as discusses their therapeutic potential for future applications. coadministration of pro-inflammatory agents (such as various interleukins, tumor necrosis factor, and gm-csf) plus th -inducing cytokines increase antibody responses, whereas pro-inflammatory agents and th -inducing cytokines decrease humoral responses and increase cytotoxic responses (which is more important in viral protection, for example). co-stimulatory molecules like b - , b - and cd l are also sometimes used. mpl (monophosphoryl lipid), a immunostimulant, is derived from the lipopolysaccharide (lps) of salmonella minnesota, r . an important characteristic of mpl adjuvant activity is to enhance the generation of specific immunity without being directly associated with an antigen. the choice of an mpl adjuvant formulation will depend on several factors such as the nature of the antigen, desired immune response characteristics, and level of tolerable local reactogenicity. aqueous dispersions of mpl in isotonic buffers when admixed with soluble protein antigens can provide a strong adjuvant effect. an advantage of these mpl plus antigen is that they tend to be well tolerated and induce little or no local tissue reaction at the injection site (qureshi et al. ) . mpl-a has been used to enhance immunity induced by dna vaccination against human immunodeficiency virus type (hiv- ). results indicate that mpl performances as an effective adjuvant for immunogenic dna injection despite reduced expression of encoding protein in muscle (sasaki et al. ) . combination of mpl with antigen-encoded dna has shown the enhanced protective neutralizing antibody response against glycoprotein of the cvs rabies virus (lodmell et al. ) . lipid a has also been admixed with plasmid dna (pdna)-coated nanoparticles and studied for their immunological potential. immunological results revealed that plasmid dna with lipid a have shown significant higher immunological response, especially cellular response (cui and mumper a, b) . studies indicated that la is potential adjuvants to further enhance immune responses; however, limited studies have been utilized this adjuvant for dna vaccination. several established methods have utilized for transferring plasmid dna into cells, including calcium phosphate precipitation, electroporation, particle bombardment, liposomal delivery, polymeric delivery, viral-vector delivery, and receptormediated gene delivery. however, compared to viral vectors, nonviral vectors are easy to make and are less likely to produce immune reactions (edelman and tacket ). in addition, there is no replication reaction required. the engineered novel nano-construct may deliver immunogens safely, with the appropriate kinetics, to the appropriate location, and possibly together with the adequate recognition and maturation stimuli (fig. ) . the use of nonviral particulate carriers for dna-based vaccination could provide better and safe delivery of encapsulated genetic material, circumvent the need for muscle involvement and facilitate instead the uptake of the fig. schematic representation of immunological response greeted by novel dna-loaded nanocarrier dna by apcs. however, transfection of apcs with encapsulated dna into particulate carrier systems will be dependent upon choice of carrier surface charge, size, and lipid/polymer composition, or presence of other biological [e.g., interleukin and interferon-γ (ifn-γ)]. toxicity, transfection efficiency, nucleic acid (na) degradation and free na release are challenging problems for all of the current nonviral gene delivery systems, including lipid and polymers carrier systems (pouton et al. ; cui and mumper a, b) . one current trend in dna vaccine formulation is the use of biodegradable polymeric microparticles and liposomes delivery systems for dna vaccines are excellent formulations for delivery and enhanced immunogenicity in several different hosts like mice, nonhuman primates and humans (herrmann et al. ; kaur et al. ). as noted earlier, genetic materials attached to a particulate carrier are more likely to bring about a successful immunological reaction and some, such as chitosan particles, can act as adjuvants in their own right. natural polymers such as gelatin or albumin have been used as particulate drug delivery systems, although they are of uncertain purity and certainly have the potential for immunogenicity (pouton et al. ; cui and mumper a, b; xiang et al. ; pichichero ) . plasmid dna is trapped on the surface of the polymers like polylactice-coglycolide, chitosan, polyethyleneimine, amine-functionalized polymethacrylates, cationic poly(β-amino esters), poloxamers, and polyvinylpyrrolidone (densmore ) . polymer-trapped plasmid dna is delivered systemically or directly to mucosal surfaces (orally or via the respiratory tract), where the complex is taken up by dendritic cells (dcs) and results in upregulation of dc activation markers and further augments systemic and mucosal immune responses. liposomes offer considerable flexibility towards vaccine optimization due to its structural versatility, including vesicle surface charge (both cationic and anionic liposomes can be made), size, and lipid content. liposome with other suitable adjuvants can protect dna from degradation by serum proteins during transfer of dna across membranes and after the release of genetic material following fusion with endosome (gao and huang ; nakanishi and noguchi ) . among the different approaches to drug delivery, lipid vesicles for both hydrophobic and hydrophilic drugs have attracted much attention. lipid-based gene delivery is the focus of several specialized high-technology companies, of which vical (san diago, ca, usa), genzyme (farmington, ma, usa), genemedicine (the woodlands, tx, usa) and megabios (burlingame, ca, usa) have products in clinical trials. some of the engineered liposomal and non-liposomal versions like ph-sensitive cationic and anionic liposomes, ph-sensitive immunoliposomes, fusogenic liposomes; genosomes (dna-liposomes/lipid complexes), lipofection tm (lipid-dna complex) and recently cochleates are investigated as the major gene vectors (fig. ) . however, most of the commercially available nonviral gene vectors used for transfection is cationic liposome-dna complexes (fenske and cullis ). liposomes are self-assembling structures comprising concentric amphipathic lipid (e.g., phospholipid) bilayers separated by aqueous compartments (baca-estrada et al. ; saupe et al. ) . in , first humoral immune responses observed in mice after injection of liposome-entrapped diphtheria toxoid (allison and gregoriadis ; manesis et al. ) . liposomal vaccines that have been investigated inhuman trials include malaria, hiv, hepatitis a, influenza, prostate cancer and colorectal cancer (katre et al. ). in a liposome-based drug delivery system, genetic material is encapsulated in the liposome and then administered to the patient to be treated. advantage of the use of liposomal dna is that it may be taken up directly by apcs such as dendritic cells, which results in transfection and mhc classes i and ii expression, which stimulates the cd + and cd + t cells by antigenic peptide and induces ctl responses and also b cells to produce antibodies, whereas vaccination with naked plasmid dna, the plasmid is taken up by the myocytes, which are transfected. unfortunately, there are a number of problems associated with the use of conventional liposomes as genetic vaccine delivery vehicles. the relatively low transfectivity of liposomes, particularly evident with insufficient quantities of polynucleotide within liposomal formulations, can be overcome by adding positively charged amphipathic lipid moieties to liposomal formulations. several phospholipids may be used for the preparation of liposomes entrapped vaccines include phosphatidylcholine, phosphatidic acid, triolein, phosphatidylglycerol, phosphatidylserine, distearoyl phosphatidylcholine, dioleylphosphatidylethanolamine, phosphatidylethanolamine, polyethyleneglycol etc. overall, by modification, these systems may provide high membrane fluidity, flexibility, endocytosis and fusiogenic behavior, that is making this system far better than other particulate carriers (fig. ). cationic liposomes are widely explored nowadays for the delivery of dna into eukaryotes. they are formed by simple mixing of positively charged lipid bilayers with negatively charged naked dna. the resulting cationic liposomes-dna complexes (lipoplexes) are taken up via endocytosis, followed by their release from an early endosomal compartment (duzgunes et al. ) . cationic lipid-dna complexes have been used successfully to deliver plasmid dna to the lungs, brain, tumors and skin, by local administration, or to vascular endothelial cells after systemic, intravenous injection (brigham et al. ) . in addition to different cationic lipids (fig. have also shown an important role in membrane perturbation and fusion for intracellular delivery of genetic material. liu et al. have shown that lipoplexes showed much higher transfection in the liver than naked dna alone (liu et al. ). gregoriadis et al. for the first time showed that intramuscular immunization of mice with prc/cmv hbs (encoding the s region of hepatitis b antigen; hbsag) entrapped into positively charged (cationic) liposomes leads to greatly improved humoral and cell-mediated immunity (gregoriadis et al. ). these cationic liposome-entrapped dna vaccines generate titers of anti-hbsag igg antibody isotype in excess of -fold higher and increased levels of both ifn-γ and il- when compared with naked dna or dna complexed with preformed similar (cationic) liposomes. further, modification of liposomal surface with polymer offers potential for oral administration of plasmid dna and able to elicit markedly enhanced transgene-specific cytokine production following in vitro restimulation of splenocytes with recombinant antigen (somavarapu et al. ) . modification of lipid/dna complexes by the polymer poly(d,l-lactic acid) was found to be consistently and significantly more effective than either unmodified liposomal dna or naked dna in eliciting transgene-specific immune responses to plasmid-encoded antigen when administered by the s.c. route (bramwell et al. ) . surface-modified mannosylated cationic liposomes were developed for targeted delivery of pdna to apcs, and the results verified that man lipoplex induces significantly higher pub-m gene transfection into dendritic cells and macrophages than unmodified lipoplex and naked dna and it also strongly induces ctl activity against melanoma, inhibits its growth and prolongs the survival after tumor challenge compared with unmodified liposomes (lu et al. ). an anionic lipid formulation called fluid liposomes was capable of delivering fluorescently labeled oligonucleotides into bacterial cells. it was composed of dppc and , -dimyristoyl-sn-glycero- -[phospho-rac-( -glycerol)] (dmpg). lack of further progress of these systems may be attributed to the poor association between dna molecules and anionic lipids by electrostatic repulsion between these negatively charged species (perrie and gregoriadis ) . liposomes have been prepared from mixtures of anionic and zwitter ionic lipids, dopg and dope, respectively, at a molar ratio of : (dopg:dope). efficient and relatively safe dna transfection using anionic lipoplexes makes them an alternative for gene delivery (patil et al. ) . similarly, endosomolytic bacterial protein listeriolysin o (llo) incorporated in an anionic liposome-entrapped polycation-condensed dna delivery system (lpdii) has been developed that demonstrated better condensation of the dna with improved transfection efficiency due to endosomolytic properties of llo (lorenzi and lee ) . combination of cationic lipoplexes and pegylated anionic liposomes has also been used to prepare anionic pegylated lipoplexes. studies demonstrated that the gene expression of the developed formulation was similar for the cationic formulation taken as a control and the anionic formulations prepared (mignet et al. ) . overall, anionic lipoplex formulation shown promise as a nonviral vector with high-transfection efficiency and low cytotoxicity. a growing amount of literature describes the role of ph-sensitive liposomes for targeting and/or release encapsulated genetic material within cellular compartment. ph-sensitive liposomes are designed to release their contents in response to acidic ph within the endosomal system, while remaining stable in plasma thus improving the cytoplasmic delivery of biopharmaceuticals. they can be generated by the insertion of dope into acidic lipids liposomes such as cholesteryl hemisuccinate or oleic acid (venugopalan et al. ) . it is reported that detergent removal method is a superior method for preparing glycosaminoglycan-resistant and ph-sensitive lipid-coated dna complexes. this method is produced stable, but acid activatable, lipid-coated dna complexes (lehtinen et al. ) . at the neutral cellular ph , these lipids undergo protonation and collapse into a non-bilayer structure of endosomal compartmentalization which in turn helps in the rapid release of dna into the cytoplasm. recently, citraconyl-dope (a chemical derivative of dope), deliver dna-based therapeutics to cancer cells, in this manner combining the targeting and the rapid endosome release (reddy and low ) . addition of ph-sensitive fusogenic peptide, gala (peptide composed of repeating sequences of glu-ala-leu-ala) in lipidic preparation is also promising method to enhance the expression of the desired proteins. studies demonstrated that addition of . μm gala to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of lipofectin, but higher concentration of gala decreased transfection efficiency (futaki et al. ; nakase et al. ) . similarly, ph-sensitive histidine-modified galactosylated cholesterol derivative (gal-his-c -chol) has also been synthesized that demonstrate much greater transfection activity than conventional liposomes in hepg hepatic cells (shigeta et al. ). further, ph-sensitive tat-modified pegylated liposomes are utilized for delivery of tumor-specific stimuli-sensitive drug and gene delivery systems (kale and torchilin ) . immunoliposomes are sophisticated gene delivery systems in which incorporation of functionalized antibodies attached to lipid bilayers used for cell targeting (maclean et al. ) . using immunoliposomes, tissue-specific gene delivery has been achieved in the brain, embryonic and breast cancer tissue. recently, immunoliposomes containing an antibody fragment were successfully used in targeted delivery of tumor-suppressing genes into tumors in vivo (xu et al. ) . chloramphenicol acetyltransferase (cat) gene-encoded plasmid was entrapped in ph-sensitive immunoliposomes comprising of h- kk antibody-coated liposomes with dope, cholesterol, and oleic acid. studies revealed that approximately % of the injected immunoliposomes were taken up by the target rdm- cells. uptake was much less when liposomes without antibody were used (wang and huang ) . similarly, these authors have also reported that compositions of liposomes have altered the distribution for targeted drugs. delivery was also dependent on the lipid composition of the liposome. the ph-sensitive lipid composition gave eightfold higher efficiency than the corresponding ph-insensitive composition (wang and huang ) . ligand-modified immunoliposomes has been used to efficiently deliver plasmid dna expressing ns -ns b (hcv-specific antigenic sequence) to antigen-presenting cells. results confirm that this is as a more efficient delivery system than direct intramuscular inoculations with naked dna (zubkova et al. ). overall, studies have shown that immunoliposomes are efficiently used for targeted delivery of genetic material, especially in treatment of genetic disorders; however, very limited work has been done for delivery of dna vaccines. stealth liposomes (polyethylene glycol(peg)-conjugated lipids) are sterically stabilized liposomal formulations. pegylation prevents the liposomal vesicles by opsonization and recognition from the reticuloendothelial system and conjunction with other polymeric delivery systems such as pll to achieve longer circulation half-lives (mannisto et al. ) . peg grafted liposomes carrying antigenic epitope of gp , a transmembrane protein of hiv- has shown higher immune response and prolonged persistence of antibodies than plain liposome-based antigenic formulations (singh and bisen ) . further, it is also reported that grafting of peg on cationic liposomes have resulted in enhanced lymphatic drainage, but there is no improvement in immune responses, when compared to non-pegylated liposomes (carstens et al. ) . similarly, immune cell-specific ligand anchored pegylated liposomes have been developed to provide selective uptake at immunological cell. ultrasound (us)-responsive and mannose-modified gene carriers, man-peg ( ) bubble lipoplexes, have been utilized for transfer of ovalbumin (ova)-expressing plasmid dna to selectively and efficiently into antigenpresenting cells. developed systems have demonstrated - -fold higher gene expressions in the antigen-presenting cells (apcs) selectively in vivo compared with the conventional lipofection method (un et al. ). virosomes are lipidic envelope devoid of genetic information, which retain the antigenic profile and fusogenic properties from their viral origin. reconstituted lipid vesicles equipped with viral glycoproteins seems to possess many ideal properties for delivery of immunogens such as no limitation of size of encapsulated immunogens, high efficiency for cytosolic delivery, simplicity in handling and brevity of incubation time (okamoto et al. ) . virosome-mediated delivery has low toxicity and high immunogenicity with various prospective applications for the treatment and prevention of cancer, neurodegenerative disorders, and infectious diseases. the use of immunopotentiating reconstituted influenza virosomes (iriv) as delivery system of dna appear to be a promising tool in vaccinology and gene therapy. irivs are spherical, unilamellar vesicles with a mean diameter of~ nm, short surface projections of - nm. irivs are prepared by a mixture of natural and synthetic phospholipids containing % egg yolk phosphatidylcholine (for enhancement of immune responses), % synthetic phosphatidylethanolamine (able to directly stimulate b cells to produce antibodies), and % envelope phospholipids originating from h n influenza virus. irivs were first utilized in the manufacture of hepatitis a vaccine. the adjuvant function of virosomes is based on their virus-like particle structure providing repetitive antigen presentation to b cells, partial protection from extracellular degradation, and a depot effect (gluck et al. ) . proteasomes this immunogenic delivery system generally uses a noncovalent interaction between the proteosomes and antigen to form the appropriate complexes for delivering apolar or amphiphilic antigens. in most cases, these trials have involved intranasal administration of the vaccine and qualified as safe and well-tolerated materials through various human clinical trials. proteasome-conjugated shigella flexneri a lps vaccine shows an immune response similar to that observed after immunization with the live pathogen (fries et al. ) . intranasal delivery of proteasome-based vaccines may be able to produce both systemic and mucosal immunity. another very similar category of vaccines is the conjugate vaccine. these vaccines consist of a relatively non-immunogenic (especially in infants) antigen linked to a more immunogenic carrier such as a protein or toxoid. the conjugate vaccines for h. influenzae type b (hib) were developed using hib polysaccharide conjugated to either diphtheria toxoid (prp-d), omp of neisseria meningitidis (prp-omp), mutant diphtheria toxoid crm (hboc) or tetanus toxoid (prp-t) to provide the hib antigen immunogenic (heath ). cochleates are phospholipid calcium precipitates with a unique structure consisting of a large continuous solid lipid bilayer sheet rolled up into a "jelly roll-like" structure (papahadjopoulos et al. ) . cochleate delivery vehicles composed of simple, natural materials (phosphatidylserine and calcium) are unique vaccine carrier and delivery formulations (mannino and gould-fogerite ) . they are nontoxic, noninflammatory, and biodegradable. cochleates are prepared through the calcium-induced fusion of negatively charged phospholipid liposomes to collapse into solid sheets that roll up or stack, excluding water. the entire cochleate structure is a series of solid layers, components within the interior of the cochleate structure remain intact provides protection from degradation when exposed to harmful environmental conditions or enzymes. the protection of encochleated materials and structural stability of the cochleate allows for efficient delivery of dna by various routes like mucosal (oral, intragastric, intranasal, and intraocular) and parenteral (intramuscular, subcutaneous, intraperitoneal, and intradermal). strong, long-lasting, mucosal and circulating, antibody and cell-mediated responses are generated. protection from challenge with live viruses following oral or intramuscular administration has been achieved (mannino et al. ) . cochleates efficiency can be improved by attachment of surface glycoproteins of enveloped viruses and can be integrated into the lipid bilayers. dna cochleates can be formed by trapping oligonucleotides or high molecular weight plasmids within or between the lipid bilayers (papahadjopoulos et al. ). virus-like particles (vlps) are small particles consisting of one or more viral coat proteins can act as an adjuvant by carrying peptide sequences inside the apc and feeding into the endogenous processing pathway (schirmbeck et al. ) . these are safe, highly immunogenic, no additional adjuvant is needed, well tolerated, noninfective, and can easily be handled in the laboratory. it uses nature's own mechanism and structural principles to trigger the immune system for protective effects by stimulating both cellular immunity by effectively stimulating cd proliferative responses and cytotoxic t lymphocyte (ctl) responses and humoral immunity by efficiently cross-linking the membrane-associated immunoglobulin molecules that constitute the b-cell receptor (chackerian ; jennings and bachmann ; buonaguro et al. ) . the immune-stimulating complex (iscom) is a highly versatile and effective particulate antigen delivery system that has been extensively studied as an adjuvant system for a range of viral, bacterial, parasite, and other antigens. iscoms are threedimensional "cage-like" structures, which have been shown to form upon detergent removal from mixtures of saponins, detergents, and cholesterol. the iscom (immunostimulating complex) is a complex consisting of protein antigen, cholesterol, phospholipid, and the saponin adjuvant quil a. a similar vaccine delivery vehicle and adjuvant has also been developed that uses the same material minus the antigen and is referred to as iscomatrix ® . the antigen can be added later to the iscomatrix ® during formulation of the vaccine. this material seems to work similarly to iscoms but provides for more general applications by removing the requirement for hydrophobic antigens (pearse and drane ) . iscoms potentiate both humoral and cellular immune responses to incorporated antigens (cox et al. ) . iscoms stimulate apcs to produce il- , il- , and il- and induce thelper cells of both th and th type and the cell-mediated immune response includes cd + class i restricted cytotoxic t cells in a variety of experimental animal models and have now progressed to phase i and ii human trials (claassen and osterhaus ; barr and mitchell ) . oral administration of iscom vaccines has been shown effectiveness and immune-potentiating effect, but this route requires the use of high and frequent dosing. a study in which iscom vaccines may be able to elicit strong mucosal immune responses when administered in the pelvic presacral space of sheep, which could be useful for immunization against viral infections of the female genital tract (thapar et al. ) . a quil a-containing iscom with modified cholera toxin a (cta -dd) used as a mucosal vaccine carrier system for the influenza virus pr antigen (helgeby et al. -ji et al. ) . nasal vaccinations with p dna vaccine and matrix-m (immunostimulatory complex adjuvant) have shown significant higher iga-producing cells in addition to th and th cytokine expression. this strategies may provide a new way for the induction of specific immunity at mucosal sites (kodama et al. ). archaeosomes are nanometric size liposomes made from the polar ether lipids of archaea found in eukaryotes and bacteria. polar ether lipids of archaeosomes are providing excellent physicochemical stability and self-adjuvanting properties for delivery of vaccine preparations. archaeosomes have demonstrated relatively higher stabilities to oxidative stress, high temperature, alkaline ph, action of phospholipases, bile salts, and serum proteins (patel and chen ; benvegnu et al. ). archaeosomes facilitated a strong antibody (th ) response to entrapped protein antigens. the antibody humoral response was superior to that obtained with conventional liposomes and was in some instances comparable to that obtained with the potent but toxic freund's adjuvant (patel and sprott ; patel and chen ) . sprott et al. have also been described the role of co-enzyme q into archaeosome-based antigen formulation. incorporation of coq into archaeosomes and conventional liposomes can enhance the phagocytosis of the resultant vesicles by macrophage cells that allow the alteration in targeting profiles to specific tissues when the vesicles are administered to an animal via different routes and further enhance the immune response to coadministered immunogens. recently, "cationic archaeosomes," based on mixtures of neutral/cationic bilayerforming lipids and archaeobacterial synthetic tetraether-type bipolar lipids, have shown better transfection efficiency and can be utilized for dna vaccination (rethore et al. ). among the variety of lipid delivery systems, polymeric delivery systems have emerged as a promising alternative because of their ease of preparation, purification and chemical modification as well as their enormous stability. polymeric nonviral carriers (polyplexs) are one of the effective means of delivering a therapeutic or other biologically active substance in controlled and sustained manner. polymeric particulate delivery system induces adjuvant effect on the incorporated antigen and reduces the frequency of vaccination required to establish long-term protection. both natural and synthetic polymers have been considered to encapsulate antigenic materials for vaccination (table ) . various polymeric delivery systems have been developed using these polymers like micellar systems, emulsions, polymerosomes, nanoparticles, microspheres, nanocapsules, dendrimers, and dendrosomes (fig. ) . however, there are several associated concerns for the use of polymers as vaccines delivery systems such as toxicity, irritancy, allergenicity, and biodegradability. the advantages of using natural polymers include their low cost, biocompatibility and aqueous solubility. however, the natural polymers may also be limited in their use due to the presence of extraneous contaminants, variability from lot to lot and low hydrophobicity. in contrast, synthetic polymers are more reproducible and can be prepared with desired degradation rate, molecular weight and copolymer composition. nevertheless, synthetic polymers may be disadvantageous due to their limited solubility, they are often soluble only in organic solvents and consequently may not release biologically active antigen (rice-ficht et al. ). polymeric vaccines may offer improved stability and activity of encapsulated antigen materials by avoiding exposure to organic solvents used during formulation and acidic ph conditions caused by degradation of the polymer (duncan et al. ) . effective application of a polymeric nanoparticulate delivery system is greatly dependent on the specific polymer used, as this will dictate the properties of the nanoparticle in vivo (hanson et al. ) . for example, polycationic polymers can interact with negatively charged dna, resulting in a improved intracellular dna delivery to occur. whereas noncondensing polymers are neutral or slightly negatively charged polymers that physically encapsulate materials and can be used to target apcs and m-cells in the mucosa (bhavsar and amiji ) . there are a number of factors that affects the physicochemical properties of polymeric delivery vehicles like molecular weight, degree of branching, cationic charge density buffer capacity, polyplex properties and the experimental conditions like the polyplex concentration, the presence or absence of serum during transfection, the incubation time and the transfection model chosen for the gene delivery experiment. to reduce its cytotoxicity and improve transfection efficiency, polyplexes have been modified by conjugating with polyethylene glycol (peg), histidine, and targeting ligands including polysaccharides, transferrin, and galactose. various biodegradable polymers like aliphatic polyesters such as poly(lactic acid) (pla), poly(glycolic acid) (pga), poly(e-caprolactone) (pcl), poly (hydroxybutyrate) (phb), and their copolymers being evaluated for their uses as vaccine adjuvants and delivery systems (panyam and labhasetwar ) . recently, poly(amino acid)s-based copolymers have also been employed for the delivery of protein, vaccine, and genetic materials such as poly-l-glutamic acid, poly-l-aspartic acid, poly-l-lysine, poly-l-arginine, poly-l-proline, poly-l-asparagine, and poly-lhistidine. polyamino acids have properties that mimic proteins, making them ideal for vaccines delivery. they provide better adjuvanticity, low toxicity, biodegradability and targeting into intracellular compartments (chiang and yeh ) . various type of polysaccharides, such as agarose, alginate, carrageenan, hyaluronic acid, dextran, chitosan, cyclodextrins, mannan, and pullulan, have been used for delivery of vaccines (table ) . at specific concentrations and temperatures, when amphiphilic molecules, or molecules containing hydrophobic and hydrophilic regions, are maintained, naturally form association colloids known as amphiphilic micelles as a result of hydrophobic interactions. poly (ethylene glycol) (peg) is commonly incorporated as the hydrophilic segment in both amphiphilic micelles (gaucher et al. ) . gelatin: a denatured protein obtained by acid and alkaline processing of collagen. insoluble in water to prepare hydrogel through chemical cross-linking, with water-soluble carbodiimides and glutaraldehyde lou et al. ( ) easy processability, good biodegradability poor mechanical properties, brittle capable of targeting fibronectinbearing surfaces associated with some tumors silk fibroin: silkworm bombyx mori produces silk to weave its cocoon, and its major components are fibroin and sericin. this is light weight, extremely strong and elastic and exhibits mechanical properties comparable to the best synthetic fibers produced by modern technology zhang et al. ( ) environmentally safe, biocompatibility, excellent mechanical properties less production, high brittleness model antigen enhance the stability, up to c over more than months fibrin: fibrin is a protein matrix produced from fibrinogen, providing an immune-compatible carrier for delivery of active biomolecules, antigens. fibrin naturally contains sites for cell binding and has been investigated as a substrate for cell linkage, distribution, relocation, and propagation khan et al. ( ) induce improved cellular interaction, used as a cell carrier as well as antigen carrier rapid degradation, instable, low mechanical stiffness elastin: elastin is synthesized by vascular smooth muscle cells and secreted as a tropo-elastin monomer that is soluble, hydrophobic and non-glycosylated. elastin is a potent regulator of vascular smooth muscle cells activity, regulations important for preventing fibro-cellular pathology gaudreau et al. ( ) conferring elasticity, precise molecular weight, low polydispersity become insoluble and aggregate at a critical temperature soybean: the most cultivated plant in the world is rich in proteins ( - %), carbohydrates ( - %),and lipids ( - %). it is a species of legume native that can be processed into protein-rich products moravec et al. ( ) abundant, renewable, inexpensive, environment friendly biodegradable application of soy-based polymers in this field is still very narrow iga, mucosal iga antibody response after administered orally to mice chitosan: fully/partially deacetylated form of chitin. degree of deacetylation of commercial chitosan is usually between and %, and the molecular weight between and , kda. chitosan exhibits a ph-sensitive behavior as a weak polybase due to the large quantities of amino groups on its chain verheul et al. ( ) enhanced immune response, mucoadhesive property poly(ester-amide)s: this polymer is made up of a soft peg segment, connected to a hard diester-diamide segment through an ether bond. it is a high performance thermoplastic elastomer. it is used to replace common elastomers-thermoplastic polyurethanes, polyester elastomers, and silicones-for these characteristics: lower density among tpe, superior mechanical and dynamic properties (flexibility, impact resistance, energy return, fatigue resistance) and keeping these properties at low temperature (lower than À c), and good resistance against a wide range of chemicals. it is sensitive to uv degradation li and hu ( ) enhanced cell mediated immunity, superior mechanical and thermal properties poly(lactide-co-glycolide)(plga): plga or poly(lactic-co-glycolic acid) is copolymer, is synthesized by means of random ring-opening copolymerization of two different monomers, the cyclic dimers ( , -dioxane- , -diones) of glycolic acid and lactic acid. during polymerization, successive monomeric units (of glycolic or lactic acid) are linked together in plga by ester linkages, thus yielding a linear, aliphatic polyester as a product moore et al. ( ) degradation products are naturally occurring metabolites and readily absorbed by neighboring cells generate acidic environment and effect the stability yersinia pestis, hiv gp dominant th response block copolymer micelles are colloidal particles with a size around - nm, which are currently under investigation as carriers for delivery of biopharmaceuticals. in contrast to cationic polymeric systems, nonionic polymers enhance gene expression through mechanisms, which most likely do not involve dna condensation and facilitated transport within cells. adjuvant-active nonionic block copolymers that are flexible, linear structures, flanked on both ends by hydrophilic polyoxyethylene (poe) with a core of hydrophobic polyoxypropylene (pop) with variable ratios (newman et al. ) . the block copolymers are useful as general surfactants and display enhanced biological efficacy as vaccine adjuvants. osmolarity, ph and buffer salts mainly affected the size and morphology of the particles. molecular weight and formulation mainly affected titer and isotype of antibody. jain et al. evaluated a system of combined poly(lactic acid) (pla) and poly(ethylene glycol) (peg) for the delivery of a recombinant hepatitis b surface antigen (hbsag). pla forms the hydrophobic core in an aqueous medium, which controlling the release of the antigen as it degrades into lactic acid. an outer shell form by peg allows for prolonged release patterns and enhanced mucosal uptake to occur (jain et al. ). hunter et al. ( ) showed that the adjuvant activity of block copolymers varies with the lengths of the chains of polyoxypropylene (pop) and polyoxyethylene (poe). pluronic block copolymers have been used extensively in a variety of pharmaceutical formulations like low molecular mass drugs and polypeptides. kabanov et al. ( ) described that these molecules can modify the biological response during gene therapy in the skeletal muscle, resulting in an enhancement of the transgene expression and therapeutic effect of the transgene. block copolymers were recently used to promote gene delivery of plasmid encoding a food allergen, bovine beta-lactoglobulin. tetronic based block copolymers have decreased blg-specific ige concentrations and reduced local inflammatory response (adel-patient et al. ) . similarly, triblock copolymers consisting of three alternating hydrophobic and hydrophilic segments are also used to delivery genetic materials. biodegradable and nontoxic triblock copolymers of pla-peg-pla and plga-peg-plga were also utilized micellar carriers for delivery of encapsulated plasmid pcdna . (+)-ma against hcv. developed carrier system has provided long-term better adjuvant effect with no side effects (yang et al. ) . similarly, copolymers of a hydrophilic poly(ethylene glycol) block and a cationic poly(aminoethyl methacrylate) (paem) block have been used for dna vaccine delivery. synthesized polyplexes based carrier systems have induced a modest up-regulation of surface markers for dc maturation and better uptake by dcs in the draining lymph nodes (tang et al. ). further, cationic block copolymers poly(ethylene glycol) (peg) with a positively charged poly(dimethylamino)ethyl methacrylate have been synthesized and utilized for hiv- tat dna molecules. results indicated that synthesized cationic block copolymers was safe and ability to deliver genetic material for cell machinery and promising candidate for dna vaccination (caputo et al. ) . similar to cationic block polymers, nonionic block copolymers of poly(ethyleneoxide)-poly (propyleneoxide) (peo-ppo) have also been utilized dna vaccination using a beta-galactosidase (betagal) encoding plasmid (mcilroy et al. ). herpes simplex virus type- genes specifying glycoproteins gb and gd have been also delivered by nonionic block copolymers. plasmid-encapsulated block polymers have protected the mice against lethal hsv- challenge when immunization was performed via the i.m. route (baghian et al. ) . dendrimers are a unique class of polymeric nanoconstructs having highly branched, three-dimensional, nanoscale architecture with very low polydispersity and high functionality. first discovered in the early s by donald tomalia and coworkers, these hyperbranched molecules were called dendrimers. dendrimers are highly branched, synthetic spherical macromolecules with layered architectures that can be considered analogous to a globular protein. they have the potential for high loading capacities due to small diameters ( . - . nm) through mechanisms such as complexation or formation of chemical bonds at terminal branch points or other active sites (wiwattanapatapee et al. ) . in addition, the low polydispersity of dendrimers should provide reproducible pharmacokinetic behavior in contrast to that of some polymers containing fractions with vastly different molecular weight within a given sample (parekh ) . several dendrimer-based products have been approved by the fda and successfully commercialized for treatment and diagnosis of diseases, including vivagel™ (starpharma) designed as a topical microbicide, superfect ® , (qiagen pvt ltd.) used for gene transfection, and alert ticket™ (us army research lab) for anthrax detection (merdan et al. ) . in the past decade, research has increased on polyamidoamine, polyethylenimines, polylysine, polypropyleneimine, polyaryl ether, polyester, polyglycerol and their derivatives for the design and synthesis of biocompatible dendrimers. dendrimers form complexes by electrostatic interaction with all forms of nucleic acids such as dna, rna, and antisense oligonucleotides. the nature of the dendrimer-nucleic acid complexes ("dendriplexes") is dependent on the stoichiometry and concentration of the dna-phosphates, dendrimer amines, bulk solvent properties (e.g., ph, salt concentration, buffer strength), and even the dynamics of mixing. high ionic strength interferes with the binding process and affects the nature of complexes formed by the different generations, for example, highergeneration ppi dendrimers in higher concentrations form water-soluble dendriplexes, whereas the g and g ppi dendrimers lead to the formation of electroneutral complexes (tang and szoka ) . dendrimer-dna complex is formed by simply mixing the components in an aqueous solution. transfection property can be improved by the use of an excess of cationic dendrimer because the negatively charged phosphate groups on the dna neutralize the positively charged amine groups on the dendrimer through electrostatic interaction and an overall positively charged system is important in cell uptake (bielinska et al. ) . immunogenicity and efficacy of dna vaccines can be improved by physical conjugation of the pamam dendrimer to the mhc class ii-targeting peptide. therefore, dendrimers can be further explored for dna-based vaccine development against malaria parasite (pietersz et al. ). in a recent study, dendriplexes, complexes of dendrons and condensed plasmids containing the gene for protective antigen (pa) of bacillus anthracis, were encapsulated in polylactide-co-glycolide (plg) particles using the double emulsion method. studies indicated that the plg-dendriplex particles produced superior levels of anti-pa igg antibodies when compared to animals immunized with the plg particles (ribeiro et al. ). conjugation of fifth-generation polyamidoamine (g -pamam) dendrimers, a dna-loading surface, with mhc class ii-targeting peptides that can selectively deliver these dendrimers to apcs under conditions that enhance their immune stimulatory potency. dna conjugated with this platform efficiently transfected murine and human apcs in vitro. subcutaneous administration of dna-peptide-dendrimer complexes in vivo preferentially transfected dendritic cells (dc) in the draining lymph nodes, promoted generation of high affinity t cells, and elicited rejection of established tumors. taken together, our findings show how pamam-dendrimer complexes can be used for high transfection efficiency and effective targeting of apcs in vivo, conferring properties essential to generate effective dna vaccines. multiple antigenic peptide (map) dendrimer system is being used for vaccine and immunization purposes. map-based delivery can prepare by addition of multiple immune-functional components, like b/t-cell epitopes, cell-penetrating peptides, and lipophilic moieties or by controlled synthesis of nanomaterials like micelles, dendrimers, and nanoparticles (fujita and taguchi ) . a tetravalent multiple antigen peptide (map) dendrimer with four identical branches of a c-terminal peptide sequence of the rat gh-bp (gh-bp - ) was synthesized and used as an immunogen in rabbits. the tetravalent rat gh-bp - map dendrimer served as an effective immunogenic antigen in eliciting specific antibodies (aguilar et al. ). similar to map dendrimers, glycopeptide dendrimers containing both carbohydrates and peptides can be also used in delivery of vaccine components (niederhafner et al. ; sebestik et al. ) . the encapsulated dendrimer-nucleic acid complex within a lipophilic shell known as dendrosomes. these are novel vesicular, spherical, supramolecular entities and possess negligible hemolytic toxicity and higher transfection efficiency. dendrosome are reported to be completely nontoxic both in vitro as well as in vivo. poly (propyleneimine) dendrosome-based genetic immunization found to be highly effective against hepatitis b when compared to dendrimer-plasmid dna complex, and the results indicate that dendrosomes hold great potential in dna vaccination. in dendrosomes, the poly(propyleneimine) dendrimer-dna complex is largely protected by multilamelarity of the vesicles. it has been reported that polyamidoamine dendrimer-based dendrosomes are efficient systems for the delivery of s sirna targeting e /e oncogenes in cervical cancer (pourasgari et al. ). in vitro superior transfection efficiency displayed by pamam dendrosomes as comparison to other nonviral gene delivery vectors. nontoxic self-assembled dendritic spheroidal nanoparticles (den ) have been used for the delivery of pcmv-betv loaded dendritic spheroidal nanoparticles (den ) have shown low toxicity, enhanced transfection efficiency, and improved the immune response against birch pollen allergy (balenga et al. ) . similarly, efficiency of dendrosome (a gene porter) is assessed in transferring recombinant human rotavirus vp cdna. studies revealed that dendrosome has lower cytotoxicity and better transfectivity in a , a human lung cell line (pourasgari et al. ). dendrosome has been used to deliver the dna vaccines encoding hiv- p -gp gene. studies have proved the efficacy of this carrier for the delivery of recombinant plasmids construct (roodbari et al. ) . polymersomes are self-assembled polymeric colloidal vesicular systems containing aqueous inner core. polymersomes are made up from amphiphilic block copolymers that allow polymersomes to stably encapsulate or integrate a broad range of active molecules. the aqueous core can be utilized for the encapsulation of therapeutic hydrophilic molecules and the membrane can integrate hydrophobic drugs within its hydrophobic part. further, the brush-like surface properties of the polymersome can provide better biocompatibility and blood circulation times. these systems have better loading efficiency, stabilities and provide sustained, controlled release of encapsulated therapeutics. further, these systems have also been used to deliver biotherapeutics, especially peptides, proteins, and nucleic acids to site-specific cellular environment due to escape from endolysosomes (levine et al. ; christian et al. ). amphiphilic diblock copolymer of poly (oligoethylene glycol methacrylate)-block-poly( -(diisopropylamino)ethyl methacrylate in association with tannic acid forms dna-loaded polymersomes. developed systems have demonstrated better cytosolic release of encapsulated nucleic acid materials (lomas et al. ) . further, calcein-loaded polymersomes have also observed for their cytosolic delivery within dendritic cell (scott et al. ) . similarly, poly(g-benzyl-l-glutamate)-k (pblg -k) polymersomes have been used for delivery of influenza hemagglutinin antigen. the immunogenicity and adjuvanticity of developed polymerosomes was better for administered the influenza antigen. in future, this nanostructured polymeric vesicular system may have huge potential for delivery of protein and dna vaccines. emulsions can be manufactured as water-in-oil (w/o) or oil-in-water (o/w) particulate carrier systems. emulsion carrier systems are similar in size to pathogens and taken up by epithelial or m cells in the mucosal surfaces for successive delivery of the vaccine component to apcs and lymphoid tissue. a nanoemulsion formulation of intranasal hepatitis b vaccine showed improved vaccine efficacy, stability and ease of distribution (makidon et al. ) . multiple emulsion formulations can also be used as vaccine carrier systems due to its longer stability and high entrapment efficiency of protein antigens without damage during emulsification procedures. types of surfactants, processing methods and stabilizers is requisite for making stable multiple-emulsions (hanson et al. ) . the emulsion adjuvant mf immunostimulator has been shown to result in the recruitment of antigen-presenting cells (apcs) to the site of injection and to increased uptake of soluble antigen by the apcs. it has been formulated by a simple mixing of the antigen with the adjuvant and has shown excellent compatibility with a variety of subunit antigens. mf shows strong immunogenicity as comparison to other adjuvant is clearly seen in pre-clinical data published by ott et al. they reported that when immunized guinea pigs and goat with glycoprotein d of herpes simplex virus (hsv) type in the presence of mf showed a -fold and ninefold increases in antibody titers compared to aluminum hydroxide, respectively (ott et al. ). an oil-in-water (o/w) emulsion, syntex adjuvant formulation (saf) is an effective adjuvant composed of a muramyl dipeptide derivative (threonyl-mdp). threonyl-mdp demonstrated a lack of side effects (pyrogenicity, uveitis, adjuvant-induced arthritis) and increased adjuvant activity. saf adjuvant used with a variety of antigens, such as influenza and malaria, and showed both cellmediated and humoral immune responses. saf, or a suitable equivalent, provides an excellent tool for vaccine research (lidgate et al. ; lidgate et al. ) . there are several different types of montanide™, including isa v, , . isa and have been used in human's vaccine formulations, while isa and v have been used only in veterinary vaccine formulations. they are composed of metabolizable squalene-based oil with mannide monooleate emulsifier and permit antigens to be released more rapidly. the montanide emulsions induce high antibody titers and ctl responses due to the formation of a depot at the site of injection. these emulsions have been used as vaccines against malaria, hiv and various cancers and found to be safe and fairly well tolerated (lawrence et al. ; toledo et al. ). various physical delivery methods are being heavily investigated because of direct transfection of apcs with the dna vaccine (porgador et al. ). the transcutaneous microneedle has the ability to bypass the stratum corneum layer of the skin, thus reaching langerhans cells-the apcs of the skin. jet-injection mechanical devices deliver dna vaccines into the viable epidermis and increased efficacy in the prevention and/or therapy of infectious diseases, allergic disorders and cancer (chen et al. ; imoto and konishi ; roberts et al. ) . the tattooperforating needle device has been used to puncture the skin and transfer dna into skin-associated cells. the bundles of fine metal needles that oscillate at a constant high frequency have shown better expression of reporter genes in mice and induction of immune responses. electroporation has been extensively studied to deliver therapeutic genes that encode a variety of hormones, cytokines, enzymes or antigens in large animal species such as dogs, pigs, cattle and nonhuman primates. several different strategies of this technology are being pursued. however, too little is currently known about several of these devices and much additional research in this area is warranted (van drunen littel-van den hurk et al. ; roos et al. ; hirao et al. ). nanotechnology is the development of engineered devices due to their small size at the micromolecular level in the nanometer range and large surface area, which enhances their action for early diagnosis of cancer and infectious diseases. advances in nanotechnology have also proved to be beneficial in therapeutic fields such as drug discovery, drug delivery and gene/protein delivery. this concept has been found to be useful in developing nanovaccines using different routes of administration like oral, nasal and parenteral. the oral route is the most popular and convenient route of administration. oral delivery refers to absorption from the buccal through the rectal mucosa. several barriers associated with genetic vaccination through the oral are generally attributed to (a) low permeability across biological membranes, (b) harsh gastric environment, (c) hepatic first-pass metabolism, and (d) chemical instability. the major drawback with oral route of administration is a higher concentration and is required for the vaccine to be effective due to dilution during the transport of the vaccine through the gastrointestinal tract. to date, most gene delivery strategies have concentrated on the parenteral route of delivery and oral administration has been largely ignored. different nano-and microparticulate delivery systems using natural and synthetic lipid and polymers have been utilized to improve the stability and immunogenicity of oral dna vaccines (bhavsar and amiji ) . oral vaccination with dna-chitosan nanoparticles has appeared interesting because of their great stability and the ease of target accessibility, besides chitosan immunostimulatory properties. studies demonstrated that % of protection against parasite infection after delivery chitosan nanoparticles loaded with dna encoding rho -gtpase protein of schistosoma mansoni (oliveira et al. ) . similarly, chitosan nanoparticles are used for dna vaccine against vibrio anguillarum through oral route. studies revealed that chitosan-dna (pvaomp ) complex showed moderate protection against experimental v. anguillarum infection after oral vaccination in asian sea bass (rajesh kumar et al. ) . the orally administered tresylmonomethoxypolyethylene glycol (tmpeg) grafted liposome complexes with modified vaccinia virus ankara (mva(iiib/beta-gal) is also capable of delivering the transgenes to mucosal tissues and enhances the env-specific cellular and humoral immune responses after repeated oral immunization of balb/c mice (naito et al. ) . mannosylated niosomes loaded with hepatitis dna have shown humoral (both systemic and mucosal) and cellular immune response upon oral administration (jain et al. ) . chitosan-coated and polyplex-loaded liposomes (plls) containing plasmid prc/cmv-hbs are developed for oral delivery of vaccines specifically for targeting to peyer's patch. chitosan-coated pll demonstrated better uptake of encapsulated dna to the distal intestine and provide better stability from enzymatic degradation (channarong et al. ) . the nasal route has been chiefly employed for producing local action on the mucosa. this route has a number of advantages, such as the high permeability of the nasal epithelium, which allows a higher molecular mass cut-off for permeation of approximately , da, as well as the rapid drug absorption rate. accurate and repeated dispensing of vaccine, mucociliary clearance, presence of peptidases, proteases and nuclease enzymes in the mucus or associated with nasal membrane, variation in extent of absorption with the mucus secretion and mucus turnover and deposition of the formulated vaccine to all areas of the nasal mucosa (especially lymphoid tissues), potential of uptake of vaccine formulations by the primary olfactory nerves in the nasal cavity, local irritation and unpleasant taste from concentrated drug reaching the mouth are major challenges associated with intranasal delivery of vaccines (oliveira et al. ; sharma et al. ). these problems can be overcome by design of appropriate antigen carriers. nanocarriers for nasal vaccines are able to facilitate the transport of the associated antigen across the nasal epithelium, thus leading to efficient antigen presentation to the immune system and provide the protection and stability of encapsulated genetic materials (koping-hoggard et al. ) . further, use of mucoadhesive agents offers a strategy for the facilitation of increased residence time and increased vaccine efficacy (alpar et al. ) . polycarbophil (pc) or polyethylene oxide (peo)-based in-situ mucoadhesive polymers have demonstrated better nasal absorption of plasmid dna (park et al. ) . several studies have proven that wide applicability of chitosan nanoparticles for the nasal delivery of dna vaccines like severe acute respiratory syndrome coronavirus (sars-cov) (raghuwanshi et al. ) , pneumococcal surface antigen a (psaa) (xu et al. ) , hepatitis b antigen-encoding plasmid (khatri et al. ) , and dna plasmid-expressing epitopes of respiratory syncytial virus (iqbal et al. ) . further, several modification on the chitosan polymers have also been made to improve the potential of chitosan nanoparticles for nasal administration of dna vaccines like preparation of low molecular weight chitosan, development of water soluble chitosan (n-trimethyl chitosan), etc. blends of poly(lactic-co-glycolic acid) (plga) and polyethylene oxide (peo) have exhibited the capacity to associate and release plasmid dna in a controlled manner. results showed that dna-loaded nanoparticles elicit significantly pronounced immune response compared to the naked plasmid dna for up to weeks (csaba et al. ) . dry-powder influenza virosomes-based vaccines have also been advantageous for mucosal immunization (de jonge et al. ). needle-free nasal immunization, using nanoemulsion is made of soya bean oil, alcohol, water and detergents emulsified into droplets of nm, has been reported to be a safe and effective hepatitis b vaccine (makidon et al. ) . the release of liquid or particles into the airflow enters one nostril via a sealing nozzle and exits through the other nostril and minimizes the risk and problems related to deposition of particles in the lung, which occurs during conventional inhalation from a nebulizer and increases the delivery of particles to the posterior part of the nasal mucosa. encapsulation of the antigen into bioactive nanoparticles is a promising approach to nasal vaccine delivery (slutter et al. ) . the ocular route holds immense potential for peptides/proteins intended for pathological ophthalmologic conditions. the eye mucosa is a possible route for mucosal vaccine because it is an important entry point for environmental antigens and infectious materials occupying most of the external ocular surface (streilein et al. ) . lymphoid follicles are found in close association with the epithelium of the conjunctival mucosa in humans, rabbits, guinea pigs, dogs, pigs, and many other mammals (chodosh et al. (seo et al. ) . ocular mucosal delivery of peptide epitopes of herpes simplex virus (hsv- ) glycoprotein d (gd) has mixed with oligodeoxy nucleotides containing unmethylated cpg motifs (cpg ). results suggested enhanced local and systemic immune response after multi-instillation of gd peptide epitopes with cpg adjuvants (nesburn et al. ) . ocular mucosal administration of iron nanoparticles with glutamic acid containing dna vaccine herpes stromal keratitis (prsc-gd-il- ) have confers protection against mucosal challenge with herpes simplex virus type in mice (hu et al. ) . vaginal mucosa is a portal of entry to many viral and bacterial pathogens. vaginal route serves as a potential site of drug administration for local and systemic absorption of therapeutically important molecules, proteins, peptides, small interfering rnas, oligonucleotides, antigens, vaccines and hormones (hussain and ahsan ) . it is one of alternative site for the systemic delivery of protein drugs because of the relatively high permeability of the vaginal epithelium, by passage of the hepatic first-pass metabolism, large surface area and rich blood supply (gupta et al. (gordon et al. ) . thermo-sensitive mucoadhesive vaginal vaccine delivery systems have also been tested for the local and systemic antibody responses to hpv l virus-like particles (park et al. ) . vaginal delivery of vaccines which is associated with vaginal infection could be better alternative to induce an immune response in the genital mucosa capable of controlling the entry of the pathogen. noninvasive gene delivery approaches could be able to deliver and express naked plasmid dna to tissue-specific localized delivery to skin. there are several advantages of needle-free noninvasive gene administration such as limited toxicity, potential cell receptor-independent uptake, minimal dna size restrictions, and the potential for multiple treatments via a relatively uncomplicated administration modality, thus improving patient compliance. topically applied formulation, especially nanosystems have been shown to enter skin, accumulate in hair follicles, diffuse via dendritic cells to draining lymph nodes, and elicit antigen-specific humoral and cell-mediated immunity (nasir ). a number of methods have been developed to perform noninvasive topical gene delivery, which includes passive diffusion of genetic materials between a skin patch and skin, as well as active processes such as iontophoresis, sonophoresis, electroporation, and chemically enhanced diffusion (mehier-humbert and guy ). topical vaccination has been achieved using topical application of naked dna with or without tape stripping and dna/lipid-based complex such as liposomes, niosomes, transfersomes, or microemulsion (cui and sloat ) . ethanol-in-fluorocarbonbased microemulsion has been for topically delivery of anthrax protective antigen (pa) protein-encoding dna vaccine (pgpa). pgpa-loaded microemulsion has significantly enhanced the anti-pa antibody responses (cui and sloat ) . similarly, dna delivery by novel lipid-based biphasic delivery system has significant deliver plasmid dna into the "viable" layers of skin (foldvari et al. ) . plasmid dna-encoding hepatitis b surface antigen (hbsag)-loaded cationic transfersomes are also utilized for topical immunization. results revealed that dna-loaded cationic transfersomes elicited significantly higher anti-hbsag antibody titer and cytokines level as compared to naked dna. it was also observed that topical application of dna-loaded cationic transfersomes elicited a comparable serum antibody titer and endogenous cytokines levels as produced after intramuscular recombinant hbsag administration (mahor et al. ) . -or -nm sized polystyrene nanoparticles have been studied to target active compounds to the hair follicle and may result in a better penetration and higher efficiency of compound uptake by skin resident cells. studies demonstrated that and nm nps and modified vaccinia ankara (mva) expressing the green-fluorescent protein penetrated deeply into hair follicles and uptake by apcs and transport to the draining lymph nodes (mahe et al. ). nanoengineered genetic vaccine formulation has been developed for topical immunization comprising of emulsifying wax (oil phase), ctab (cationic surfactant), mannan (dc ligand), dioleoylphosphatidylethanolamine (endosomolytic agents), and cholesterol. all pdna-coated nanoparticles, especially the mannan-coated pdna-nanoparticles with dope, have shown significant immune response (igg titers; -fold over "naked" pdna alone) (cui and mumper ) . diffusion patches and tape stripping techniques are used for delivery of small (< da) and large molecules, respectively. liquid jet injector is an approach in which dna vaccine is delivered around the langerhans cells by a high-speed injector. (chen et al. ) reported that particle-mediated gene-gun dna immunization use similar mechanical devices to deliver dna vaccines into viable epidermis (chen et al. ) . microneedle arrays is a set of needles of microscale length with their nanoscale tips coated with dna and can accurately, efficiently and safely deliver biomolecules to the viable cells of the epidermis. recently, tran et al. ( ) developed a unique nanoliposomal ultrasound-mediated device for delivering small interfering rna (sirna) specifically targeting melanocytic tumors present in the skin and they observed that decrease early melanocytic lesion development in the skin and prevent the spread of cutaneous metastases of melanoma (tran et al. ) . these results suggested that skin may provide an appealing, noninvasive route of delivery for dna vaccines and other therapeutic genes. table represents positive and negative aspects of various routes of administration, which are very helpful for selection of particular route. novel vaccine carriers, adjuvant, vehicles, and particle-based delivery strategies are being evaluated in a variety of vaccines, including those against diseases such as cancer, malaria, aids, hepatitis, etc., in which a cellular and/or mucosal immune response is desired. various immunity responses were generated by different adjuvant like mf and mpl ® generated th responses, vlps, virosomes, nondegradable nanoparticles, and liposomes generated cellular immune responses in humans. viral vectors, iscoms and montanide™ isa , and various nanoparticulate immunopotentiators and antigen delivery vehicles have shown ctl responses. the desirable responses can be achieved by using combination of various adjuvants. systemic antibodies produced in humans when viral-vectored vaccines as well as proteasomes given in. the clinical trials required for vaccine approval are often very long and difficult. furthermore, since many vaccines are often administered to healthy individuals, and frequently to infants, it is critical that they are proven safe and well tolerated in nonhuman primates before entering human trials. while the development of novel vaccine delivery systems and adjuvant has been aided by nanotechnology, it must be necessary to perceived potential problems such as their high surface area and reactivity, the ability to cross biological membranes, slow biodegradability of some materials, its safety and tolerability before its approval. many challenges must be met before new classes of vaccines become available like ability to stimulate humoral, cellular and mucosal immune responses, longer duration response, easily metabolized of vaccine components in body, cost-effective production, and lesser risk and less invasive approaches for the administration of vaccinations. as these challenges are met, the prevention and therapy of many previously untreatable diseases should become increasingly possible. block copolymers have differing adjuvant effects on the primary immune response elicited by genetic immunization and on further induced allergy map dendrimer elicits antibodies for detecting rat and mouse gh-binding proteins comparison of immune response generated against japanese encephalitis virus envelope protein expressed by dna vaccines under macrophage associated versus ubiquitous expression promoters dna vaccines: technology and 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chitosan-dna nanoparticles expressing pneumococcal surface antigen a protects mice against nasopharyngeal colonization by streptococcus pneumoniae immunogenicity of multiple-epitope antigen gene of hcv carried by novel biodegradable polymers construction of dna and rna based on bifunctional replicon vector derived from semliki forest virus enhanced potency of individual and bivalent dna replicon vaccines or conventional dna vaccines by formulation with aluminum phosphate stabilization of vaccines and antibiotics in silk and eliminating the cold chain t-cell vaccines that elicit effective immune responses against hcv in chimpanzees may create greater immune pressure for viral mutation key: cord- -nu ok w authors: varshney, deeksha; ekbal, asif; nagaraja, ganesh prasad; tiwari, mrigank; gopinath, abhijith athreya mysore; bhattacharyya, pushpak title: natural language generation using transformer network in an open-domain setting date: - - journal: natural language processing and information systems doi: . / - - - - _ sha: doc_id: cord_uid: nu ok w prior works on dialog generation focus on task-oriented setting and utilize multi-turn conversational utterance-response pairs. however, natural language generation (nlg) in the open-domain environment is more challenging. the conversations in an open-domain chit-chat model are mostly single-turn in nature. current methods used for modeling single-turn conversations often fail to generate contextually relevant responses for a large dataset. in our work, we develop a transformer-based method for natural language generation (nlg) in an open-domain setting. experiments on the utterance-response pairs show improvement over the baselines, both in terms of quantitative measures like bleu and rouge and human evaluation metrics like fluency and adequacy. conversational systems are some of the most important advancements in the area of artificial intelligence (ai). in conversational ai, dialogue systems can be either an open-domain chit-chat model or a task-specific goal-oriented model. task-specific systems focus on particular tasks such as flight or hotel booking, providing technical support to users, and answering non-creative queries. these systems try to generate a response by maximizing an expected reward. in contrast, an open-domain dialog system operates in a non-goal driven casual environment and responds to the all kinds of questions. the realization of rewards is not straightforward in these cases, as there are many factors to model in. aspects such as understanding the dialog context, acknowledging user's personal preferences, and other external factors such as time, weather, and current events need consideration at each dialog step. in recent times, there has been a trend towards building end-to-end dialog systems such as chat-bots which can easily mimic human conversations. [ , , ] developed systems using deep neural networks by training them on a large amount of multi-turn conversational data. virtual assistants in open-domain settings usually utilize single-turn conversations for training the models. chitchat bots in such situations can help humans to interact with machines using natural language, thereby allowing humans to express their emotional states. in dialogue systems, generating relevant, diverse, and coherent responses is essential for robustness and practical usages. generative models tend to generate shorter, inappropriate responses to some questions. the responses range from invalid sentences to generic ones like "i don't know". the reasons for these issues include inefficiency of models in capturing long-range dependencies, generation of a large number of out-of-vocabulary (oov) words, and limitations of the maximum likelihood objective functions for training these models. transformer models have become an essential part of most of the state-of-the-art architectures in several natural language processing (nlp) applications. results show that these models capture long-range dependencies efficiently, replacing gated recurrent neural network models in many situations. in this paper, we propose an efficient end-to-end architecture based on the transformer network for natural language generation (nlg) in an open-domain dialogue system. the proposed model can maximize contextual relevancy and diversity in generated responses. our research reported here contributes in three ways: (i) we build an efficient end-to-end neural architecture for a chit-chat dialogue system, capable of generating contextually consistent and diverse responses; (ii) we create a singleturn conversational dataset with chit-chat type conversations on several topics between a human and a virtual assistant; and (iii) empirical analysis shows that our proposed model can improve the generation process when trained with enough data in comparison to the traditional methods like retrieval-based and neural translation-based. conversational artificial intelligence (ai) is currently one of the most challenging problems of artificial intelligence. developing dialog systems that can interact with humans logically and can engage them in having long-term conversations has captured the attention of many ai researchers. in general, dialog systems are mainly of two types -task-oriented dialog systems and open-domain dialog systems. task-oriented dialog systems converse with the users to complete a specific task such as assisting customers to book a ticket or online shopping. on the other hand, an open-domain dialog system can help users to share information, ask questions, and develop social etiquette's through a series of conversations. early works in this area were typically rule-based or learning-based methods [ , , , ] . rule-based methods often require human experts to form rules for training the system, whereas learning-based methods learn from a specific algorithm, which makes it less flexible to adapt to the other domains. data from various social media platforms like twitter, reddit, and other community question-answering (cqa) platforms have provided us with a large number of human-to-human conversations. data-driven approaches developed by [ , ] can be used to handle such problems. retrieval based methods [ ] generate a suitable response from a predefined set of candidate responses by ranking them in the order of similarity (e.g., by matching the number of common words) against the input sentence. the selection of a random response from a set of predefined responses makes them static and repetitive. [ ] builds a system based on phrase-based statistical machine translation to exploit single turn conversations. [ ] presented a deep learning-based method for retrieval-based systems. a brief review of these methods is presented by [ ] . lately, generation based models have become quite popular. [ , , , ] presented several generative models based on neural network for building efficient conversational dialog systems. moreover, several other techniques, for instance generative adversarial network (gan) [ , ] and conditional variational autoencoder (cvae) [ , , , , , ] are also implemented for dialog generation. conversations generated from retrieval-based methods are highly fluent, grammatically correct, and are of good quality as compared to dialogues generated from the generative methods. their high-quality performance is subjected to the availability of an extensive repository of human-human interactions. however, responses generated by neural generative models are random in nature but often lack grammatical correctness. techniques that can combine the power of both retrieval-based methods and generative methods can be adapted in such situations. on the whole hybrid methods [ , , , ] first find some relevant responses using retrieval techniques and then leverages them to generate contextually relevant responses in the next stage. in this paper, we propose a novel method for building an efficient virtual assistant using single-turn open-domain conversational data. we use a self-attention based transformer model, instead of rnn based models to get the representation of our input sequences. we observe that our method can generate more diverse and relevant responses. our goal is to generate contextually relevant responses for single-turn conversations. given an input sequence of utterance u = u , u , ..., u n composed of n words we try to generate a target response y = y , y , ..., y m . we use pre-trained glove [ ] embeddings to initialize the word vectors. glove utilizes two main methods from literature to build its vectors: global matrix factorization and local context window methods. the glove model is trained on the non-zero entries of a global word to word co-occurrence matrix, which computes how frequently two words can occur together in a given corpus. the embeddings used in our model are trained on common crawl dataset with b tokens and . m vocab. we use -dimensional sized vectors. we formulate our task of response generation as a machine translation problem. we define two baseline models based on deep learning techniques to conduct our experiments. first, we build a neural sequence to sequence model [ ] based on bi-directional long short term memory (bi-lstm) [ ] cells. the second model utilizes the attention mechanism [ ] to align input and output sequences. we train these models using the glove word embeddings as input features. to build our first baseline, we use a neural encoder-decoder [ ] model. the encoder, which contains rnn cells, converts the input sequence into a context vector. the context vector is an abstract representation of the entire input sequence. the context vector forms the input for a second rnn based decoder, which learns to output the target sequence one word at a time. our second baseline uses an attention layer [ ] between the encoder and decoder, which helps in deciding which words to focus on the input sequence in order to predict the next word correctly. the third model, which is our proposed method, is based on the transformer network architecture [ ] . we use glove word embeddings as input features for our proposed model. we develop the transformer encoder as described in [ ] to obtain the representation of the input sequence and the transformer decoder to generate the target response. figure shows the proposed architecture. the input to the transformer encoder is both the embedding, e, of the current word, e(u n ), as well as positional encoding pe(n) of the nth word: there are a total of n x identical layers in a transformer encoder. each layer contains two sub-layers -a multi-head attention layer and a position-wise feedforward layer. we encode the input utterances and target responses of our dataset using multi-head self-attention. the second layer performs linear transformation over the outputs from the first sub-layer. a residual connection is applied to each of the two sub-layers, followed by layer normalization. the following equations represent the layers: where m is the hidden state returned by the first layer of multi-head attention and f is the representation of the input utterance obtained after the first feed forward layer. the above steps are repeated for the remaining layers: where n = , ..., n x . we use c to denote the final representation of the input utterance obtained at n x -th layer: similarly, for decoding the responses, we use the transformer decoder. there are n y identical layers in the decoder as well. the encoder and decoder layers are quite similar to each other except that now the decoder layer has two multihead attention layers to perform self-attention and encoder-decoder attention, respectively. r y = [y , ..., y m ] y m = e(y m ) + p e(m) ( ) to make prediction of the next word, we use softmax to obtain the words probabilities decoded by the decoder. in this section, we present the details of the datasets used in our experiments, along with a detailed overview of the experimental settings. our dataset comprises of single-turn conversations from ten different domains -data about user, competitors, emotion, emergency, greetings, about bixby, entertainment, sensitive, device, and event. professional annotators with a linguistics background and relevant expertise created this dataset. the total dataset comprises of , utterance and response pairs with an average of . and . words for utterance and response, respectively. we first split the data into a train and test set in a : ratio. we then use % of the training data for preparing the validation set. the dataset details are given in table . some examples from the dataset are shown in table . we use two different types of models for our experiments -recurrent and transformer-based sequence-to-sequence generative models. all data loading, model implementations, and evaluation were done using the opennmt [ ] as the code framework. we train a seq seq model where the encoder and decoder are parameterized as lstms [ ] . we also experiment with the seq seq model with an attention mechanism [ ] between the decoder and the encoder outputs. the encoder and decoder lstms have layers with -dimensional hidden states with a dropout rate of . . the layers of both encoder and decoder are set to with -dimensional hidden states with a dropout of . . there are multihead attention heads and nodes in the feed-forward hidden layers. the dimension of word embedding is empirically set to . we use adam [ ] for optimization. when decoding the responses, the beam size is set to . automatic evaluation: we use the standard metrics like bleu [ ] , rouge [ ] and perplexity for the automatic evaluation of our models. perplexity is reported on the generated responses from the validation set. lower perplexity indicates better performance of the models. bleu and rouge measure the ngram overlap between a generated response and a gold response. higher bleu and rouge scores indicate better performance. to qualitatively evaluate our models, we perform human evaluation on the generated responses. we sample random responses from our test set for the human evaluation. given an input utterance, target response, and predicted response triplet, two experts with post-graduate exposure were asked to evaluate the predicted responses based on the given two criteria: . fluency: the predicted response is fluent in terms of the grammar. . adequacy: the predicted response is contextually relevant to the given utterance. we measure fluency and adequacy on a - scale with ' ' indicating an incomplete or incorrect response, ' ' indicating acceptable responses and ' ' indicating a perfect response. to measure the inter-annotator agreement, we compute the fleiss kappa [ ] score. we obtained a kappa score of . for fluency and a score of . for adequacy denoting "good agreement. in this section we report the results for all our experiments. the first two experiments (seq seq & seq seq attn) are conducted with our baseline models. our third experiment (c.f fig. ) is carried out on our proposed model using word embeddings as the input sequences. table and table show the automatic and manual evaluation results for both the baseline and the proposed model. our proposed model has lower perplexity and higher bleu and rouge scores than the baselines. the improvement in each model is statistically significant compared to the other models . for all the evaluation metrics, seq seq attn has the highest score among the baselines, and our model outperforms those scores by a decent margin. for adequacy, we find that our seq seq model achieves the highest score of . among the baseline models. our proposed model outperforms the baselines with a score of . . for fluency, we observe that the responses generated by all the models are quite fluent in general. to observe our results in more details, we perform an error analysis on the predicted response. in table as seen in the example, the predicted response would not be the best fit reply to the utterance "you are online" as the response falls out of context for the given utterance. in this paper, we propose an effective model for response generation using singleturn conversations. firstly, we created a large single-turn conversational dataset, and then built a transformer-based framework to model the short-turn conversations effectively. empirical evaluation, in terms of both automatic and humanbased metrics, shows encouraging performance. in qualitative and quantitative analyses of the generated responses, we observed the predicted responses to be highly relevant in terms of context, but also observed some in-corrections as discussed in our results and analysis section. overall we observed that our proposed model attains improved performance when compared with the baseline results. in the future, apart from improving the architectural designs and training methodologies, we look forward to evaluating our models on a much larger dataset of single-turn conversation. neural machine translation by jointly learning to align and translate deep retrieval-based dialogue systems: a short review variational autoregressive decoder for neural response generation measuring nominal scale agreement among many raters long short-term memory an information retrieval approach to short text conversation generating informative responses with controlled sentence function adam: a method for stochastic optimization opennmt: open-source toolkit for neural machine translation adversarial learning for neural dialogue generation rouge: a package for automatic evaluation of summaries njfun-a reinforcement learning spoken dialogue system reinforcement learning of questionanswering dialogue policies for virtual museum guides bleu: a method for automatic evaluation of machine translation glove: global vectors for word representation data-driven response generation in social media a survey of statistical user simulation techniques for reinforcement-learning of dialogue management strategies a hierarchical latent variable encoder-decoder model for generating dialogues neural responding machine for short-text conversation improving variational encoder-decoders in dialogue generation an ensemble of retrieval-based and generation-based human-computer conversation systems a neural network approach to context-sensitive generation of conversational responses sequence to sequence learning with neural networks attention is all you need a neural conversational model the generalization of student's' problem when several different population variances are involved retrieve and refine: improved sequence generation models for dialogue partially observable markov decision processes for spoken dialog systems diversity-promoting gan: a cross-entropy based generative adversarial network for diversified text generation learning to respond with deep neural networks for retrieval-based human-computer conversation system a hybrid retrieval-generation neural conversation model unsupervised discrete sentence representation learning for interpretable neural dialog generation learning discourse-level diversity for neural dialog models using conditional variational autoencoders the design and implementation of xiaoice, an empathetic social chatbot acknowledgement. the research reported in this paper is an outcome of the project "dynamic natural language response to task-oriented user utterances", supported by samsung research india, bangalore. key: cord- -q y fewk authors: lemaire, d.; barbosa, t.; rihet, p. title: coping with genetic diversity: the contribution of pathogen and human genomics to modern vaccinology date: - - journal: braz j med biol res doi: . /s - x sha: doc_id: cord_uid: q y fewk vaccine development faces major difficulties partly because of genetic variation in both infectious organisms and humans. this causes antigenic variation in infectious agents and a high interindividual variability in the human response to the vaccine. the exponential growth of genome sequence information has induced a shift from conventional culture-based to genome-based vaccinology, and allows the tackling of challenges in vaccine development due to pathogen genetic variability. additionally, recent advances in immunogenetics and genomics should help in the understanding of the influence of genetic factors on the interindividual and interpopulation variations in immune responses to vaccines, and could be useful for developing new vaccine strategies. accumulating results provide evidence for the existence of a number of genes involved in protective immune responses that are induced either by natural infections or vaccines. variation in immune responses could be viewed as the result of a perturbation of gene networks; this should help in understanding how a particular polymorphism or a combination thereof could affect protective immune responses. here we will present: i) the first genome-based vaccines that served as proof of concept, and that provided new critical insights into vaccine development strategies; ii) an overview of genetic predisposition in infectious diseases and genetic control in responses to vaccines; iii) population genetic differences that are a rationale behind group-targeted vaccines; iv) an outlook for genetic control in infectious diseases, with special emphasis on the concept of molecular networks that will provide a structure to the huge amount of genomic data. braz j med biol res ( ) infectious agents remain a major cause of morbidity and mortality worldwide in spite of advances in hygiene, drug development, and vaccines. the emergence of drugresistant strains of infectious agents (including bacteria, viruses and parasites) and emerging diseases caused by either newly identified infectious agents or newly identified pathogen strains (e.g., severe acute respiratory syndrome corona virus, h n avian flu, h n swine flu) are a global health concern. vaccine development therefore appears to be essential to limit the spread of emergent and re-emergent diseases. however, the development of vaccines faces major difficulties highlighted by the absence of efficient vaccines against major health concern infectious diseases, such as malaria, tuberculosis, and aids ( ) . the difficulties range from i) the incapacity of inducing host protective responses due to inadequate adjuvant, dose, route of immunization, or candidate vaccine antigens with low immunogenicity, high rate of genetic variation, or limited accessibility to the immune system, to ii) high interindividual variability in the response to the vaccine observed as highly variable adverse event risk or protective efficacy, which are partially determined by the genetic background and the immune status of vaccine recipients. new high-throughput technologies, new statistical and bioinformatics tools, and new biological concepts such as molecular networks should help in overcoming some of these difficulties. in particular, the availability of complete microbe genome sequences, including the sequences of multiple strains of a pathogen, may help in identifying new candidate antigens ( ) . furthermore, new high-throughput technologies may lead to a better understanding of genetic variation in resistance to infectious disease and in response to vaccination. here, we will present recent advances in genomic-based vaccine approaches, genetic control in www.bjournal.com.br braz j med biol res ( ) infectious diseases, and we will discuss the possibility of vaccines for target groups or personalized vaccines. the first vaccines resulted from empirical studies and were mainly composed of whole organisms (live attenuated or killed) or inactivated toxins. the majority of vaccine studies developed later also used conventional approaches to select for appropriate antigens. antigen selection was a key step in vaccine development in the pre-genomic era but was time-consuming and had many limitations. the properties aimed for in these molecules were: ) being accessible to the immune system and inducing protective immunity and ) being conserved within the species and not containing regions of high variability ( ). the selection of good candidate vaccine antigens was primarily based on the capacity of induction of protective antibodies, which probably explains why effective vaccines currently available mainly induce a protective humoral immune response ( ). more recently, molecular biology methods have been applied to the development of new vaccines, primarily to enable large-scale production of subunit or peptide-based vaccine antigens. nevertheless, only a small number of available vaccines are based on recombinant antigens. the strategies of conventional vaccinology pose limitations for the development of effective vaccines against a variety of infectious diseases. the development of vaccines against pathogens that show important antigenic variation or that cannot be cultivated in the laboratory remains a great challenge. currently, new approaches using high-throughput technologies are being used for the identification of new vaccine candidate molecules ( ) . the publication of the haemophilus influenzae genome, the first pathogen to have its complete genome sequence published as a result of an approach to genome analysis using new technologies of high-throughput sequencing ( ) , has opened the mind of scientists to a range of new possible approaches to the study of microorganisms and has marked the beginning of a new era in vaccine development: the identification of pathogen candidate antigens based on the knowledge of the genome of the pathogen and on the understanding of microbial biology and host-pathogen interactions, an approach called reverse vaccinology ( ) . this approach has been favored in recent years by the use of high-throughput technologies in the fields of genomics, proteomics and immunomics, among other "omics", which has expanded enormously. the number of available viral and parasitic genome sequences is growing continuously ( ) , and important advances have been made to make new biostatistics and bioinformatics tools available for candidate gene prospection. the current "genomic era" is marked by a major impact on the ability to find new candidate vaccine antigens and to develop effective vaccines. one of the approaches that may be used to analyze the dna data sequences in order to identify vaccine candidate molecules is the in silico analysis of complete genomes. it does not require cultivation of the pathogen and it uses a variety of computer resources to process dna sequence data and the available information about protein functions, cell transport and localization, antigen processing, and immunogenicity. computational analysis may then enable the identification of which dna sequences encode the proteins that have some relevant features of potential vaccine targets ( ) . the impact of in silico analysis on vaccine development can be exemplified by the first study published using this approach ( ) . a capsular polysaccharide-based vaccine against neisseria meningitidis serogroups a, c, w , and y is available, but not against serogroup b as its structure is identical to the polysialic acid structure present on human cells ( ) . the entire genome sequence of a virulent n. meningitidis serogroup b strain, which accounts for the majority of meningococcal disease burden in the united states and europe ( ) , was analyzed to identify candidates for a new vaccine. the bacterial genome was screened using bioinformatics tools to identify relatively conserved, surface-exposed outer membrane protein antigens: of open reading frames (orfs) were thus selected. immunoproteomics methods were then used to select the most immunogenic meningococcal antigens that would elicit a protective immunity. five of genome-derived candidate antigens are components of a rationally designed vaccine against n. meningitidis serogroup b ( ) and a formulation of four of these antigens ( cmenb, novartis, switzerland) has induced high titers of bactericidal antibodies in a phase i/ii clinical trial ( ) . the multivalent vaccine formulation has been proposed to overcome the issue of antigenic diversity between strains of n. meningitidis, and it would be expected to stimulate immune responses capable of recognizing multiple antigenic variants. the promising results of this new vaccine candidate may be the critical "proof of concept" stage for this novel approach that has significantly shifted the paradigm of vaccine development from microbiological to sequence-based approaches. genome-wide screening using functional selection methods such as dna microarrays, gene knockout libraries, or screening of cdna expression libraries using patients' sera has been employed in the prospection of vaccine candidates (reviewed in ref. ) . these approaches allow the evaluation of the possible immunogenic and immunoprotective roles of all molecules (either surface expressed or secreted by pathogens), thereby enabling the identification of candidate antigens that were not identified by the methodologies applied in conventional vaccinology. this means that even molecules that are expressed at very low levels or only under certain conditions can be tested for their potential as candidate vaccines. in brief, genomics coupled with other new "omics" fields of study such as www.bjournal.com.br braz j med biol res ( ) transcriptomics, proteomics, and immunomics enables the identification of vaccine candidate molecules based on pathogen genome analysis. high-throughput sequencing technologies have also been used to understand the molecular biology of several viruses and parasites. recently, genomic-based approaches have allowed the identification of potential novel viral and parasitic targets, which will be included in vaccines against infectious diseases, such as severe acute respiratory syndrome (sars; ), aids ( ) , trypanosomiasis ( ) , and malaria ( ) . pathogens have developed multiple strategies to evade the immune response, and one of the most commonly used is antigenic variation of immune response targets. the genetic variability observed in most microorganisms, especially those that are pathogenic, accounts for the capacity of microbial pathogens to adapt to genetically distinct hosts, different antibiotics, or immune system defense mechanisms ( ) . the high intra-species variability is an important limiting factor to the development of vaccines against several diseases such as hiv, hepatitis c virus (hcv), plasmodium, and meningococcus. genotypic differences among infecting mycobacterium tuberculosis strains have also been pointed out as potential factors influencing the efficacy of the bacillus calmette-guérin (bcg) vaccine against tuberculosis ( ) . this vaccine has been extensively used and studied, and shows wide variation in efficacy among populations and geographic regions worldwide ( ) . entire microorganism genome sequences can be obtained relatively inexpensively and quickly, a fact that makes it feasible to have whole genome data available from multiple isolates of a single pathogen. this technological advance has provided the basis for the exciting, newly proposed concept of pangenome ( ) . according to this concept, a bacterial species could be characterized by its global gene repertoire, which contains genes shared by all the strains (core genome) and genes shared by some strains but not all (dispensable genome). additionally, genetic variants in genes that are found in all strains can be characterized by dna sequence analysis. this concept has been used to characterize intraspecies diversity in a novel approach for the discovery of new vaccine candidate molecules: pangenomics reverse vaccinology ( ) . thus, the development of effective, multivalent and broad coverage genome-based vaccines will probably result from approaches that include genomes from multiple isolates from each microbial pathogen. yet, underrepresented strains not targeted by the multivalent vaccine may become a matter of concern after broad vaccine coverage is established. serotype replacement, a phenomenon by which minor bacterial serotypes become predominant over time, has been documented after the introduction of the heptavalent anti-pneumococcal conjugate vaccine. it can be attributed to the fact that strains not targeted by the vaccine are able to escape vaccine selective pressure ( ) . the ongoing efforts of data mining for all the information in pathogen genomes already made available should effectively help to identify new potential vaccine targets for most infectious diseases ( figure ). however, these new approaches using powerful technologies (genomic and pangenomic reverse vaccinology; functional and structural genomics) have advantages and limitations that should be considered in the rational design of a vaccine development project. in general, the methodologies used for identification and analysis of vaccine candidate antigens are based on the assumption that surface and secreted proteins will induce a protective humoral immune response. that is true for almost all extracellular pathogens, but the presence of antibodies is not always associated with protection against intracellular pathogens. then, eliciting both antibody and t-cell immune responses can be an important property of the candidate antigens to be evaluated. in fact, despite the amount of available data about the genome of pathogens and the human genome, the genes, molecules, and mechanisms involved in pathogen virulence or in host defense are still not clear. understanding the mechanisms of microbial infections and the immune response against them remains a major challenge in vaccine development. this could explain why relatively slow progress has been made in the development of new efficient vaccines in spite of the emergence of high-throughput sequencing technologies and advances in bioinformatics. much research is still needed for novel vaccine development: the contribution of each microbial gene expressed to the disease and to the activation of a protective immune response should be understood. the current data show that there is a wide variety of new molecules that can potentially constitute new vaccine targets; the choice of the selection methods for these new vaccine antigens is, thereby, a critical step and depends on the accumulated knowledge in the fields of bioinformatics, genomics, immunology, pathology, microbiology, parasitology, and virology. host-pathogen interactions are also influenced by the host background. thus, different animal models or human populations, age, specific nutrient deficiencies, co-infection/ co-morbidities, among other host conditions can influence the immune responses to a given pathogen or vaccine. there is accumulating evidence for a genetic control of infectious diseases. rare primary immunodeficiencies have been shown to cause susceptibility to either multiple infectious agents or to a single type of infectious agent; these are very rare and mendelian inherited. twin studies have provided further evidence of a genetic control, and complex segregation analyses that explicitly model the effect of genes and the environment have revealed either multigenic predisposition or a genetic predisposition based on major genes having a big influence on the phenotypes related to the disease ( ) . several candidate genes have been associated with infectious diseases ( ) . the candidate gene approach further supports the role of human genetics in resistance or susceptibility to infectious diseases. more recently, rna interference has been used to exploit human cell models of infection with various agents to elucidate pathogen virulence mechanisms capable of subverting host pathways involved in immune regulation and pathogen killing response ( ) , an approach that can be used to discover and associate differences in preferential immunoregulatory networks involved in host-pathogen interactions in susceptible versus resistant hosts. these approaches are, however, restricted to well-known genes. several genome-wide linkage analyses have been performed to map new resistance or susceptibility genes. major genes have been mapped to chromosomes q -q ( ) and q ( ) for schistosomiasis, chromosomes q ( ) and q/xq ( , ) for tuberculosis, chromosomes p ( , ) and q ( ) for leprosy, and chromosome q for visceral leishmaniasis ( ) . blood infection loads of plasmodium falciparum or mild malaria have been genetically linked to chromosomes q -q ( ), p ( ), p - ( ) , and q - ( ) . the identification of genes underlying complex diseases remains a challenging task, and few genes have been identified. nevertheless, il (chromosome q -q ), tnf and ncr (chromosome p -p ), park and pacrg (chromosome q ) polymorphisms have been associated with schistosomiasis ( ), mild malaria ( , ) , and leprosy ( ), respectively. the first genome-wide association study in infectious disease was published by fellay et al. ( ) , who found an association of hla-b and hla-c with hiv virus load, and of rnf and znrd with disease progression. more recently, several genome-wide association studies have been published for malaria, hepatitis b, and hepatitis c. the best signals of association were found at the hbb locus for severe malaria ( ) , at the hla-dp locus for hepatitis b ( ) , and at the il b locus for hepatitis c ( , ) . all the studies were based on single nucleotide polymorphism (snp) arrays: , snps were genotyped in the hiv, malaria, and hepatitis b studies, while , snps were analyzed in the hepatitis c study. twin studies have recently established that genetic variation influences the response to hepatitis a and b, diphtheria, tetanus, measles, mumps, rubella, polio, h. influenzae type b, pertussis, and bcg vaccines ( ) . such studies have led to estimate the heritability, defined as the ratio of genetic variance to total variance, of immune responses ( ) . early papers have reported a heritability of and % for the antibody response to the hepatitis b surface antigen vaccine in adults ( ) and young children ( ) , respectively; it should be stressed that high levels of antibody against hepatitis b surface antigen correlate with protection against infection and persistent carriage ( ) . the heritability of vaccine responses is generally high ( ) , reaching % for the antibody response against measles vaccine ( ) . it should be stressed that the immune mechanisms responsible for protection are largely unknown for a variety of infectious diseases. also immunological correlates of protection are needed to monitor the efficacy of vaccines and to study the genetics of protective responses to vaccines ( ) . the influence of the genetic background has been further supported by twin-based and population-based association studies, which have revealed the association of hla and non-hla candidate genes with the response to hepatitis a and b, influenza, measles, rubella, and bcg vaccines ( , ) . however, few candidates have been simultaneously tested by each research group, except for the study by hennig et al. ( ) , who analyzed candidate genes. it is likely that there are genes involved in both resistance to a particular infectious disease and response to the corresponding candidate vaccines. therefore, genes that have been associated with resistance or susceptibility to infection or disease can be considered candidates potentially involved in the response to the vaccine. the genes that will be identified through genome-wide approaches in infectious diseases will be of particular interest; conversely, the genes that affect the response to vaccination may be analyzed for their potential role in resistance or susceptibility to the corresponding disease. it may be the time to move from the candidate gene approach to genome-wide linkage and/or association approaches in order to identify genes controlling the response to vaccination (figure ). it is very likely that populations with different genetic backgrounds will differ in their genetic control of infectious diseases: for a particular infectious disease, the involvement of some loci may be evidenced in a population living in africa and may not be detected in another one living in asia. ethnic groups living in the same area can also differ in their ability to control infection or disease. for example, the fulani, whose genetic background is clearly different from that of other ethnic groups in west africa, have been compared to sympatric ethnic groups for several phenotypes related to malaria resistance. the fulani produce higher antibody levels than the mossi and the rimaibe, and have lower parasite densities and fewer and milder malaria attacks, indicating that the fulani are more resistant against malaria than the mossi and the rimaibe ( ) . similarly, it is known that populations differ in their response to vaccination, suggesting that populations differ in the frequency of the protective alleles. poland et al. ( ) have reported that native (innu and inuit) and caucasian schoolchildren differed in their production of antibodies against measles antigens after vaccination in canada. additionally, malawi and uk populations strikingly differed in the level of prowww.bjournal.com.br braz j med biol res ( ) tection following bcg vaccination (i.e., % in malawi and % in the uk), and in their ifn-γ production in response to mycobacterium antigens ( ) . furthermore, native children (navajo, white mountain apache and alaska) and children in the general us population differed in the incidence of disease due to h. influenzae type b both before and after vaccination, indicating that native children were more susceptible than other children in the us, and that their response to vaccination was also weaker ( ) . further understanding of the genetic basis of variation in vaccine response may lead investigators to consider different vaccine strategies for groups having different genetic backgrounds. the combination of high throughput methodologies and multifactorial analyses may help in the identification of genetic markers that affect immune and physiological responses leading to serious adverse effects, thus allowing the identification of groups at risk of vaccine-induced adverse events, which would be entitled to preventive therapies or alternative vaccine formulations ( ) . in the same way, the exposure to the infectious agents, current immune status (e.g., immunocompromised subjects), gender, and age could be taken into account to minimize the rate of vaccine failure or vaccine adverse events. such a targeted-group vaccination approach requires more information about the human groups of interest. efforts are particularly needed to study various populations in africa, where genetic diversity is the most important in the world, and data are lacking. in these populations, the international scientific and medical community would be able to screen for millions of polymorphisms within each potential target group. it has been also suggested that profiles based on individual information, such as genetic information, age, and gender, would allow prediction of the need to receive a given vaccine (i.e., being susceptible to infection), the number and amount of doses needed, the likelihood of response, the likelihood of adverse events, and so on, thereby making it possible to develop a personalized vaccination approach. at this stage, developing a health system in which each individual would be genotyped at the genome scale would become a political and economic choice. before coming to the era of personalized vaccination, however, we should develop methods that will eventually allow us to predict the individual response to vaccination on the basis of individual information, including genome-wide genotypes. the challenge is less technological than conceptual. the emergence of new technologies, such as the microarray technology and more recently the next generation sequencing technology, gives the illusion that genes controlling a complex phenotype would be easily identified. this is due to the fact that we can obtain a huge amount of biological data for one sample. for example, the whole-genome transcriptome can be analyzed by using one array for an individual, and more than , snps can be simultaneously genotyped by using snp arrays for the same individual (the last generation of affymetrix chips allows the analysis of , million snps). the nextgeneration sequencers (solexa/illumina and solid/abi) allow the analysis of the transcriptome, of small rnas, of dna methylation, of snps, and of copy number variation at the genome scale ( ) . all these technologies can produce a huge amount of data that need to be stored, transferred, carefully analyzed and interpreted. the statistical analysis is a significant challenge because a number of statistical tests are performed; to tackle this multi-testing problem, new approaches have been proposed ( , ) . such approaches have been used in genome-wide studies and have provided significant results from the statistical point of view. however, it is clear that most biological information is missing. this lack is highlighted by the results obtained in genome-wide linkage and association in infectious diseases. for instance, fellay et al. ( ) estimated that the polymorphisms identified explained % of the virus load variance in the study population, suggesting that the effect of other genes had not been detected. strikingly, a genome-wide association study only detected the association between protection against severe malaria and hemoglobin s after correcting for multi-tests ( ) , while several other genes and genetic variants have been associated with severe malaria in independent populations ( ) . it is clear that the biological interpretation of such data needs an integration of various biological data stored in different databases, and statistical approaches that make use of these data. the central concept that can drive the development of new approaches is the molecular network. figure represents this concept in the context of infectious diseases. this concept has stimulated the development of gene prioritization tools to propose candidate genes lying within a chromosomal region linked to the disease. the approaches used imply that most genes involved in a disease share functional properties and/or can be mapped in the same gene or protein network. the most frequent approach is to rank candidates on the basis of their similarity to a set of training genes that have been demonstrated to be involved in the disease (reviewed in ref. ) . the biological concept of the gene network implies the existence of gene-gene interactions that are generally not taken into account in genome-wide linkage and association analyses. genegene interaction can be explicitly included in the models used in linkage and association analyses. statistical approaches have been proposed to detect such interactions also in studies of vaccine responses ( ) . furthermore, the influence of tlr and tirap polymorphisms on clinical disease caused by m. tuberculosis has been found to depend on pathogen genotypes, suggesting the existence of bacterial gene-human gene interactions ( ) . obviwww.bjournal.com.br braz j med biol res ( ) ously, the gene-gene interaction studies at the genome scale pose additional multitest problems that must be tackled. in addition, such approaches are generally limited to low order interactions, and efforts are now needed to develop statistical approaches that make use of the whole biological network. the analysis of genetic variation and gene expression levels could also provide help in understanding how polymorphisms could perturb a transcriptional network in infectious diseases ( ) . microarray studies that are based on mice infected with either virulent or nonvirulent pathogens have identified gene expression patterns associated with some infectious diseases, indicating the existence of at least one transcriptional network perturbed by a virulent strain. moreover, gene expression profiles have been shown to discriminate between genetically cerebral malaria-resistant versus genetically cerebral malaria-susceptible mice at early and late stages of infection with p. berghei anka ( , ) . this further indicates that genetic variants direct at least one transcriptional network towards a state conducting to cerebral malaria and death in susceptible mice after infection. in humans, blood cell gene expression profiles have been associated with the clinical status of patients infected with different pathogens ( ) , and have been used to monitor the response to bcg vaccination in children ( ) . although very few genomic studies have been performed in the field of vaccination it has been anticipated that the biological network concept would be useful. in this way, poland et al. ( ) proposed "the immuneresponse network theory", whereby the response to the vaccine can be viewed as the cumulative result of gene interactions. the findings in naturally infected individuals and in mouse models of infectious diseases support this hypothesis. this urges the effort to perform genome-wide studies in vaccinated mice and humans, and to compare the results with those obtained in natural infections. the same authors have suggested that the network combined with individual genomic profiles may lead to predicting the response to vaccination. this is a big challenge because it will require biological networks that capture all the relevant information, including the effect of genetic and other biological information (age, gender, etc.). this further implies that one would be able to predict the effect of single or combined perturbations such as polymorphisms on molecular phenotypes (figure ). for this pur-pose, regulatory network modeling methods would be useful: models constructed on available data to predict molecular and cellular phenotypes will provide working hypotheses that can be tested, leading to the improvement of the initial model. interestingly, immune response system-level tools have been made available ( ) . thus, systems biology is a new rational approach that gives a more comprehensive understanding of networks or interacting components in the immune response to pathogen antigens. this concept has been recently reviewed ( ) . it can be applied to identify signatures of protection that would be evaluated in the de- vaccinomics) . this emergent research strategy in vaccine development was already applied to identify signatures of protection to influenza ( ) and yellow fever vaccines ( ) . nevertheless, the current tools can manage small biological networks; the challenge is to develop new approaches that allow the modeling of large regulatory networks. in conclusion, genomics of infectious agents will accelerate the identification of new relevant vaccine candidate antigens, including antigens that are conserved between strains or collectively cover the diversity of strains and that are the targets of protective immune responses. the genome-based approaches should help in developing vaccines against old, but so far uncontrolled, infectious diseases that are expected in developing countries, and may play a critical role in the design of vaccines against emerging infectious diseases as well as non-infectious diseases. the identification of relevant molecular targets will require further research on protective immune responses and the development of immunological tools. recent investigations that have systematically searched for viral protein-human protein interactions open new avenues for understanding host-pathogen interactions and therapeutic strategies. further research on the mechanisms of action of adjuvants and the use and restraints of relevant animal models will be mandatory. the human genetic variation should also be taken into account when analyzing the response to vaccination. vaccine strategies may have to be adapted to target groups in order to optimize vaccination efficacy and reduce the risk of adverse events. similarly, the possibility of a personalized vaccination strategy (appropriate dose, schedule and even molecular components of the vaccine?) has been suggested, which would be based on individual information, including genome-wide genetic information. this implies the ability to predict an individual response to the vaccine. the challenge is to develop new tools that are necessary to accurately construct and model molecular networks, the perturbation of which causes either the absence of response or adverse events. a vision for vaccines against hiv, tuberculosis and malaria the genomes on line database (gold) in : status of genomic and metagenomic projects and their associated metadata genome-based vaccines the top five "game changers" in vaccinology: toward rational and 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malaria has affected the human genome and what human genetics can teach us about malaria prioritizing genes for pathway impact using network analysis ectopic kidney with varied vasculature: demonstrated by ct angiography the influence of host and bacterial genotype on the development of disseminated disease with mycobacterium tuberculosis interactome networks and human disease gene expression analysis reveals early changes in several molecular pathways in cerebral malaria-susceptible mice versus cerebral malaria-resistant mice gene-expression profiling discriminates between cerebral malaria (cm)-susceptible mice and cm-resistant mice assessing the human immune system through blood transcriptomics transcriptional profiling of mycobacterial antigen-induced responses in infants vaccinated with bcg at birth curating the innate immunity interactome systems biology approaches to new vaccine development systems biology approach predicts immunogenicity of the yellow fever vaccine in humans systems biology of vaccination for seasonal influenza in humans key: cord- -fbotc authors: fagone, paolo; shedlock, devon j.; kemmerer, stephen; rabussay, dietmar; weiner, david b. title: electroporation-mediated dna vaccination date: - - journal: clinical aspects of electroporation doi: . / - - - - _ sha: doc_id: cord_uid: fbotc there are many positive attributes to dna vaccination that make it a conceptually desirable platform. in clinical studies, however, standard dna injection alone generally induces low levels of transgene-specific immunity when compared to other vaccine approaches. in order to boost the immunogenicity of this platform, next-generation dna vaccines require additional techniques such as the administration of electroporation. this new method involves the generation of a brief electric field in tissue around a local injection site that results in the transient poration, or permeabilization, of the cellular membranes. as a result, antigen-specific immune responses are greatly enhanced and are likely due to increased dna uptake and antigen expression. thus, electroporation-mediated dna vaccination represents a promising new strategy for the elicitation of strong immune responses directed against the expressed antigen(s) and not the vector, and ongoing studies are currently underway to optimize the working parameters of this technique. here, we review the uses of this technology in conjunction with vaccination and suggest future directions for its further exploration. the discouraging results from the recent human hiv trial performed by merck and collaborators, named "step," raises serious questions about current vaccine approaches using replicationdefective viral vectors [ ] . this study was an international phase ii "test of concept" trial in uninfected volunteers at high risk for acquiring hiv infection which showed that vaccination using a recombinant adenovirus serotype vector was ineffective at preventing virus infection and even increased the rate of transmission in individuals exhibiting prior immunity to the viral vector. while this vaccine was safe and immunogenic in both humans and nonhuman primates, eliciting long-lasting and multifunctional cd + t cell responses that were partially protective in rhesus macaques, the discovery that the vaccine could possibly heighten hiv infection was both unexpected and alarming, resulting in the immediate discontinuation of vaccinations as recommended by the independent data safety monitoring board for step. since preexisting immunity against a vaccine vector may compromise its efficacy in humans, future vaccine approaches should aim to utilize vectors that exhibit minimal or no reactivity in immunocompetent vaccines. dna vaccination consists of the administration of genetic material encoding a desired antigen that when expressed in the vaccine, is capable of eliciting an immune response. compared with other approaches, the advantages of dna vaccination are many [ ] ; no infectious agents are involved and vaccines are unable to revert into virulent form like live vaccines. they can prime both cytotoxic [ ] and humoral responses [ ] , and dna vectors are easily manipulated, can be tested rapidly, produced at high yield in bacteria, and are readily isolated. they are also more temperaturestable than conventional vaccines, easily stored and transported, and do not require a cold chain. furthermore, dna vaccines could potentially induce immunity in newborns in situations that usually neutralize conventional vaccines via the presence of high levels of maternal antibodies [ ] . the introduction of exogenous dna into cells or tissue can be achieved using dna conjugates [ ] [ ] [ ] [ ] [ ] , virus-derived vectors [ ] , or naked plasmid dna (pdna) [ ] . naked pdna shows variable and low transfection efficacy when administered by conventional means, such as needle injection or topical application. however, several strategies aimed to improve pdna vaccine immunogenicity have been developed, including codon optimization [ ] , mrna optimization [ ] , addition of leader sequences [ ] , and construction of consensus immunogens [ ] . while these strategies help to boost the overall immunogenicity of a dna vaccine, they may not be applicable to all antigens. recently, electroporation, or electropermeabilization, has gained great interest in multiple research areas including gene therapy and vaccinology [ ] . although the precise mechanism of action has not yet been well defined, it is hypothesized that cell membranes in host tissue receiving electroporation, normally impermeable to charged molecules, form pores or functionally equivalent structural changes upon application of an external electric field which facilitate the influx of macromolecules. thus, higher transfection efficacy of naked pdna as a result of electroporation is thought to be the major contributor to the increased immunogenicity of electroporation-mediated dna vaccination. in addition, it has been shown that electroporation increases vaccine potency by activating antigenpresenting cells (apcs) via danger signals and local inflammation [ ] and by recruiting immune cells to the site of dna administration [ , ] . furthermore, direct transfection of apcs could also be facilitated by electroporation. currently, intensive investigation is focused upon utilizing electroporation of muscle and skin as an effective method for dna vaccine delivery to small and large animals, and in humans. the safety and feasibility of electroporation in humans has recently been demonstrated, but not finally proven [ ] . thus, the paramount question for dna vaccines at this time is whether a sufficient level of efficacy can be reached with the present methodology, or if further improvements or breakthroughs in vaccine design and/or electroporation delivery will be necessary. in , the first transgene expression detected in skeletal muscle after injection of naked mrna or pdna raised the possibility of using this method for certain gene therapies and dna vaccinations [ ] . subsequently, transgene expression was also obtained in the same way in a wide variety of other tissues, but transgene expression was generally too low and variable to be useful for the envisioned purposes [ ] . attempts to sufficiently enhance pdna uptake, and thus transgene expression, with cationic lipids or the gene gun have also proven unsuccessful to date. the first publications on a substantial increase in transgene expression (about -fold) when electroporation was applied in vivo after pdna injection appeared as late as [ ] , although electroporation had been used for in vitro cell transfections since [ ] . in addition, as pointed out by bettan et al. [ ] , when using gene electrotransfer, a higher interindividual reproducibility in gene transduction can be observed. skeletal muscle (fig. . ) has been the most frequently targeted tissue in both gene therapy and dna vaccine studies, either with or without electroporation. some reasons why muscle cells (also known as myocytes) and muscle tissues continue to be attractive targets for transgene expression include: muscle tissue is easily accessible, plentiful, and well vascularized; the latter facilitates circulation of the antigens produced by the transfected muscle cells. more discussion of gene electrotransfer to muscle can be found in chap. . in vivo electroporation has been used to deliver dna vaccine encoding antigens from numerous infectious agents, summarized in table . . enhanced immune responses to electroporation-mediated dna vaccination have been observed both in small and large animals such as mice [ ] , pigs [ ] , and monkeys [ ] . widera et al. [ ] demonstrated in mice that upon electroporative treatment, the delivery of a weakly immunogenic hepatitis b virus (hbv) surface antigen (hbs ag) dna vaccine resulted in an increased humoral immune response, characterized by rapid onset and higher titers of anti-hbs ag antibodies. in addition, the authors observed in the same study that the potency of an hiv gag pdna vaccine was increased as shown by the lower dosage of dna required to induce higher antigen-specific antibody levels and increased cd + t cell responses. similarly, in a study carried out with a bovine herpes virus- truncated glycoprotein d dna vaccine, tsang et al. [ ] showed that the onset of the primary humoral response was earlier in the group treated with dna followed by electroporation, and that this group produced higher antibody levels than those in the group receiving i.m. dna immunization or a recombinant protein vaccine only; similar results were obtained earlier with [ , ] an otherwise inert microparticulate adjuvant [ ] . interestingly, the efficiency of transfection by electroporation was not increased by doubling the dose of dna administered; however, the duration of the antigen-specific antibody response was increased at a higher rate in comparison to the immunization with the same dose of plasmid without electroporation. moreover, electroporation increased the degree of consistency among the individuals in the dna-plus-electroporation group as seen in the weeks of follow-up. finally, a high correlation between the duration of the primary immune response and the magnitude of the secondary antibody response was observed, implying that electroporation could represent an effective approach to elicit a longer memory antibody response. capone et al. [ ] have demonstrated that gene electrotransfer efficiently increased the cellular immune response both in mice and rhesus macaques vaccinated with a plasmid encoding a nonstructural region of hepatitis c virus (hcv). in particular, they showed by ex vivo interferon (ifn)-g elispot assay that electroporation in mice induced a fivefold more potent t cell response than dna administration alone, and that the elicited response was directed against all six of the antigen pools spanning the hcv ns -ns b region. to assess whether electroporation treatment elicited similar responses in a nonhuman primate model, they immunized rhesus macaques three times with the vaccine and collected peripheral blood mononuclear cells at periodic intervals to test the t cell effector function. the immune responses observed in the electroporation-treated group showed a faster kinetic, with all the animals responding after the second challenge and reaching a peak after the third. moreover, all animals treated with electroporation showed both cd + and cd + t cell responses, in comparison to the naked dna group which showed a weaker cd + response and no cd + response. finally, gene electrotransferimmunized macaques maintained anti-hcv effector t cells for the entire observation period of months, indicating that the gene electrotransfer efficiently elicited a strong memory t cell response. dna vaccination in association with electroporation represents an effective strategy to elicit strong, broad, and long-lasting b and t cell responses. although muscle is the most common target for dna vaccine immunizations [ ] , the presence of apcs in both the skin layers makes it an attractive target for nucleic acid vaccination, since direct transfection of apcs may be important for t cell priming upon skin dna immunization [ ] (fig. . ) . in a murine model using a viral [ ] have demonstrated that a single intradermal (i.d.) injection (without electroporation) of naked dna encoding the influenza nucleoprotein gene is sufficient to induce production of antigen-specific antibodies and cytotoxic t lymphocytes that persist for at least weeks and are protective against a lethal challenge with a heterologous strain of influenza virus. furthermore, immune responses to i.d. dna vaccination have been recorded to be significantly enhanced by in vivo electroporation [ ] ; analysis of the antibody response to an hbs ag-encoding plasmid delivered i.d. upon electroporation in mice has revealed a strong enhancement of the t h response, which is mainly characterized by a strong cell-mediated response, compared to that elicited by protein immunization, which showed an exclusively t h pattern, characterized by a dominant humoral responses. also, in a nonhuman primate model study carried out in rhesus macaques [ ] , the i.d.-plus-electroporation group developed % more ifn-g-producing cells and twice more memory t cells than the group not treated with electroporation. higher antibody responses were recorded in the i.d.-plus-electroporation group when compared to the i.m.-plus-electroporation group. altogether, these results support the idea that electroporation following dna injection, both in muscle and skin, represents an effective approach to large animal immunization. several authors have hypothesized that inflammation caused by electroporation is important to prime the immune response to dna vaccination [ , , ] . local inflammation was previously proposed to augment immune responses in studies where pdna was coinjected with bupivicaine-hcl [ ] [ ] [ ] . the localized tissue damage induced by the electric field is thought to recruit cd + cells, increasing the number of infiltrating immune cells at the injection site [ ] . indeed, electroporation caused the activation of proinflammatory signals including the expression of chemokines such as mip- a, mip- b, mip- g, ip- , mcp- , and xcl [ ] . liu et al. [ ] characterized the extent and nature of the cellular infiltrates at the site of electroporative vaccine delivery in mice and found both polymorphonuclear and mononuclear cells localized in the perivascular spaces and throughout the muscle tissue. in particular, they observed a significant increase in b cells, cd + and cd + t cells, and a dramatic increase in macrophages and dendritic cells compared to vaccination alone. no difference, however, was recorded among cell populations of blood, spleen, and draining lymph nodes of the mice treated with or without electroporation, suggesting that only local factors are involved in the augmentation of immune responses following electroporation. also, these authors observed that cell infiltrates were transient and resolved within weeks. thus, improved antigen presentation may represent one of the mechanisms by which electroporation may elicit a more potent immune response. typically, innate immune recognition of the adjuvant component of vaccine formulations has been shown to be critical for their immunogenicity [ ] . many adjuvants are ligands for toll-like receptors (tlrs), like monophosphoryl lipid a and cpg dna [ , ] , while some conventional adjuvants, such as aluminum hydroxide and incomplete freud's adjuvant are free of tlr ligands [ ] . therefore, these examples demonstrate that multiple innate immune recognition and signaling pathways are required for adjuvants to function [ ] . in the case of dna vaccines, it has been controversial as to the main vaccine component contributing most to the induction of both innate and adaptive immune responses; while cpg motifs expressed within the plasmid backbone can stimulate innate immunity through tlr , the induction of adaptive immune responses were unaffected in the absence of this innate receptor [ , ] . however, it has recently been shown that the doublestranded structure of dna, independently of cpg sequences, possesses immunomodulatory effects when administered intracellularly [ ] , which can trigger tlr-independent, tank-binding kinase i (tbk )-and inf regulatory factor -dependent innate activation of both immune and nonimmune cells to produce type i infs and their inducible genes [ , ] . recently, ishii et al. [ ] have reported that the enhancement of dna vaccine immunogenicity achieved by electroporation may be due to increased transfection rates resulting from this technique, which better contributes to local inflammation by activating cells to produce ifn through the tbk -dependent signaling pathway. these data suggest that tbk is a key signaling molecule for dna vaccination immunogenicity by regulating innate immune signaling, which is critical for the induction of adaptive immune responses, and that the enhanced immunogenicity of pdna by electroporation may be a result of more pdna interacting with intracellular tbk . in accordance with this hypothesis, peng et al. [ ] postulated that local inflammation is more important than the actual quantity of expressed transgene in determining the magnitude of the immune response, demonstrated by higher antibody titers and cd + t cell proliferation rates observed by applying electric pulses - days prior to i.m. dna immunization. in this case, it can be postulated that both increased cross-presentation and direct transfection of infiltrating apcs resulting from increased local inflammation may contribute to the augmented immune response in electroporation-mediated dna vaccination. it appears that the mechanisms by which electroporation enhance the responses to naked plasmid vaccination is by an increase in dna transfection and possibly include local inflammation, which may be augmented by the magnitude or duration of transgene expression. indeed, babiuk et al. [ ] observed that the highest level of lymphocytic infiltration was only in muscle tissue treated with electroporation, which elicited higher levels of transgene expression, as was expected considering that antigen production is critical for the retention of the cellular infiltrates at sites of local inflammation. although two dna vaccines have been recently approved in the usa and canada for the vaccination of horses against west nile virus [ ] and salmon against infectious hematopoietic necrosis virus [ ] , no dna vaccine has been approved for use in humans. however, encouraging results from preclinical trials using electroporation technology with dna vaccination in large animal models has prompted much interest in the technique and its safety. currently, tolerability in humans has been demonstrated in healthy volunteers [ ] , anti-dna antibodies have not been detected in patients electroporated after muscle dna injection, and the integration of pdna into host chromosomes following electroporation-mediated delivery has not been observed [ ] . together, these results have been sufficient for the regulatory approval of several clinical trials [ ] . as reported on clinicaltrials.gov, seven electrotransfer dna vaccine trials for cancer and three clinical trials using dna vaccine against infectious agents in association with electroporation are currently open. ongoing clinical studies using electroporation-mediated dna vaccination against infectious agents include three phase i studies involving muscle electroporation. the first will test safety and immunological effects of pennvax ™ -b, an hiv vaccine encoding gag, pol, and env, in hiv-infected individuals (vgx pharmaceuticals, inc.); the second will assess safety, tolerability, and immunogenicity of human papillomavirus (hpv) dna plasmid (vgx- ™ ) delivered by electroporation in adult females postsurgical or ablative treatment of grade or cervical intraepithelial neoplasia (vgx pharmaceuticals, inc.); the third one, a phase i/ii trial is testing tolerability and efficacy of i.m.-administered chronvac-c ™ in combination with electroporation in chronic hcv genotype infected and naïve patients with low viral load (tripep ab). the amount of pain and distress associated with electroporation in humans has been of a tolerable level for the anticipated benefit [ ] . to date, electroporation-mediated dna vaccination in humans is performed administrating an injection volume of . - . ml followed by short ( - ms), low electric field strength ( - v/cm) pulses ( - pulses) . given that these conditions are efficient for the dna vaccination of large animals, such as nonhuman primates, they should be sufficient in humans. electroporation results in a sharp, but quick pain that is comparable to receiving a short electrical shock. while this sensation is transient, administration of short-acting sedative drugs or painkillers before treatment has been considered. accordingly, as reported by daud et al. [ ] , in a clinical trial using an interleukin- -encoding plasmid delivered by electroporation in patients with metastatic melanoma, in order to limit patients' discomfort, lidocaine was either administered topically or injected around each tumor site, and intravenous analgesic and/or anxiolytic drugs were offered to the patients before electroporation. notably, previous studies have shown that pain is not a limiting factor as patient discomfort is limited to the period of electrical stimulation, and subjects have usually returned for repeated treatments without asking for sedation. also, after muscle electroporation, like muscle injection, a mild ache may be experienced at the site of electroporation for some days, and similar to that following a strenuous workout. several factors determine the strength of pain associated with electroporation, although there is a high interindividual variability in the perception of pain. among these factors are the number, length, spacing, and thickness of the electrode needles, but primarily the electric pulse parameters dictate the pain threshold [ ] . exclusion criteria for electroporation treatment may include the presence of metal implants near the site of electrical delivery, the presence of a pacemaker, and in the case of muscle electroporation, obesity, since treatment efficacy may be decreased if muscle tissue is not reached for vaccine and/ or electric pulse delivery. electroporation-mediated dna vaccine delivery requires a pulse generator that controls the parameters of individual pulses or pulse trains (amplitude, duration, number, polarity, wave form, frequency), and electrodes usually integrated into an applicator. electrodes are in direct contact with the subject to be treated and it is their geometry (shape, size, and distance from each other) that ultimately determines the shape and strength of the electric field and the electrical currents in the target tissue ( fig. . ) . thus, both the properties of the pulse(s) and the electrodes are responsible for the desired enhancement of dna delivery as well as undesirable side effects. proper design of pulses and electrodes will maximize the effectiveness of a given dna vaccine and minimize unwanted side effects, such as long-lasting histological changes, pain, and muscle contractions. further discussion of electroporation parameters and equipment can be found in chaps. and . skin is a potentially interesting target tissue for dna vaccines because of its natural role in the immune defense of the body and its ready accessibility. discussion of gene electrotransfer of dna in general and to skin can be found in chaps. and , respectively. the goal of any vaccination strategy is to prime a broad and long-lasting immune response that is capable of robust effector responses upon antigenic restimulation [ ] [ ] [ ] and protect against infectious agents, while minimizing toxicity of the vaccine. since complications may arise due to the use of viral-based vectors, such as preexisting immunity regulating vaccine effectiveness and the possibility of reversion into virulence that may decrease vaccine efficacy or safety, alternative approaches should be explored for future vaccine approaches. dna vaccination appears an even more attractive candidate for future vaccines since it is safe, immunogenic, and does not stimulate vector-specific immunity. extensive literature supports the hypothesis that electroporation represents a valid approach to vaccine administration in that it increases the consistency and potency of vaccination, inducing higher levels of both antibody-mediated and cytotoxic t cell responses. in this way, electroporation may help augment translocation of exogenous dna into the nucleus. furthermore, it has been proven to be effective in enhancing immune responses to antigens regardless of the degree of transgene expression achieved. recent evidence suggests that innate immune recognition of the adjuvant component of vaccines and the danger signals provided by the method of vaccination may be important in determining the magnitude of the resultant immune response. electroporation may enhance local inflammation at the site of immunization by facilitating the transfection of greater amounts of pdna, which may be more readily available to interact with intracellular signaling proteins that trigger the secretion of inflammatory cytokines and their . needle-type electrodes consist of a variable number of (primarily) stainless steel needles arranged in different configurations. picture depicts a -needle array firing rotating chord patterns, a -needle array fired in opposing pairs, crossing or triangular patterns, and a -needle array firing pin to pin inducible genes. while electroporation-mediated dna vaccination is currently the topic of intensive research, more theoretical studies and practical trials are required to optimize the delivery of the vaccine into the target tissue, and the electrical parameters facilitating dna uptake while calibrating local tissue damage and reducing pain associated with vaccine delivery through electroporation. the failed hiv merck vaccine study: a step back or a launching point for future vaccine development? dna vaccination: antigen presentation and the induction of immunity heterologous protection against influenza by injection of dna encoding a viral protein genetic immunization is a simple method for eliciting an immune response vaccination with hemagglutinin or neuraminidase dna protects balb/c mice against influenza virus infection in presence of maternal antibody in vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin i increased expression of dna cointroduced with nuclear protein in adult rat liver liposome mediated gene transfer direct introduction of genes into rats and expression of the genes receptor-mediated gene delivery and expression in vivo human immunodeficiency virus type vaccine development: recent advances in the cytotoxic t-lymphocyte platform "spotty business the mechanism of naked dna uptake and expression multiple effects of codon usage optimization on expression and immunogenicity of dna candidate vaccines encoding the human immunodeficiency virus type gag protein inactivation of the human immunodeficiency virus type inhibitory elements allows rev-independent expression of gag and gag/protease and particle formation induction of potent th -type immune responses from a novel dna vaccine for west nile virus new york isolate (wnv-ny ) antigenicity and immunogenicity of a synthetic human immunodeficiency virus type group m consensus envelope glycoprotein gene therapy progress and prospects: electroporation and other physical methods electric pulses applied prior to intramuscular dna vaccination greatly improve the vaccine immunogenicity increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of dna vaccines recruitment of antigen-presenting cells to the site of inoculation and augmentation of hiv- dna vaccine immunogenicity by in vivo electroporation electroporation for drug and gene delivery in the clinic: doctors go electric direct gene transfer into mouse muscle in vivo in vivo gene electroinjection and expression in rat liver gene transfer into mouse lyoma cells by electroporation in high electric fields high-level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle increased dna vaccine delivery and immunogenicity by electroporation in vivo electroporation improves the efficacy of dna vaccines in large animals anti-hbv immune responses in rhesus macaques elicited by electroporation mediated dna vaccination a single dna immunization in combination with electroporation prolongs the primary immune response and maintains immune memory for six months enhancement of the effectiveness of electroporation-augmented cutaneous dna vaccination by a particulate adjuvant modulation of the immune response induced by gene electrotransfer of a hepatitis c virus dna vaccine in nonhuman primates high expression of naked plasmid dna in muscles of young rodents predominant role for directly transfected dendritic cells in antigen presentation to cd + t cells after gene gun immunization intradermal gene immunization: the possible role of dna uptake in the induction of cellular immunity to viruses cutaneous transfection and immune responses to intradermal nucleic acid vaccination are significantly enhanced by in vivo electropermeabilization combined effects of il- and electroporation enhances the potency of dna vaccination in macaques early events of electroporation-mediated intramuscular dna vaccination potentiate th -directed immune responses inflammatory responses following direct injection of plasmid dna into skeletal muscle dna immunization with hiv- tat mutated in the trans activation domain induces humoral and cellular immune responses against wild-type tat specific immune induction following dna-based immunization through in vivo transfection and activation of macrophages/antigen-presenting cells facilitated dna inoculation induces anti-hiv- immunity in vivo in vivo electroporation enhances the immunogenicity of hepatitis c virus nonstructural / a dna by increased local dna uptake, protein expression, inflammation, and infiltration of cd + t cells translating innate immunity into immunological memory: implications for vaccine development signaling to nf-kappab by toll-like receptors the vaccine adjuvant monophosphoryl lipid a as a trif-biased agonist of tlr adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling tlr −/− and tlr +/+ mice display similar immune responses to a dna vaccine vaccination with plasmid dna activates dendritic cells via toll-like receptor (tlr ) but functions in tlr -deficient mice a toll-like receptor-independent antiviral response induced by double-stranded b-form dna recognition of cytosolic dna activates an irf -dependent innate immune response tank-binding kinase- delineates innate and adaptive immune responses to dna vaccines recombinant canarypoxvirus vaccine carrying the prm/e genes of west nile virus protects horses against a west nile virus-mosquito challenge efficacy of an infectious hematopoietic necrosis (ihn) virus dna vaccine in chinook oncorhynchus tshawytscha and sockeye o. nerka salmon clinical evaluation of pain and muscle damage induced by electroporation of skeletal muscle in humans abstracts from the american society of gene therapy th annual meeting the uptake and fate of exogenous cellular dna in mammalian cells national institutes of health phase i trial of interleukin- plasmid electroporation in patients with metastatic melanoma applicator and electrode design for in vivo dna delivery by electroporation cd + t cells are required for secondary expansion and memory in cd + t lymphocytes requirement for cd t cell help in generating functional cd t cell memory defective cd t cell memory following acute infection without cd t cell help sphingosine-mediated electroporative dna transfer through lipid bilayers increased immune responses in rhesus macaques by dna vaccination combined with electroporation persistent antibody and t cell responses induced by hiv- dna vaccine delivered by electroporation enhancement of dna vaccine potency in rhesus macaques by electroporation a therapeutic siv dna vaccine elicits t-cell immune responses, but no sustained control of viremia in sivmac -infected rhesus macaques the relative immunogenicity of dna vaccines delivered by the intramuscular needle injection, electroporation and gene gun methods protection against influenza virus infection in balb/c mice immunized with a single dose of neuraminidase-expressing dnas by electroporation protection against influenza virus infection in mice immunized by administration of hemagglutinin-expressing dnas with electroporation protection against avian influenza h n virus challenge by immunization with hemagglutinin-or neuraminidase-expressing dna in balb/c mice electroporation-mediated hbv dna vaccination in primate models non-cytolytic antigen clearance in dnavaccinated mice with electroporation a single hbsag dna vaccination in combination with electroporation elicits long-term antibody responses in sheep enhancement of immune responses to an hbv dna vaccine by electroporation skin targeted dna vaccine delivery using electroporation in rabbits. i: efficacy enhancing b-and t-cell immune response to a hepatitis c virus e dna vaccine by intramuscular electrical gene transfer search for potential target site of nucleocapsid gene for the design of an epitope-based sars dna vaccine construction of a eukaryotic expression plasmid encoding partial s gene fragments of the sars-cov and its potential utility as a dna vaccine induction of specific immune responses by severe acute respiratory syndrome coronavirus spike dna vaccine with or without interleukin- immunization using different vaccination routes in mice analysis of humoral immunity of hepatitis d virus dna vaccine generated in mice by using different dosage, gene gun immunization, and in vivo electroporation immunogenicity of a dna vaccine for coxsackievirus b in mice: protective effects of capsid proteins against viral challenge in vivo electroporation of skeletal muscles increases the efficacy of japanese encephalitis virus dna vaccine dna vaccination against foot-and-mouth disease via electroporation: study of molecular approaches for enhancing vp antigenicity comparative studies of the capsid precursor polypeptide p and the capsid protein vp cdna vectors for dna vaccination against foot-and-mouth disease virus immunogenicity of novel consensus-based dna vaccines against chikungunya virus potentiation of an anthrax dna vaccine with electroporation smallpox dna vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge intramuscular immunization with a monogenic plasmid dna tuberculosis vaccine: enhanced immunogenicity by electroporation and co-expression of gm-csf transgene improved humoral immunity against tuberculosis esat- antigen by chimeric dna prime and protein boost strategy improved cellular and humoral immune responses against mycobacterium tuberculosis antigens after intramuscular dna immunisation combined with muscle electroporation multivalent dna vaccine protects mice against pulmonary infection caused by pseudomonas aeruginosa markedly enhanced immunogenicity of a pfs dna-based malaria transmission-blocking vaccine by in vivo electroporation identification of minimal cd + and cd + t cell epitopes in the plasmodium yoelii hepatocyte erythrocyte protein kda co-delivery of plasmid-encoded cytokines modulates the immune response to a dna vaccine delivered by in vivo electroporation in vivo electroporation improves immune responses to dna vaccination in sheep we would like to thank karuppiah muthumani and shaheed abdulhaqq for critical review of the manuscript and members of the dbw laboratory for helpful discussion.competing financial interests: the laboratory of d.b.w. has grant funding and collaborations, advising, or consulting including serving on scientific review committees for commercial entities and therefore notes possible conflicts associated with this work with pfizer, inovio, bms, virxsys, ichor, merck, althea, vgxi, j&j, aldevron, and possibly others. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. key: cord- -jd wxobz authors: běláková, jana; horynová, milada; křupka, michal; weigl, evžen; raška, milan title: dna vaccines: are they still just a powerful tool for the future? date: - - journal: arch immunol ther exp (warsz) doi: . /s - - - sha: doc_id: cord_uid: jd wxobz vaccination is historically one of the most successful strategies for the prevention of infectious diseases. for safety reasons, modern vaccinology tends toward the usage of inactivated or attenuated microorganisms and uses predominantly subunit vaccines. the antigens need to be clearly defined, pure, stable, appropriately composed, and properly presented to the immune system of the host. differing ratios of various proportions between specific cd (+) and cd (+) t cell responses are essential for conferring the required protection in the case of individual vaccines. to stimulate both cd (+) and cd (+) t cells, the antigens must be processed and presented to both antigen-presentation pathways, mhc i and mhc ii. protein antigens delivered by vaccination are processed as extracellular antigens. however, extracellularly delivered antigen can be directed towards intracellular presentation pathways in conjugation with molecules involved in antigen cross-presentation, e.g. heat shock proteins, or by genomic-dna vaccination. in this overview, current knowledge of the host immune response to dna vaccines is summarized in the introduction. the subsequent sections discuss techniques for enhancing dna vaccine efficacy, such as dna delivery to specific tissues, delivery of dna to the cell cytoplasm or nucleus, and enhancement of the immune response using molecular adjuvants. finally, the prospects of dna vaccination and ongoing clinical trials with various dna vaccines are discussed. immunization with plasmid dna (dna-based vaccination) is a relatively novel technique for the efficacious stimulation of a specific cellular and humoral immune response to protein antigens. dna vaccines deliver transgenes, which code for antigens, directly into the cells of immunized host organisms. such antigens are expressed in a similar way as antigens during viral infection [ ] . antigens are processed identically as proteins synthesized in cytoplasm [ , ] and peptide fragments are presented to the immune system on cell-surface mhc i molecules. if the dna vaccine-coded proteins are secreted from the cell, they could be processed by mhc ii and could elicit a specific antibody response [ , ] . dna-based immunization is a new and attractive strategy in the prophylaxis and treatment of infections caused by extracellular and intracellular pathogens. the method of application, dose, boosting schemes, and species used are factors that influence the strength and nature of the elicited immune response. vaccination with plasmid dna may offer several important advantages over traditional vaccines, e.g. the relative stability of dna, the specificity of the antigen produced, and the possibility of guiding the type of elicited specific immune response [ ] . one of the major benefits of dna vaccines is that host cells express a vaccine-coded antigen and thus the antigen presents epitopes which may resemble native viral epitopes more closely than is the case with other vaccination approaches. intracellularly processed epitopes are presented to the host immune system in a way similar to that of a natural viral infection (mhc i presentation followed by cd + t cell responses), but without the risk associated with the administration of infectious agents [ ] . dna vaccines encoding several glycoproteins, i.e. multivalent vaccines, can be delivered to the host in a single dose. a few micrograms to milligrams of plasmid dna are sufficient for inducing a vigorous immune response. dna vaccines can also be manufactured in a relatively cost-effective manner and stored with relative ease [ ] . the temperature-stable storage of dna in a lyophilized form is feasible and more practical for transport and distribution as well [ ] . another important advantage of dna vaccines is their therapeutic potential in ongoing chronic viral infections. dna vaccines are very promising tools for an effective induction of a protective immune response against viral infections (hbv, hcv, hiv) and parasitic infections (malaria, leishmaniasis) [ , , ] . dna vaccines are principally derived from bacterial plasmids [ ] . conventional plasmid dna vaccines consist of two different parts: ) a eukaryotic cistron coding for the target antigen and consisting of a strong promoter/enhancer, cdna coding for the target antigen(s) (full-length or truncated), and a polyadenylation/termination signal, and ) sequences necessary for the manipulation-construction and amplification of plasmid dna in a prokaryotic host (e. coli) and consisting of the origin of replication (usually from e. coli), a multiple cloning site, and an antibiotic-resistant gene used as the selection marker during bacterial amplification [ , , , ] . the majority of eukaryotic promoters used are derived from the human cytomegalovirus (hcmv) or the rous-sarcoma virus (rsv). hcmv and rsv were found to give the highest levels of antigen expression after intramuscular (i.m.) dna injection [ ] . other promoters tested are tissue-specific promoters, e.g. the myocyte-specific desmin promoter [ ] , the actin promoter, major creatine kinase promoter, alphaglobin promoter, chicken beta-actin promoter, and adenovirus promoter. these have been tested primarily for i.m. dna application. efficacious expression of protein from dna vaccines is dependent on the presence of dna vaccine in the nucleus. the effective targeting of plasmid dna to the cell nucleus is dependent on the presence of dna sequences recognized by proteins or peptides called nuclear localization signals (nlss) [ ] , which form a dna-nls complex recognized by cytoplasmic proteins called importins which dock dna-nls complexes to the nuclear membrane receptor/transporter (nuclear pore complex) as a nuclear import substrate [ ] . nuclear dna transport is typical for viral dna. the most effectively recognized dna sequence is localized within the sv early promoter enhancer region of simian virus dna. this dna sequence was shown to be effective in the nuclear transport of plasmid dna [ ] . other dna sequences have been identified and tested, but their recognition through cellular nlss followed by the transport of plasmid to the nucleus is generally poor. an alternative approach is based on the chemical linkage of plasmid dna with an nls. a variety of nlss were tested (large sv t-antigen, m sequence of heterogeneous nuclear ribonucleoprotein, adenovirus fiber protein residues), but they generally obtained unsatisfactory results in in vivo experiments [ ] . this was most likely linked to the size-exclusion limit for active nuclear transport of such artificial dna-nls complexes. studies of the viral dna nuclear transport mechanism are promising because some viral dna, although more than ten times larger than standard vaccination dna plasmids, are effectively transported to the nucleus [ ] . polya-termination signals, which provide stabilization of mrna transcripts, are commonly taken from bovine growth hormone or from sv [ , ] . dna vaccines stimulate both exogenous (mhc class ii-restricted) and endogenous (mhc class i-restricted) antigen-presentation pathways. mhc i-restricted cytotoxic t lymphocytes (ctl) may be induced after dna vaccination by: a) directly transfected somatic cells (myocytes, keratinocytes, or any mhc ii-negative cells) which present expressed antigen on mhc i, b) directly transfected professional antigen-presenting cells (apcs) such as dendritic cells (dcs) which, besides mhc i presentation of the expressed antigen, effectively stimulate naive t cells via a variety of co--stimulatory molecules and cytokines, or c) by cross--priming when protein expressed by dna-transfected somatic cells is taken up by the surrounding professional apcs and presented to t cells [ , ] . the cross--priming phenomenon, discovered by bevan in , is at present used as a general explanation for the re-presentation of exogenously derived cell-associated antigens on both mhc i and mhc ii molecules [ , ] . when the dna vaccine reaches the cell nucleus, transcription is initiated and subsequent translation of the coded protein takes place on cytosolic ribosomes. the proteasome complex processes the expressed protein and released peptides are transported to the endoplasmic reticulum through the membrane transporter complex tap- and tap- . inside the endoplasmic reticulum, the peptides are bound to mhc i molecules [ , ] , which subsequently migrate to the cytoplasmic membrane and present the peptides to the surrounding cd + t cells [ ] . professional apcs, such as macrophages, b cells, and dcs, play a central role in the regulation of the immune response to any vaccine. in contrast to somatic cells, apcs can present the antigen to both mhc i and mhc ii molecules and thus stimulate t-helper cells (cd + ), which control other t cell or b cell responses [ , ] . dcs are much more effective than other apcs because of their unique ability to prime naive t cells. thus dcs are most likely the key cells initiating the immune response to the dna vaccine [ ] . intradermal (i.d.) and gene-gun application would directly target the plasmids to langerhan's cells, which represent immature dcs located in the stratum spinosum of the epidermis. dcs can migrate to lymphatic organs (the spleen and the lymph nodes) where they activate antigen-specific t lymphocytes. apart from high levels of mhc i and mhc ii, dcs express the stimulatory molecules b . and b . (cd and cd , respectively) that are required for immune response initiation [ ] . if non-apcs such as myocytes take up the dna vaccine (following i.m. injection), the expressed antigen is delivered to t cells by cross-presenting dcs [ , , , ] . cross-presentation and cross-priming are responsible for inducing the immune response to apoptotic and/or necrotic bodies [ ] , which may also be associated with dna vaccination. thus dna immunization results in the induction of both humoral (antibody) and cellular (t cell) immune responses. the major advantage of dna vaccines is their ability to generate a strong cellular immunity with preference for mhc i-restricted cd + cytotoxic t cells and mhc ii-restricted t helper type (th ) cell responses [ ] . on the other hand, dna vaccines can induce th responses and results from a number of studies indicate that dna vaccination can be effective in inducing long-term antibody responses [ , ] . this effect may depend on the type of antigen coded by the vaccine [ ] . effective dna vaccination should generate a long--term memory immune responses. there are several possible ways for dna vaccines to induce a long-term response: a) antigen is continuously expressed at a low level sufficient for antigen presentation and b) plasmid dna as well as antigen are completely gone and the response is antigen independent. memory cells generated by dna vaccines probably differ qualitatively from those achieved by other forms of vaccination, such as protein plus adjuvant [ , ] . memory cd + t cell and b cell populations are the most relevant for vaccine development. the most critical factors for current dna vaccines are: ) the efficacy with which the dna vector reaches the target cells' nuclei (transfection efficacy) and ) the amount of actual protein synthesized in dna vaccine--transfected cells. it has been estimated that injection of microgram doses of dna plasmid results in the production of only nanograms of protein [ ] . it is difficult to quantify the number of plasmids that enter the cell nucleus and the number of plasmids that are degraded before they enter the nucleus. it is estimated that more than % of the dna plasmids never reach the cytoplasm and only about . % of cytoplasmatically localized dna plasmids enter the nucleus, where gene expression is initiated [ ] . identifying and overcoming each hurdle along the dna vaccine entry pathway (low uptake across the plasma membrane, inadequate release of dna molecules within the cell's cytoplasm resulting in reduced dna stability, and a lack of nuclear targeting) can improve dna vaccine efficacy [ ] . dna vaccination was described in when wolff et al. [ ] demonstrated induced gene expression after direct i.m. injection of naked plasmid dna into experimental mice. naked dna was then injected into various tissues with the aim of comparing the intensity of proteosynthesis and the host immune response. besides i.m. injection the most frequently tested injection routes were i.d., intravenous, subcutaneous, epidermal, intraepidermal, intraperitoneal, injection into lymphatic follicles, and injection into the thyroid gland [ , , , ] . different routes of administration lead to markedly different levels of protein expression as well as different levels of intensity and quality (th , th , antibody) of the immune response. the skin was found to be one of the best sites for immunization due to the ease of skin injection and the high concentration of dcs (in the skin langerhans cells), macrophages, and lymphocytes, which are necessary for the induction of the immune response [ ] . in contrast, muscle is generally not equipped with dcs and elicitation of the immune response probably relies on cross-presentation, the effectiveness of which is limited by the dose of available antigen [ ] . thus methods which increase antigen expression by increased uptake of the dna by muscle cells could dramatically improve the applicability of i.m. dna vaccination. dna delivery methods can be classified into two general types: ) mechanical and electrical strategies for introducing naked dna into cells, including microinjection, particle bombardment, and the use of electroporation, and ) dna delivery systems which can be classified into biological viral dna delivery systems and chemical non-viral delivery systems (dna-binding polymers and liposomes). physical methods for increasing naked dna delivery dna vaccination into skin and muscle. wang et al. [ ] tested the effect of i.m. injection followed by electroporation on plasmid uptake in mice. the electroporation increased muscle cell plasmid uptake by approximately -to -fold. direct i.d. or i.m. dna injection vaccination very often results in the induction of a th response characterized by interferon (ifn)-γ synthesis and predominately igg a antibodies in mice [ , ] . the doses applied by needle injection are normally in the range of about - µg of naked plasmid dna. in the case of gene-gun dna application, only . - µg of plasmid dna is sufficient to induce antibody or ctl responses. the gene-gun or biolistic system uses compressed heli-um to propel micrometer-sized colloidal gold particles coated with precipitated plasmid dna (dna-coated microparticles) directly into the epidermal cells. in mice, intraepidermal gene-gun dna inoculation generates a prominent th response with interleukin (il)- production and an excess of the igg isotype [ , , ] . the approximately log of the dna dose may explain the dominance of the th response because low plasmid doses result in a low cpg motif moiety, which is important for a th response elicitation [ , ] . needle-free jet injection (biojector) is another dna delivery approach which has been investigated extensively as a method of i.d. immunization of laboratory animals, such as mice, pigs, rabbits, dogs, and monkeys [ , ] . gramzinski et al. [ ] analyzed the effect of various routes for immunizing with a dna vaccine encoding hepatitis b surface antigen (hbsag). experiments confirmed that needle-free injection of a dna vaccine (biojection of dna) induces a greater immune response to hbsag antigen than i.d. or i.m. injection using the classic needle-syringe approach [ ] . similar results were obtained in the earlier experiments of baizer et al. [ ] . the needle-free injection of dna has been tested in several human clinical trials as well [ ] , e.g. a gag-pol candidate hiv dna vaccine [ ] . the delivery of dna vaccines to the liver. a high level of dna vaccine expression in liver cells was achieved by the rapid injection of naked plasmid dna in relatively large volumes via the tail vein, the portal vein, the hepatic vein, and the bile duct in mice and rats [ , ] . this liver-specific approach has been designated "hydrodynamic delivery" and it is increasingly being used as a research tool for elucidating the mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of diseases in laboratory animal models [ , , ] . the procedure has also been shown to be effective in large animals such as dogs and non-human primates [ , ] . the hydrodynamic approach is proving to be a very useful research tool not only for gene expression studies, but also more recently for the delivery of small interfering rna [ , , ] . the application of dna vaccine to the liver is associated with enormous protein expression followed by a strong antibody-and cell-mediated immune response. when the same dose of dna was administered by hydrodynamic application or by i.m. or i.d. injection, the antigen-specific antibody levels induced by the hydrodynamic application were approximately times higher (raska et al., unpublished result) . dna vaccine minimization to minicircle dna. minicircles are small circular dna molecules that are derived from parental dna plasmids by specific recombination. antibiotic resistance genes, selection markers, and bacterial origins of replication are fully removed by the specific recombination [ , ] . minicircles contain only the gene of interest, making them promising tools for dna vaccination and gene therapy. chen et al. [ ] have shown that minicircles can express high ( -and -fold more) and persistent levels of target protein in mouse liver compared with their parent plasmids. this could be attributed to a higher transfection efficacy and the low to minimal content of cpg in minicircle dna. avoidance of bacterial dna also increases the safety of dna application because no antibiotic-resistant genes can be passed to pathogenic bacteria present in host tissues. non-viral carrier systems: dna/polyplexes. dna carriers tested for dna vaccination are various molecules which complex with dna by ) electrostatic forces between negatively charged dna molecules and a positively charged carrier (or cationic ions on a negatively charged carrier), ) analogous to the natural dna-protein interaction, or ) artificial covalent linkage between dna and a carrier [ ] . the dna/carrier complexes protect dna from serum dnases, increase transmission of dna through the cytoplasmic membrane of target cells, allow targeting to specific tissues, and some of them induce the escape of dna entrapped in endosomes by promoting endosomal disruption (weak bases such as chloroquine, the proton-sponge effect of many polymers) [ ] . non-viral dna vaccine delivery systems are based on ) electrostatic complexation of dna with cationic polymers (poly-l-lysine, protamine sulfate, polyethyleneimine, chitosan, polyethylene glycol, poly--(d,l-lactide-co-glycolide)), complexes commonly termed dna/polyplexes, ) electrostatic complexation and condensation of dna with artificial cationic lipids or lipopolyamines (dc-chol, dotma, dotap, dospa, dogs) which are mixed together with zwitterionic helper lipids responsible for the fusion of complexes with the target cell membrane (chol, dope, dppc), complexes commonly termed dna/lipoplexes or dna/lipopolyplexes, ) complexation of dna with artificial anionic lipids (dmpg) through electrostatic interaction mediated by na + and k + ions which are supplemented with zwitterionic helper lipids (dppc), complexes commonly termed fluid dna/liposomes, ) association of dna with proteins or peptides (histones, peptide tyrosine-lysine-alanine-(lysine) -tryptophan-lysine, fab fragments of anti-dna antibodies, cationic viral proteins µ and vp ) which in combination with cationic polymers or lipids facilitate nuclear targeting, and ) complexation of dna with dendrimers with a very low degree of polydispersity (pamam), complexes commonly termed dna/dendrimers (table ) [ , , , , , , ] . cationic liposomes and polymers are accepted as effective vectors for gene delivery with low immunogenicity, unlike viral vectors [ ] . liposomes are often used for systemic (intravenous, i.m., i.d.) or topical (nasal, oral) dna administration [ , , , , , ] . the final dna/lipopolyplex structures, dna concentration, ratio of cationic moiety to dna, and supplements such as condensing agents, endosmolytic agents, or nuclear targeting molecules are the most critical factors in the transfection efficacy of each formulation [ , , ] . the development of modified dna/lipopolyplexes suitable for intravenous application and targeting to tissues and organs such as heart, lung, liver, spleen, or kidney is desirable and necessary. the current focus is on decreasing the toxicity of complexes and increasing transfection efficacy and tissue specificity. to target specific tissue, the carriers are modified by linkage to various ligands allowing dna complexes to be recognized by specific tissue and cell populations. targeting to the liver is possible by linking liposomes or other polymers (polylysine, pei, polyamidoamine dendrimers) with a sugar motive recognized by the liver--specific asialoglycoprotein receptor, which is expressed on hepatocytes. such ligands are composed of trimeric terminal galactose [ , , ] . lipid-based dna/complexes are a promising approach in systemic application because of the minimal toxicity of particular lipids (dppc) in contrast to cationic polymers [ ] . on the other hand, lipid-based dna complexes are larger in size and are thus limited in their penetrability into liver--specific compartments (disse spaces), which are the port for hepatocyte targeting [ , , , ] . therefore an effort has been made to decrease the size of final complexes by protamine sulfate or poly-l-lysine, which increased the transfection efficacy in some in vitro experiments by up to times [ , ] . targeting the dna/complexes to dcs, mainly by interaction with the mannose receptor on the dc surface, is immunologically very promising. a specific ligand, mannose, was linked to a variety of polymers and lipids used for dna/polymer preparation which were tested in vitro or in vivo according to their dc specificity and transfectability. unfortunately, the high transfection efficacy commonly achieved in in vitro experiments is still dramatically diminished under in vivo conditions [ , ] . viral carrier systems. one highly efficacious delivery system for dna vaccines, or, more precisely, genetic vaccines, is based on recombinant viral vectors derived either from attenuated viruses used for preventive vaccination (vaccinia, poliovirus, hepatitis b virus, measles virus) or from viruses such as human adenovirus (hadv), adeno-associated virus (aav), alphavirus, vesicular stomatitis virus (vsv), or poxviruses other than vaccinia [ ] . in contrast to plasmid dna vaccines, virally vectored genetic vaccines induce a specific immune response not only against the expressed transgene, but also against the viral capsid and/or envelope and this response is often effective even after the first immunization. therefore, repeated immunization, often necessary for elicitation of a satisfactory immune response against the transgene product, could be inefficient. virally vectored genetic vaccination is generally performed as the second immunization after dna priming, i.e. a heterologous vaccination approach [ ] . the choice of transgene, viral vector, dose, route of application, prime/boost regimen, and number of immunizations are the most important factors influencing appropriate antigen-specific humoral and cd + and cd + cellular responses. furthermore, delivery efficacy of viral vectors could be dramatically hampered by preexisting immunity to the capsid or envelope proteins in a population. only a few viral vectors (alphavirus, vsv, and some serotypes of hadv or aav) are considered to be unrecognized by preexisting immunity of vaccines. finally, similarly to dna plasmid, a protective immune response elicited in experimental animals by virally vectored genetic immunization may not necessarily be observed in subsequent human clinical trials. this could be attributed to the above-mentioned preexisting immunity, the restricted range of viral hosts, restricted viral tissue tropism, or to many other, not yet well-described factors. poxvirus-derived vectors are some of the most frequently tested. they are safe and genetically stable double-stranded (ds) dna vectors whose entire life cycle occurs in the cytoplasm of somatic cells. they do not enter the nucleus and therefore do not integrate into the host genome. poxviruses are capable of carrying large amounts of foreign genetic material. two vaccinia--derived vectors, the new york vaccinia virus [ , , ] . edible bait vaccine, raboral vr-g, is licensed worldwide for the prevention of rabies in animals. vectors derived from avian poxviruses such as canarypox virus (alvac) and fowlpox virus (fwpv- ) are expected not to be recognized by preexisting immunity in humans. poxvirus-vectored transgenes induce good specific humoral and cd + and cd + cellular responses [ , , ] . recombinant adenoviruses are extensively tested for both vaccination and gene therapy. they contain dsdna in a rigid icosahedral non-enveloped nucleocapsid. the wild-type of adenovirus does not integrate into the genome. nevertheless, hadvs are oncogenic for animals and thus the safety of hadv therapy is still an open question [ ] . replication-incompetent recombinant hadvs are able to carry transgenes of up to kb. adenoviruses are used because of effective cellular uptake and transgene expression. wild and recombinant adenoviruses induce intense inflammatory responses followed by an induction of serotype-specific immunity, which could hamper the effectiveness of a subsequent vaccination with the same serotype [ ] . among the about known hadv serotypes, hadv- is the most explored for recombinant vaccine application. hadv- has one of the weakest pathogenicities associated with mild upper respiratory disease and fever in humans. recombinant hadvs were tested in animal models as a potential vaccine for hiv, malaria, sars, and ebola. heterologous dna-prime/recombinant adeno-boost immunization induced both humoral and cd + and cd + cell responses in experimental animals. a weak response was detected in human volunteers. because the prevalence of hadv- is relatively high in the population, altered surface proteins or chimeric hadv- carrying the surface proteins of another serotype are developed for avoiding the hampering effect of preexisting immunity [ ] . aav are members of the parvoviridae family of single-stranded (ss) dna non-enveloped viruses of the genus dependovirus [ ] . recombinant aavs are nonreplicative but persist within the cells as non-episomal, mainly circular dna. the integration frequency determined in rodent and rabbit muscle tissue is less than approximately - , which is two orders lower than spontaneous mutations in human genes. recombinant aavs could package kb of ssdna, including itr (transgene < . kb) [ ] . within the cell, the ssdna genome is transcriptionally active after conversion to a dsdna template, which takes in vivo a few weeks, a necessary delay before the induction of an immune response. therefore, dsaavs were developed. the maximum capacity for transgene insertion is . kb [ ] . in mice and rhesus macaques, a single i.m. injection of serotype aav expressing hiv antigens induced robust specific antibody and cd + cell responses [ ] . a significantly lower response in humans could be attributed to many factors, including preexisting immunity, because the presence of specific neutralizing antibodies ranges from - % according to age group and geographic location. specific antibodies recognize other serotypes, but their neutralizing activity seems to be less prominent, for example aav- with an affinity for muscle and hepatocytes, aav- for airway epithelium, aav- for muscle, and aav- for hepatocytes. beside vaccination, aav vectors are one of the most frequently tested vectors for gene replacement therapy [ ] . alphavirus vectors were earlier used for in vitro recombinant protein expression. their wild precursors belong to the togavirus family of positive ssrna enveloped viruses which replicate entirely in cytoplasm. a high level of transgene expression from this viral promoter is achieved due to the self-replicating nature of viral rna and the efficient inhibition of translation of host mrna by the viral replicase. because of limited prevalence, the preexisting immunity to the vector seems to be minimal. in experimental animals, immunization with vectors based on sindbis, semliki forest, and venezuelan equine encephalitis viruses induced a cellular and humoral immune response to the transgene. in the case of hiv- antigens the immune response in non-human primates was able to significantly reduce the viral load after challenge [ , ] . the vsv belongs to the rhabdoviridae family of enveloped negative-sense ssrna viruses. vsv-derived vectors are able to generate viral particles that express foreign transmembrane protein on the membrane surface. because vsv proteins do not down-regulate the interferon response of an infected cell, vsv could exhibit promising adjuvant activity. although wild-type vsv could induce neuropathy after intranasal application to mice, recombinant vsvs were proved to be safe in non-human primates after intranasal and i.m. application. immunization of non-human primates with vsv expressing hiv and siv antigens demonstrated efficacy in subsequent challenge with the highly pathogenic shiv . p virus. thus the potential neural toxicity of vsv needs to be finally solved before entering into the clinical trials [ ] . a few additional viral vectors were designed for genetic vaccination. one viral vector of future vaccines will be probably based on the attenuated measles virus, an enveloped negative-sense ssrna virus. at present, genetically stable recombinant vaccine strains are available for cloning up to three transgens (edmonston, schwartz). in animal experiments a recombinant measles vector expressing hiv- envelope antigen induced neutralizing antibodies and envelope-specific cd + and cd + cell responses after a single dose [ ] . although about % of the population experienced a measles infection or vaccination, it is estimated that years after the measles experience the immune response does not preclude successful immunization with recombinant measles vaccines. furthermore, it is expected that within to years measles will be eradicated [ ] . dna/polyplexes for targeting dna vaccine to mucosal surfaces. mucosal immunity establishes the first line of the defense against pathogens which attack the body via mucosal surfaces [ ] . induction of the mucosal immune response, either cellular or humoral, requires local mucosal application of antigen or an antigen-coding dna vaccine. although some mucosal response is detectable after a systemic dna vaccination, i.m. injection and gene-gun delivery of plasmid dna have a limited ability to induce mucosal immune responses [ ] . therefore, various mucosal dna application routes have been tested to achieve sufficient antigen production on mucosal surfaces (intranasal and intratracheal application, inhalation of dna vaccine in aerosol form, application of dna on external genital mucosa, and oral administration) [ , , ] . complexing dna with various polymers enhances dna uptake on mucosal surfaces. dna/polyplexes adhered to the mucosal cells either through specific receptors or through electrostatic interaction between a negatively charged mucosal cell surface and positively charged dna/polyplexes. when dna/lipoplexes were delivered orally or intranasally, they induced a significant mucosal immune response, including secretory iga responses [ , ] . an alternative means of enhancing the efficiency of dna vaccines is the use of genetic adjuvants. genetic adjuvants are most often genes coding for cytokines, chemokines, or co-stimulatory molecules. the cdna can be either administered in separate dna plasmids (monocistronic dna vaccine) or the cdna is cloned into parental dna vaccine plasmid under separate promoters or under promoters shared with antigen-coding dna sequence separated by an internal ribosome entry site element (bicistronic dna plasmid) [ ] . such molecules supply t cells or dcs with the second, antigen-independent stimulatory signal. several co-stimulatory molecules have been tested for enhancement of a) apc activation (cpg, cd l, mip- α), b) ctl response (il- , il- , il- , il- , il- , gm-csf, ifn-γ, cd l, icam- , lfa- ), c) th -type antibody production: igg a in mice (il- , il- , il- , il- , gm-csf, ifn-γ, cd l) or th -type antibody production: igg in mice (il- , il- , il- , tgf-β), and d) cellular response associated with ifn-γ induction (il- , il- , il- , il- , gm-csf, ifn-γ, cd- l, icam- , lfa- ) [ , , , , , , , ] . two additional co-stimulatory molecules of the tumor necrosis factor receptor superfamily expressed on activated t cells are antiapoptotic, i.e. - bb (cd ), expressed on cd + t cells, and its cd + t cell analogue ox (cd ), which protect activated t cells from death and thus enhance the antigen-specific t cell response. stimulating these receptors may be useful in dna vaccine development [ ] . during mucosal dna vaccination, specific parameters are critical for the use of molecular adjuvants. the proinflammatory cytokines il- α, il- , and il- were tested for antibody and mucosal ctl responses. il- has the potential to increase antigen-specific ctl activity and is thus particularly interesting due to its potential role in regulating the homeostasis of memory t cells [ ] . il- and il- were shown to be able to markedly increase iga reactivity to co-expressed heterologous antigen. monocyte chemoattractant protein- was effective in increasing mucosal iga secretion and ctl responses [ ] . rantes, lymphotactin, mip- β, mip- , human neutrophil peptides (hnp- , hnp- , hnp- ), ifn-γ, and ifn-β are important candidates for use as adjuvants [ , ] . another approach used for modification of the immune responses to dna vaccines includes the addition of heterologous gene fragments encoding localization or secretory signals or fusion of the antigen-coding cdna with the sequence coding for ligands which drive the antigen to sites appropriate for immune induction. for example, a number of studies have shown that higher titers of antigen-specific igg (igg in mice) were elicited when the antigen was secreted rather than localized on the cell membrane or within the cell [ , ] . the cellular localization of heterologous antigen may play a role in modulating the immune response, although the role may depend upon the nature of the antigen and/or the model system used. another strategy consists of fusing the antigen-encoding gene with the ubiquitin-encoding gene, thereby accelerating cytoplasmic degradation of the antigen by targeting it to proteasomes and improving class i antigen presentation [ ] . enhancement of the immune response could additionally be reached by fusion of antigen with the hsp -binding viral j-domain during the construction of dna vaccines. this molecule stabilizes fusion antigen by binding to the hsp protein, which in addition serves as an instruction molecule to induce an increase in the cd + t lymphocyte and b lymphocyte response [ ] . further, dna vaccine potency may be improved through fusion of the antigen-coding dna with the endosomal/lysosomal sorting signal sequence (derived from lysosome--associated membrane protein type ; lamp- ), which directs the expressed antigen towards mhc class ii molecules. thus the cd + t cell response could be significantly enhanced [ ] . the above-mentioned use of the bicistronic vector is the most common practice for heterologous production of the binary protein complex, but these methods are primarily in the research stage [ ] . bicistronic vectors could be useful in constructing multivalent vaccines derived from two, and possibly more, different antigens originating from one or more microorganisms. an element common to the majority of plasmids are the cytosine-phosphate-guanosine dinucleotides (flanked by two ´ purines and two ´pyrimidines recognized in mice) [ ] , called cpg motifs, which are unmethylated when the plasmids are amplified in a bacterial host. hypomethylated or unmethylated cpg nucleotides are specific to bacterial dna, but are very rare in eukaryotic dna. cpgs play an important role as an immunomodulatory component of dna vaccines [ ] . they are recognized by the toll-like receptor (tlr) localized in the cytoplasm [ ] . cpgs are potent stimulators of b cell proliferation and antibody secretion. cpg induce apcs (macrophages and dcs) to secrete th -type cytokines il- and ifn-α/β, which activate natural killer cells and t cells (cd + ) [ , ] . rankin et al. showed that cpg motifs or sequences which are effective in mice are ineffective in humans [ ] . this highlights the species diversity within tlr substrate specificity. the amount of cpgs in plasmid backbones could be changed by simple recombinant technology. thus the addition of cpgs to dna plasmids could theoretically decrease the dna vaccine dose used for a single immunization. however, from murine experiments it is clear that too many cpg motifs can actually reduce immunogenicity [ ] . the major safety concerns here are: ) the integration of the plasmid dna into the host genome, thereby increasing the risk of malignancy by activating protooncogenes or inactivating oncosuppressors, ) transfer of antibiotic-resistant genes to surrounding bacteria, ) the induction of an immune response to transfected cells, resulting in the development of an autoimmune disease, ) the stimulation of cytokine responses that alter the host immune homeostasis, and ) the induction of tolerance rather than immunity [ , , , ] . at the present time there is still no evidence for plasmid dna integration into the host genome although traces of plasmid dna are detectable in host cells up to one year after dna vaccination. current methods used for the detection of plasmid integration into genomic dna are not sufficiently sensitive or specific and this question remains open [ ] . the spread of antibiotic resistance is another important question. dna plasmid could be detected far from the original site of injection (in the case of i.m. or i.d. application). the responsible carriers were transfected lymphocytes and macrophages. such cells, of course, could spread antibiotic resistance genes to bacteria. there is, however, no direct experimental evidence to date for this phenomenon [ ] . both plasmid integration and antibiotic resistance transfer could be effectively minimized by minicircle dna technology, as mentioned above [ ] . autoimmune response a) against dna plasmid, mediated by anti-dna antibodies or b) against transfected cells, mediated by a type i immune response was experimentally modeled in mice [ ] . dna vaccination was able to moderately increase the dna-specific serum antibody titers for a limited period of time, but clinical signs of autoimmunity were not confirmed. cpg motifs, on the other hand, may induce a truly harmful autoimmune response when injected together with autoantigens such as the myelin basic protein (inducing encephalomyelitis) or a chlamidia-derived antigen (inducing myocarditis) in experimental models. the above effects were not found in healthy animals treated with therapeutic dna vaccine doses and no sign of toxicity was found in human volunteers exposed to dna vaccines during clinical trials [ , , ] . the use of cytokine genes in modern multicistronic dna vaccines could theoretically disrupt the immune homeostasis and increase susceptibility to other infections. it could also be associated with exacerbation of autoimmune or allergic diseases. experimental observations have confirmed that the cytokines are released locally and that serum cytokine levels are unchanged [ ] . induction of tolerance rather than immunity after dna vaccination is a problem for both extreme age groups. very young animals (mice under days of age) and very old ones (mice older than years) respond weakly to dna vaccination. in newborn mice, dna vaccine encoding circumsporozoite protein of the malaria plasmodium induced tolerance which was long lasting [ ] . thus dna vaccination schedules for both extreme age groups would need to be separately tested, possibly modified by use of cytokines or addition of costimulatory molecules. the majority of ongoing human dna vaccination trials are focused on assessing vaccine safety and immunogenicity [ , ] . as estimated from the us database at www.clinicaltrials.gov as of june , approximately one hundred dna vaccination clinical trials have been registered (irrespective of their trial phase). a large proportion of dna vaccines use viral vectors as delivery systems. the dna vaccine gendicine, produced by sibiono genetech, uses an adenovirus vector for delivery of dna encoding the p suppressor. it is registered by the chinese fda for treatment of head and neck squamous cell carcinoma [ ] . another adenovirus-vectored p -encoding dna vac-cine, advexin, produced by introgene therapeutis, is being tested in phase iii clinical trials for the same indications in the us. if we focus on plasmid (naked) dna vaccines, approximately clinical trials had been registered by www.clinicaltrials.gov as of may . ten have attained phase ii. the majority of dna vaccines are focused on hiv- infection. other infectious diseases include hepatitis b, malaria, ebola hemorrhagic fever, west nile virus infection, and avian flu. the other trials deal with various cancers (hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, chronic lymphocytic leukemia, breast cancer, ovarian cancer, prostate cancer, bladder cancer, synovial sarcoma, leiomyosarcoma, lung cancer, and melanoma). all ongoing clinical trials have confirmed minimal toxicity and good tolerance to dna vaccines. however, the still poor immune response to the majority of clinically tested dna vaccines is a great challenge for further optimization using novel antigen, delivery systems, and prime-boost-based schedules. dna vaccines remain a promising approach for inducing both humoral and cellular immune responses. one of the more interesting aspects of dna vaccination is the mechanism of inducing antibody-and/or cell--mediated immune responses. this is associated either with antigen presentation on vaccine transfected cells or with cross-presentation of the antigen by dcs, which are the only known cells capable of inducing both cd + and cd + t cell response. dna vaccines are effective inducers of host immune protection against viral, bacterial, fungal, and parasitic infections and may be suitable for cancer therapy. multicistronic dna vaccines that, for example, coexpress cytokines may be able to modulate any autoimmunity or allergic reactions. one basic obstacle to applying dna vaccines in human medicine is their still relatively poor immunogenicity, linked to low transfection efficacy and low antigen production. thus the development of effective delivery systems is the main research challenge in this area of immunology. dna vaccination: transfection and 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non-muscle cells rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo detection of integration of plasmid dna into host genomic dna following intramuscular injection and electroporation tricistronic viral vectors co-expressing interleukin- (il-- ) and cd (b - ) for the immunotherapy of cancer: preclinical studies in myeloma the mechanism of naked dna uptake and expression long-term persistence of plasmid dna and foreign gene expression in mouse muscle direct gene transfer into mouse muscle in vivo oral administration of recombinant adeno-associated virus elicits human immunodeficiency virus-specific immune responses expression of naked plasmid dna injected into the afferent and efferent vessels of rodent and dog livers systemic linear polyethylenimine (l-pei)-mediated gene delivery in the mouse this work was supported by grant msm (ministry of education, youth, and sport, czech republic). key: cord- -v syiwie authors: rotz, lisa d.; layton, marcelle title: case study – united states of america date: - - journal: biopreparedness and public health doi: . / - - - - _ sha: doc_id: cord_uid: v syiwie the united states (us) considers the intentional use of a biological agent a serious national security threat. over the last decade, federal, state, and local governments in the us have made concerted efforts to enhance preparedness within the public health, medical, and emergency response systems to address this threat. these activities span a wide range of areas from the enactment of new legal authorities and legislative changes to significant financial investments to enhance multiple detection and response system capabilities and the adoption of a national command and control structure for response. many of these investments, although prompted by the concern for bioterrorism, have served to strengthen public health, medical, and emergency response systems overall and have proven invaluable in responses to other large-scale emergencies, such as the h n influenza pandemic. the intentional use of a biological agent is also something that the united states considers a serious threat and the federal, state, and local governments in the us have made concerted efforts to enhance preparedness and capabilities within the public health, medical, and emergency response systems to address this threat. in , this concern became a reality for the us when several letters containing anthrax spores were sent through the postal system to individuals and organizations [ ] . this resulted in cases of anthrax ( inhalational and cutaneous) with fi ve deaths. although this event may not have been the type of "mass-casualty" situation most bioterrorism preparedness planning activities were targeting, it still resulted in signi fi cant response efforts and cost; over , individuals were offered antibiotic prophylaxis because of possible exposures, over one million clinical and environmental specimens were tested, and hundreds of millions of dollars were spent on decontamination of the buildings where the letters were processed or opened [ , ] . although many infectious agents are capable of causing human illness, some are much more capable of causing signi fi cant morbidity and mortality if successfully used as a bioterrorism agent. in , the centers for disease control and prevention (cdc) developed a process to prioritize biological threat agents based on evaluation of the following threat agent characteristics: ( ) public health impact from ability to cause illness or death, ( ) ability to be produced and delivered in a way that could expose a large number of people, ( ) existing public perceptions of a biological agent that could contribute to heightened fear and panic, and ( ) requires signi fi cant special preparedness efforts in order to diagnose, treat, or prevent illness [ ] . based on these characteristics, biological threat agents were prioritized into three different tiers. category a (highest threat tier) included bacillus anthracis (anthrax), variola virus (smallpox), yersinia pestis (plague), francisella tularensis (tularemia), clostridium botulinum toxin (botulism), and the filo and arenaviruses (e.g., ebola and marburg virus) that cause viral hemorrhagic fevers. category b and c were lower threat tiers and included agents such as burkholderia mallei and b. pseudomallei , rickettsia prowasekii (category b), and emerging threats such as nipah virus (category c). following the release of homeland security presidential directive (hspd- ) in april , the us department of homeland security (dhs) became responsible for issuing biannual assessments of biological threats in order to guide the prioritization of ongoing investments in research, development, planning, and preparedness [ ] . the united states has made signi fi cant investments in terrorism preparedness and response coordination over the last two decades that includes the implementation of new policies, legislation, and legal authorities in addition to signi fi cant funding investments. in , presidential directive added a terrorism annex to the federal response plan and de fi ned responsibilities of federal agencies in responding to terrorism [ ] . the homeland security act of established the department of homeland security (dhs), a new cabinet level of fi ce whose primary mission is to prevent or reduce vulnerability of the united states to terrorism at home; coordinate homeland security responsibilities between the federal government and state, local and private entities; and minimize damage resulting from attacks and assist in the recovery. in , homeland security presidential directive (hspd- ) established a nationwide system to coordinate responses to emergencies between local, state, and federal governments and responding organizations and to administer this all hazards national response plan through a national incident management system (nims) that provides for uni fi ed command and better multi-agency coordination [ ] . other presidential directives and legislation enacted in the us since the world trade center and anthrax letter events in have provided stronger legal frameworks and public health capacity to prevent, prepare, and respond to intentional acts of biological terrorism. the public health security and bioterrorism preparedness response act (phsbpra) established new requirements for possession, use, and transfer of selected biological agents and toxins (select agent list) that could pose threats to human, animal, and plant health and safety as well as established other authorizations and appropriations necessary to carry out essential public health and medical preparedness and response activities [ ] . this act authorized more than . billion us dollars in grants to state and local governments and healthcare facilities to improve planning, training, detection, and response capacity as well as funding to expand the federal strategic national stockpile of medications and vaccines and upgrade food inspection capacity and cdc facilities that deal with public health threats. the project bioshield act (july ) and pandemic and all-hazards preparedness act (december ) also speci fi cally provided for new authorities and funding to address signi fi cant gaps that existed for the development, acquisition, and utilization of medical countermeasures (e.g. antimicrobials, vaccines, chemical antidotes) for chemical, biological, radiological, and nuclear (cbrn) threats. in , the cdc asked the center for law and public health at georgetown and johns hopkins universities to draft a model state public health law (the model state emergency health powers act or model act) for state and local jurisdictions to use in addressing either bioterrorism or naturally occurring disease outbreaks [ ] . the model act (available at http://www.publichealthlaw.net/msehpa/msehpa. pdf ) outlines fi ve major public health functions to be allowed by law including preparedness, surveillance, management of property, protection of persons, and communication. in addition to ensuring suf fi cient authority to collect disease surveillance data, conduct contact tracing, and provide preventive measures to those at risk, public health laws must enable local health of fi cials to implement quarantine measures, if needed, to control a contagious disease outbreak with epidemic potential that could lead to severe morbidity or mortality (e.g. smallpox). this authority should be linked with speci fi c, scienti fi cally appropriate criteria that would be met before quarantine could be implemented. in addition, public health laws should provide for due process measures to protect those affected. ideally, quarantine strategies would be determined and operational procedures would be in place prior to an emergency. ongoing broad-based investments to improve response planning and coordination, surveillance, training, information systems, and communications have been made that serve to improve public health capacity for all threats and hazards. starting in , the us government began providing funding to state, local, and territorial health departments to build stronger capacity for surveillance and epidemiology, laboratory diagnostic capacity, communications, countermeasure distribution, and emergency response planning, exercise, and evaluation. the initial investment into these public health system upgrades started at million us dollars per year with a primary focus on addressing bioterrorism threats. following the events of , funding to support enhancements in the national public health infrastructure increased to approximately . billion per year. the current state of progress towards speci fi c preparedness goals identi fi ed for cdc funded preparedness and response activities in the state, local, and us insular areas is provided in the " report -public health preparedness: strengthening the nation's emergency response state by state" which can be found online at http://emergency.cdc.gov/ publications/ phprep/ . additionally, more targeted investments have been made that address surveillance, detection, and illness prevention or treatment needs for speci fi c high priority threats. examples of these targeted initiatives include the laboratory response network (lrn), the strategic national stockpile (sns), and an environmental monitoring system called biowatch. in , the cdc and other partners formed the laboratory response network (lrn) [ ] . the lrn is a network of approximately national and international public health, veterinary, agriculture, food, military, and environmental laboratories that have increased diagnostic capability for the rapid identi fi cation of multiple biological and chemical threat agents in multiple sample types. participation in the network is voluntary and these pre-existing laboratories work under a single operational plan and adhere to policies on safety, security, and bio-containment. lrn members agree to perform testing using lrn procedures and are provided training, equipment, rapid detection assays and reagents, protocols, and secured communication and data reporting systems to increase testing and laboratory response capabilities in a standardized and coordinated fashion. there are three types of laboratory designation within the lrn: national, reference, and sentinel. national labs have unique capabilities and resources that allow them to handle highly infectious agents and perform strain-level identi fi cation and other agent characterization testing. reference laboratories are mostly based at state and large city health departments and have the capability to perform rapid con fi rmatory testing for certain agents and toxins while sentinel laboratories (primarily hospital and commercial clinical laboratories) can perform routine clinical testing on patient specimens with additional training and protocols for noti fi cation and rapid referral of isolates in the event that they are unable to rule-out a biothreat agent. in addition to the central role the lrn played in detecting and responding to the anthrax letter event, the commitment to infrastructure support and standardized platform testing capacity within the lrn has also proven extremely bene fi cial in assisting with more rapid and broader deployment of tests developed in response to other emerging public health threats such as the severe acute respiratory syndrome (sars) and the h n avian in fl uenza pandemic. lrn laboratories are also trained on chain-of-custody requirements and protocols which allow them to serve as a local testing resource for law enforcement linked samples where there is a concern for biological threat agents. approximately % of the us population lives within miles of an lrn laboratory, which provides for more rapid access to con fi rmatory diagnostic testing to evaluate potential illness from or exposures to threat agents. the sns (formerly the national pharmaceutical stockpile) program began in to acquire and store a stockpile of medications, vaccines, and other medical supplies whose rapid availability is vitally important for response to a large-scale event involving certain biological, chemical, or radiological agents [ ] . without a pre-purchased and stored stockpile, most of these medications and vaccines would not be readily available through other sources in appropriate amounts or in a timeframe that would allow for the prevention or effective treatment of illness. partnerships with storage and transportation companies have been created that provide strategically located storage facilities, allowing rapid delivery of sns materiel to any location in the us or its territories within h of the federal decision to deploy. certain medical countermeasures may be eligible for the shelf-life extension program managed by the food and drug administration and the department of defense, which allows for expiration date extension based on potency and other test results. in addition, agreements with pharmaceutical companies and medication distribution partners have allowed for rotation of certain medications back into the commercial supply chain for use prior to their expiration in order to help mitigate replacement costs. although the sns was originally developed as a medical countermeasure response resource for intentional biological, chemical, and radiological emergencies, it has been deployed and used multiple times to support the medical needs of other public health emergencies, including hurricanes katrina and rita, the recent h n in fl uenza pandemic, the world trade center and the anthrax letter attacks. the successful distribution of the sns is dependent on the capacity of state and local jurisdictions to rapidly dispense these countermeasures to the public. planning for the timely provision of antibiotics and/or vaccines to large populations requires the involvement of public health, emergency management, and the local medical community. mass prophylaxis plans need to consider the speci fi c challenges of potentially vulnerable populations, such as children, pregnant women, and those who are isolated and without resources and social supports, such as the homeless and homebound. contingency plans for setting up community-based points of dispensing (pods) for mass prophylaxis have been developed by most state and local jurisdictions, with a focus on ensuring suf fi cient staf fi ng resources, equipment and space requirements, and expediting patient fl ow. the capacity of health of fi cials to rapidly vaccinate the community was recently tested in the united states during the h n pandemic and demonstrated the need for fl exibility and coordination in distribution of vaccine, including school-based programs, community health centers, pharmacies, and large health department sponsored vaccination clinics. multiple initiatives have been supported to further strengthen public health disease surveillance and reporting that include an emphasis on traditional disease reporting as well as the utilization of non-traditional data that may provide an earlier indication of community health events or more likely assist with situational awareness assessments during an identi fi ed event [ ] . traditional public health surveillance for illness associated with potential bioterrorism agents relies on enhancing the medical and laboratory communities' familiarity with these agents, with the goal of improved reporting of suspected or con fi rmed illnesses, as well as reporting of unusual disease manifestations or illness clusters. most local and state health codes require that physicians, hospitals, and laboratories report a de fi ned list of noti fi able infectious diseases. state public health agencies have added cdc category a and b agents to their reportable disease lists. these lists are available at http:// www.cste.org/dnn/programs andactivities/publichealthinformatics/phistate reportablewebsites/tabid/ /default.aspx. in addition, recognizing the need to detect newly emergent diseases that are not yet listed on the health code, most states also require reporting of any unusual disease clusters or manifestations. early recognition of a bioterrorism-associated event depends in large part on astute clinicians and laboratorians recognizing one of the index cases based on a suspicious clinical, radiologic, or laboratory presentation (e.g. a febrile illness associated with chest discomfort and a widened mediastinum on chest radiograph in an otherwise healthy adult suggests inhalation anthrax). isolated cases presenting at separate hospitals will not be recognized as a potential outbreak unless they are reported promptly to the local health department, where the population-based aberrations in disease trends are more likely to be noticed. previous examples of astute clinicians recognizing and reporting unusual disease clusters or manifestations that led to the detection of a more widespread outbreak include an outbreak of hantavirus in the southwestern us [ ] , legionnaires' disease associated with the whirlpool on a cruise ship [ ] , an outbreak of cyclospora associated with contaminated raspberries imported from guatemala [ ] , and the initial outbreak of west nile virus in new york city in [ ] . similarly, the initial detection of anthrax in was due to a physician who recognized that large gram-positive rods in a patient's cerebrospinal fl uid could be b. anthracis [ ] . by reporting this suspected case of meningeal anthrax, rapid con fi rmation was facilitated in a state public health lrn reference laboratory. weeks later, a suspected case of inhalation anthrax was recognized and promptly reported to and con fi rmed by public health authorities in new york city [ ] . with the continued emergence of new zoonotic disease threats, including those related to bioterrorism, local, state, and federal public health agencies have taken steps to improve communication between human and animal health communities. noti fi able disease requirements have been expanded to include reporting by animal health specialists of suspected or con fi rmed illness in an animal that might be caused by a potential biothreat agent. because many medical providers and laboratorians in the united states have limited experience with most potential bioterrorist agents, early diagnosis may be delayed. therefore, the fi rst indication that a large-scale bioterrorist attack has taken place might be an increase in nonspeci fi c symptoms at the community level. surveillance for these increases in nonspeci fi c syndromes (e.g. respiratory, gastrointestinal, or neurologic) constitutes the cornerstone of syndromic surveillance used for emergency response purposes. many health jurisdictions have begun collecting and monitoring other types of health-related information such as symptom complexes presented during emergency room visits (e.g. lower respiratory tract illness, gastrointestinal illness, rash with fever), healthcare utilization information (e.g. emergency room visits, calls), or other data that may be affected by a community-wide health event (e.g. school absenteeism, fl u or diarrhea over-the-counter medication sales) [ , ] . though the approaches and cost for implementing syndromic surveillance vary, the tools and concepts for syndromic surveillance are adaptable and have been successfully implemented in both developed and developing countries to address routine surveillance, outbreak monitoring, and health security needs [ ] . while initially conceived for early detection for bioterrorism, these systems also can be used to monitor natural infectious disease outbreaks and trends in noninfectious events of public health importance. information from syndromic systems has proven to be useful for detecting, monitoring, and characterizing seasonal outbreaks of in fl uenza, winter gastroenteritis (e.g. norovirus and rotavirus) and asthma. furthermore, syndromic systems were utilized extensively in the us during the novel h n in fl uenza pandemic of , along with other methods, to estimate the scale of community-wide in fl uenza transmission. an additional concept to speci fi cally improve early detection of an intentional biological agent release is the use of environmental monitoring systems. if an agent can be detected quickly following an aerosol release, response timelines can be signi fi cantly improved, allowing for more time to intervene and potentially prevent illness in a signi fi cant portion of the exposed population. in , the united states implemented biowatch, an environmental monitoring system that consists of a network of samplers that collect air on a continuing cycle [ ] . filters from the monitors are removed on a frequent basis and screened in a laboratory for the presence of several biological threat agents. biowatch is currently operational in multiple us cities. environmental monitoring in this fashion requires a signi fi cant fi nancial commitment and is a complex system to operate as experience with this type of system was limited prior to its implementation. natural environmental presence of the target organisms and/or very closely related organisms and the size of the area to be monitored present ongoing challenges for establishing system sensitivities and speci fi cities that appropriately balance the potential value of early detection of a bioterrorism attack with the risk of inappropriately responding to a positive test that is caused by naturally occurring organisms in the environment. a separate system of detectors has also been deployed that monitors the us mail system, the method of "dissemination" used in the anthrax letter attacks. the us postal biohazard detection system (bds) has been operational since [ ] . unlike traditional disease and syndromic surveillance systems for human and animal health which monitor for both intentional and naturally occurring disease, these environmental systems are single purpose with the primary focus being early warning of bioterrorism. one of the more effective preparedness planning tools are tabletop and fi eld exercises, with involvement of representatives from key local, state, and federal agencies, as well as representatives from the local medical and laboratory communities. these exercises provide the opportunity to test assumptions in existing plans, and work out issues related to decision-making authority and respective roles and responsibilities among the various disciplines that would be involved in responding to a bioterrorist attack or other local emergency. post exercise debrie fi ngs should be conducted to highlight gaps in preparedness that can then be addressed through follow-up planning meetings and revision of written plans, if indicated, and re-evaluated with repeat exercises. depending upon the size and scope, responses to public health emergencies may involve resources and responsibilities that span multiple agencies at the local (city or county), state, and federal government levels. emergency events begin at the community level (single or multiple communities) and local personnel and resources (medical, public health, emergency services, police, fi re, etc.) provide the initial response. if local resources are overwhelmed or authorities require special assistance or resources that are not locally available, assistance from the state or federal level can be requested. this may be done through a direct assistance request to an agency or agencies (e.g. a request to cdc to assist with a food outbreak investigation or test samples) or through the formal declaration of an emergency that activates state and federal emergency support functions (e.g. declaration of state of emergency that activates the federal emergency management agency (fema) and other federal assistance as needed through the national response framework (nrf) and the associated emergency support functions (esf)). emergency responses and their coordination in the us primarily involve civilian agencies and authorities, with the military providing support as needed. central to the ability to successfully coordinate a response to a large-scale emergency is the ability to integrate information fl ow, resources, and personnel into an organizational structure that is similar across all responding agencies, whether the emergency is primarily public health in nature or due to some other cause. this incident management system or incident command system (ics) structure, has been used for many years by traditional fi rst responder agencies such as fi re and law enforcement and was formally identi fi ed as the national emergency response structure in [ ] . ics has also been adopted and used to a much greater extent by federal, state, and local public health agencies responding to public health emergencies. cdc utilized the ics to coordinate its response to public health emergencies such as the h n pandemic and multi-state foodborne outbreaks but has also bene fi ted from better integration of its response activities into larger-scale, multi-hazard emergency responses such as hurricane katrina and the recent haiti earthquake. the us department of health and human services (dhhs) has the lead for coordinating the federal public health and medical services support functions outlined in esf ( http://www.fema.gov/pdf/emergency/nrf/nrf-esf- .pdf ). these support functions include response activities in the following areas: ( ) assessment of public health/medical needs, ( ) health surveillance, ( ) medical care personnel, ( ) health/medical/veterinary equipment and supplies, ( ) patient evacuation, ( ) patient care, ( ) safety and security of drugs, biologics, and medical devices, ( ) blood and blood products, ( ) food safety and security, ( ) agriculture safety and security, ( ) all-hazard public health and medical consultation, technical assistance, and support, ( ) behavioral health care, ( ) public health and medical information, ( ) vector control, ( ) potable water/wastewater and solid waste disposal, ( ) mass fatality management, victim identi fi cation, and decontaminating remains, and ( ) veterinary medical support. several agencies exist within dhhs that help carry out these activities, including cdc, the food and drug administration (fda), the national institutes for health (nih), and the substance abuse and mental health services administration (samhsa) among others. in addition, dhhs manages the national disaster medical system (ndms) which includes disaster medical, surgical, and mortuary response teams as well as veterinary response teams. in addition to providing medical response to a disaster area, ndms also coordinates patient movement into hospital care in unaffected areas for de fi nitive medical care with the support of the department of defense (dod). in addition to dod, multiple other agencies and departments provide support to dhhs for esf functions, including the department of agriculture (doa), dhs, fema, the department of transportation (dot), the department of veterans affairs (va), the american red cross (arc), and others. once a bioterrorist event is recognized and then con fi rmed by laboratory testing, there will be a need for large-scale mobilization of surveillance and epidemiologic investigations. the focus of these investigations will be ( ) tracking the number of cases to de fi ne the scope of the incident and ( ) performing epidemiologic investigations to determine the common source(s) and site(s) of exposure. this information will be most critical in the event of a covert bioterrorist event to determine where and when the attack occurred, and who else may have been potentially exposed (either at the event or due to downwind distribution of the aerosol) and thus require prophylaxis. as active surveillance would need to be initiated rapidly once a bioterrorist event is recognized, many local and state health departments have developed materials and plans to facilitate the ability to rapidly implement an investigation, including template surveillance instruments and protocols for urgently mobilizing and deploying active surveillance surge teams to hospitals in the affected area. response to public health emergencies that result from an intentional biothreat agent, such as the us anthrax letter attacks, have an added investigational and coordination complexity due to the necessary law enforcement component of the event [ ] . if an event is known to be secondary to an intentional act, local law enforcement of fi cials and the federal bureau of investigation (fbi) have a greater leadership role in coordinating the investigation and communication, however, public health and other responding entities are still responsible for carrying out their usual surveillance and emergency response activities. in this scenario, activities such as interviewing victims to determine the common site and/or sources of exposure, specimen or sample collection and testing, and public messaging would be coordinated with the fbi or other law enforcement of fi cials in order to preserve evidence and investigative information that may be essential for attribution and conviction of the perpetrators. some activities such as sample collection or victim interviews may even need to be planned and conducted jointly by public health and law enforcement of fi cials. although law enforcement has the responsibility for conducting the criminal investigation, their primary mission is also the preservation of life and health and investigative activities are targeted towards accomplishing that goal in addition to obtaining the evidence needed to identify and convict those responsible. many local, state, and federal public health and law enforcement authorities in the us have recognized the investigation and communication coordination that would be required in a bioterrorism or other intentional chemical, radiological, or toxin induced event that affects the health of individuals or communities and have established working relationships for preparedness as well as formalized agreements for information sharing and joint investigative activities in this type of event. a model for a memorandum of understanding (mou) that can be used to create formalized working agreements between public health and law enforcement of fi cials was developed by a working group convened by the cdc and the us department of justice. this model mou has been distributed to state and local authorities and a copy can be requested through the cdc public health law practice website at http://www a. cdc.gov/phlp/mounote.asp . the united states considers bioterrorism a serious threat to its national security and has made concerted efforts over the last decade to bolster public health and other response capacity capabilities. many of these efforts, though initially begun to address the needs for bioterrorism preparedness, have proven bene fi cial for public health in responding to other emergencies, including those due to naturally occurring disease threats such as pandemic in fl uenza. speci fi cally, efforts that focused on improving: ( ) laboratory diagnostic capacity, ( ) surveillance data sources, analysis, and reporting, ( ) risk communication ( ) emergency response planning and training, and ( ) overall response coordination have proven extremely bene fi cial for supporting public health responses to all types of health threats. in most state and local health departments in the us, bioterrorism surveillance and response capacity is fully integrated into the general infectious disease and all hazards emergency response infrastructure. the same staff that surveil for and respond to both routine and emergency infectious disease outbreaks would be called upon to respond to a bioterrorism attack. this dual-use capacity is more ef fi cient and ensures that front line public health staff maintain and exercise the skills required to detect and respond to disease threats, regardless of whether intentional or natural. the h n pandemic provided one of the best training opportunities for what might be encountered in the event of a large scale bioterrorist outbreak, including the need to implement enhanced surveillance to provide greater real time situational awareness, with the initial reliance on the public health laboratory system for reference testing, and the implementation of a large scale vaccination campaign. although bioterrorism is not accorded the same level of concern everywhere, investments that help build or support stronger public health and medical systems provide the foundation for responding to all health threats and are essential, should an unthinkable event such as a large-scale bioterrorism attack occur. index case of fatal inhalational anthrax due to bioterrorism in the united states collaboration between public health and law enforcement: new paradigms and partnerships for bioterrorism planning and response emergency preparedness and response: the laboratory response network partners in preparedness emergency preparedness and response: strategic national stockpile leading causes of death syndromic surveillance: adapting innovations to developing settings hantavirus pulmonary syndrome: a clinical description of patients with a newly recognized disease encyclopedia of quantitative risk analysis and assessment the model state emergency health powers act: planning for and response to bioterrorism and naturally occurring infectious diseases overview of syndromic surveillance: what is syndromic surveillance an outbreak in of cyclosporiasis associated with imported raspberries anthrax bioterrorism: lessons learned and future directions outbreak of legionnaires' disease among cruise ship passengers exposed to a contaminated whirlpool spa investigation of bioterrorism-related anthrax implementing syndromic surveillance: a practical guide informed by the early experience responding to detection of aerosolized bacillus anthracis by autonomous detection systems in the workplace fatal inhalational anthrax with unknown source of exposure in a -year-old woman in new york city the outbreak of west nile virus infection in the new york city area in the public health security and bioterrorism preparedness and response act public health assessment of potential biological terrorism agents the biowatch program: detection of bioterrorism antimicrobial postexposure prophylaxis for anthrax: adverse events and adherence hspd- : biodefense for the st century hspd- : management of domestic incidents terrorism incident annex to the federal response plan key: cord- -migs rxb authors: garaialde, diego; bowers, christopher p.; pinder, charlie; shah, priyal; parashar, shashwat; clark, leigh; cowan, benjamin r. title: quantifying the impact of making and breaking interface habits date: - - journal: nan doi: nan sha: doc_id: cord_uid: migs rxb the frequency with which people interact with technology means that users may develop interface habits, i.e. fast, automatic responses to stable interface cues. design guidelines often assume that interface habits are beneficial. however, we lack quantitative evidence of how the development of habits actually affect user performance and an understanding of how changes in the interface design may affect habit development. our work quantifies the effect of habit formation and disruption on user performance in interaction. through a forced choice lab study task (n= ) and in the wild deployment (n= ) of a notificationdialog experiment on smartphones, we show that people become more accurate and faster at option selection as they develop an interface habit. crucially this performance gain is entirely eliminated once the habit is disrupted. we discuss reasons for this performance shift and analyse some disadvantages of interface habits, outlining general design patterns on how to both support and disrupt them.keywords: interface habits, user behaviour, breaking habit, interaction science, quantitative research. we interact with a wide variety of devices and interfaces on a daily basis. the frequency of these interactions means that users are likely to develop habits -fast, automatic behaviours that emerge in stable contexts -around these interfaces. although habit research has become increasing popular in fields such as hci (cowan, bowers, beale, & pinder, ; pinder, vermeulen, cowan, & beale, ; pinder, vermeulen, wicaksono, beale, & hendley, ; renfree, harrison, marshall, stawarz, & cox, ; stawarz, cox, & blandford, ) , there has been less focus on how habits are formed within interfaces and how their development affects user performance. research has instead concentrated on behaviour change interventions to impact health, work related habits (gardner, de bruijn, & lally, ; stawarz et al., ; stawarz, rodriguez, cox, & blandford, ) , or on problematic habitual use of technology (elhai, levine, dvorak, & hall, ; van deursen, bolle, hegner, & kommers, ) . common design guidelines such as consistency (shneiderman & plaisant, ) , or usability concepts such as learnability (nielsen, ) , allude to desirable interface properties that are beneficial for habits to develop, yet do not attempt to quantify how habit development benefits user performance. even with optimal intentions to follow these guidelines, changes are regularly made to interfaces that incorporate new features and design norms. when this occurs, previously learned habits may become disrupted, meaning users have to relearn how to perform desired actions. currently there is limited quantitative evidence specifically addressing how these disruptions affect performance. the contribution of the current paper comes from providing quantitative evidence of how the process of forming and disrupting habits affects user performance in a forced choice interaction task, similar to those seen in notification dialogs or alert boxes. to do this we conducted two studies (one in the lab and one in the wild) examining the building of interface habits. our studies indicate that as people develop a habit, they become quicker and more accurate at option selection. a concentrated set of repetitions over trials in the lab was enough to create strong improvements in speed and accuracy. in addition, more dispersed daily interactions within a -day period in the wild were also enough to see significant improvements in speed. yet once these habits are disrupted, all performance benefits are significantly reduced. our work contributes to the field by demonstrating both the benefits of habit formation and the significant cost of disrupting these habits when an interface is changed. we show that simple interface habits, like option selection, may be easily developed. when designing interfaces, designers should be cautious about interface changes, incremental or otherwise, unless clear consideration is given to the potential disruption to users' habits. around % of our daily activities frequently occur in consistent contexts which are conducive to habit formation (wood, quinn, & kashy, ) . although habits are commonly defined in the literature as "learned sequences of acts that have become automatic responses to specific cues" (verplanken & aarts, , p ) , there is currently a debate in the literature as to what truly constitutes a habit. in an effort to reduce the conflation between habits as behaviours and habits as causes of behaviours, gardner ( a) defines a habit as "a process by which a stimulus automatically generates an impulse towards action, based on learned stimulus-response associations" (p. ), which isolates the description to that of impulses to act, regardless of whether an act is completed. critically these actions and/or impulses have a degree of automaticity, and tend to be instigated during stable contexts (orbell & verplanken, ) . automaticity means that these impulses lead behaviours to be performed without intention or using limited conscious awareness (aarts & dijksterhuis, ) . these become automatic when context-response associations are created in procedural memory, bypassing the need to analyse the context when searching for an appropriate response (wood & neal, ) . this benefits users by decreasing the cognitive load required to complete frequently repeated actions (law, wehrt, sonnentag, & weyers, ) leaving more cognitive resources for other activities. while many actions are initially performed with goal-driven intentions, over time repeated exposure to stable contextual cues reinforces the association within procedural memory (wood & neal, ) . this diminishes the need for the initial goal-driven motivation (wood & rünger, ) making a habit primarily driven by environmental context rather than a specific goal. as highlighted by gardner ( a) the process of learning stimulus-response associations is critical in forming a habit, with the strength of the stimulus-response associations dictating the strength of the developed habit. interacting with technology often involves similar actions through a familiar interface in a consistent environment. frequent interaction with these interfaces makes it likely that habits develop within this context. recent work shows that habits form a significant part of our interaction with mobile devices. a longitudinal study on mobile device use found that users repeatedly perform checking habits, short interactions where users check on one or two applications, dispersed evenly throughout the day (oulasvirta, rattenbury, ma, & raita, ) . the way these form is similar to the cue-behaviour-reward process seen in developing other habits (wood & rünger, ) . checking behaviours become tightly associated to particular contextual triggers, with behavioural execution leading to positive rewards such as desired information or positive social interactions (oulasvirta et al., ) . the receipt of these rewards can be unpredictable and the level of reward highly variable. this is likely to promote stronger associations, increasing the frequency and persistence of a behaviour being executed (egel, ; morford, witts, killingsworth, & alavosius, , williams, . checking behaviours on mobile devices tend to be supported by interactive notifications, by which mobile devices frequently attempt to gain the user's attention. rather than just displaying information, these notifications commonly include a request for the user to select a function from a set of options, much like a dialog box. these types of notifications can become strong cues that instigate particular habitual responses to clear them. notifications act as frequent calls for users to perform a consistent action and can act as gateway to further app use (oulasvirta et al., ) , making them optimal habitual response cues. any design changes to these cues are likely to disrupt the habits that users form around an interface. design changes are believed to force users out of automatic behaviour, removing their attention from the task, and instead focusing it back on the design of the interface (raskin, ) . to designers, it may seem that the concept of user habituation has already been considered in the design of interfaces through popular design guidelines and heuristics (nielsen, ; shneiderman & plaisant, ; thimbleby, ) . design guidelines such as consistency, predictability, and standardisation of presentation (shneiderman & plaisant, ) allude to the idea that an ideal interface environment allows users to develop and maintain an interaction habit. whilst these rules have proven invaluable to contemporary interface design, they alone do not give us a sense of the potential impact that habit development has on performance. furthermore, there is no indication of the time taken to create these behaviour patterns or the extent to which performance is impacted if these patterns are disrupted. the current paper aims to explore these aspects of interface habit development in more detail. the current research contributes key insight on fundamental user behaviours by quantifying how the process of habit formation and disruption through design affect the speed and accuracy of interactions. crucially we investigate this both within lab based and in the wild settings. we present two studies that focus on forced choice tasks, reflecting the characteristics of alert and other dialog selection screens seen in most user interfaces (e.g. "ok" vs "cancel" in alerts, or "reply" vs "mark as read" in messaging applications). study examines how the process of formation and disruption of habits affect performance in a simple two-alternatives forced choice task in the lab under controlled conditions. study extends from study by investigating whether the effects replicate to a common real-world context in the form of a smartphone interactive dialog notification as the forced choice task. the following hypotheses are considered in this study: hypothesis a -response time will improve from baseline levels when participants begin to form an interface habit. hypothesis b -response time will be significantly negatively impacted when habit formation is disrupted. hypothesis a -response accuracy will improve from baseline levels when participants begin to form an interface habit. hypothesis b -response accuracy will be significantly negatively impacted when habit formation is disrupted. . . participants. nineteen participants ( men, women) with a mean age of years were recruited from a european university. the study was given exemption from ethical review as it did not meet any of the requirements for full review due to the low risk involved. participants were invited to participate via email and social media. a € gift voucher was given to each participant as an honorarium for participation. participants completed a forced choice task on a laptop and were asked to make a choice of whether the correct name for the object in the image was displayed on the right or left of the screen. two images were used in the study from the snodgrass and vanderwart corpus (snodgrass & vanderwart, ) , with the order of their appearance randomised. for each trial, participants were instructed to select the direction using the arrow keys (either "left" or "right") that corresponded to the location of the correct name. this type of task was used to ensure that participants did not have any pre-existing habits before taking part in the study, which may be the case if using existing dialog notification designs or common interface forced choice tasks. participants completed a total of trials balanced across the three phases of the experiment ( trials per phase).while as little as trials are enough to accurately measure reaction time in a two choice task (hultsch, macdonald, & dixon, ) , trials is usually required to achieve a reaction time plateau (parkin, kerr, & hindmarch, ) , indicating a degree of automaticity. the presentation of the two images was balanced so that each image appeared an equal number of times in each phase (i.e. times per image) and that the labels appeared an equal number of times in the top left and right-hand side of the screen. the order of presentation of the images and appearance of the labels were randomised for each participant. an example of the stimuli is shown in figure . baseline phase, the corresponding label for each image appeared randomly in either the top left or right-hand corner of the screen. this condition identified a baseline response time for the task. in the habit phase, the labels remained static and only the images were presented randomly (e.g. lion label was always on the left). in the disruption phase, the side on which the label was displayed was randomised again, thus disrupting any learned response that the participants may have developed. participants were not made aware of changes in phase, which happened in the background after trials were completed. this type of response time task is typical in tests of procedural memory (e.g. knopman & nissen, ) , which have established that improvements in reaction time arise from an increase in automaticity. making the placement of the labels consistent in the habit phase facilitates users to map between the context (the image) and the response (the direction matching the label) in procedural memory, supporting repeated encoding of context-response patterns that are needed for automaticity to form (wood & rünger, ) . although there is debate about whether a habit should be defined as the behaviour or the impulse to perform a behaviour (see gardner, b ), in the current task the users' impulse and actions were aligned, allowing both definitions to apply. the most common way to measure habit is through self-report measures such as the self-report habit index (verplanken & orbell, ) , or the self-report behavioural automaticity index (gardner, abraham, lally, & de bruijn, ) , which is a validated subscale of the srhi focusing specifically on automaticity. yet these types of measures can have downsides. awareness decreases as automaticity develops (macleod & dunbar, ) , making subjective reflection on the automatic process potentially less reliable. because of this, measures like response time and accuracy have been used as successful alternatives in gauging automaticity (logan, ; poldrack et al., ) . we therefore measure how long people take to select the label (response time) and the accuracy of their response (response accuracy) in each of the trials. participants were welcomed to the lab, given information on the purpose of the study and gave informed consent. they then received task instructions and were asked to complete all three phases of the experiment (baseline, habit and disruption). the trials were displayed on a laptop using psychopy version . (peirce, ) . after completing all phases, participants were debriefed about the purpose of the study and thanked for taking part in the research. each participant completed trials, for each of the three phases, with entries in total. using a-priori screening suggestions (baayen & milin, ) , entries with impossible reaction times for a discrimination task (< ms & > s) were removed. this initial screening removed trials ( . %), leaving trials. to assess the effect of phases in the response time analysis, trials where participants gave an incorrect answer were removed ( . %; , remaining), following general convention for this type of analysis (howell, the response time data was analysed using a linear mixed-effects model (lme) using the lme package (bates, mächler, bolker, & walker, ) in r (r core team, ) version . . feather spray. this analysis allowed us to assess the impact of the fixed effects (in this case the within participant variable: experiment phases) on the participant's response time, whilst controlling for individual differences in participant performance through random effects (for comprehensive review see barr, levy, scheepers, & tily, ) . in accordance with barr et al. ( ) , we ran the maximal model with by-participant random slopes, simplifying the model where needed to achieve convergence. the lme model includes experiment phases as a fixed effect ( levels -baseline, habit & disruption) and by-participant random slopes (random effect). as both the hypotheses refer to comparisons between the habit and the other conditions, the habit condition is used as the intercept for all comparisons. upon looking at the distribution and the homoskedacity of residuals from the original analysis run on the untransformed data, the model appeared highly stressed at longer reaction times, suggesting a strong influence of outliers on the model. we therefore used an inverse making & breaking interface habits transformation and model criticism of data outliers based on model residuals to limit the impact of outliers on the results (for in-depth discussion see baayen & milin, ) . using this method, ( . %) problematic trials were removed, leaving left for analysis. as suggested in (baayen & milin, ) the transformed reaction times were multiplied by negative to align estimates with the expected direction of interaction and to ensure the effect was visible at two decimal places. table shows the results of the lme analysis after inverse transformations and negative multiplication. figure , shows the predicted response times transformed back into the original scale to allow for greater readability and a more intuitive inspection of the differences between groups (produced by the effects package version _ _ fox, ) . the analysis showed significantly longer response times in the baseline phase when compared to the habit phase (t = . , p < . ), supporting hypothesis a that habit formation leads to a significant improvement in speed compared to baseline levels. there was a similar statistically significant increase in the response times for the disruption phase when compared to the habit phase (t = . , p < . ), supporting hypothesis b that disrupting a habit leads to a decrease in speed. to assess the effect of condition on response accuracy, a logistic mixed-effects model was run. this analysis assesses the impact of the fixed effects (in this case experiment phases) on the log odds of selecting the correct item (incorrect: , correct: ), whilst controlling for individual differences in performance through random effects. descriptive statistics of response accuracy are included in table . there was a significant decrease in correct answers during baseline when compared to the habit phase (z = - . , p < . ), supporting hypothesis a that response accuracy is improved as habits form. there was also a significant decrease in correct answers during disruption when compared to the habit phase (z = - . , p < . ), supporting hypothesis b that disrupting a habit negatively impacts response accuracy. the experimental evidence of study shows that, like other habits, allowing participants to form interface habits leads to significant gains in performance, as users became both more accurate and quicker at selecting the desired option. importantly, we found that when habits becomes disrupted, any of the performance improvements are erased, returning to baseline levels. overall the results highlight how moving context-response patterns to procedural memory is an effective way to improve performance and accuracy. when these patterns can no longer be used (i.e. habits are disrupted), performance gains are drastically reduced. the findings show strong effects of the interface habit formation process on performance that could be highly relevant to user interface behaviours. yet these effects were elicited in a lab study, where users were interacting in a one-off session on a laptop-based interface. the task also lacked ecological validity, in that it did not resemble the everyday types of forced choice task encountered by users. the controlled lab setting is also not representative of the distraction-filled environment that is common when needing to make notification or dialog box choices. study therefore extends from study by running a similar study in the wild on smartphones, replacing study 's forced choice task with a more ecologically valid dialog notification task. participants ( men, women) with a mean age of years took part in the experiment as part of a wider study on user behaviour towards mobile notifications. the study was given full ethical approval before starting recruitment. participants were recruited from two european universities through email. they were given a £ voucher as honorarium for their participation in the study. to be eligible to take part, participants needed to be over years of age and own an android device running version . . or newer. the experiment application presented a series of notificationlike dialogs to the user; an example is shown in figure . to ensure that any effect seen was not confounded by users' existing notification habits, we altered the design of the notifications used. rather than using common text action buttons, participants were asked to select an image specified in the notification text from two images displayed. we also reversed the usual convention of two text action options placed horizontally to two image action options placed vertically. the images used were taken from images by snodgrass and vanderwart ( ) . the images appeared in a random order within a dialog that was placed on top of the existing device interface, obfuscating the user's current task. the dialog required the user to select the correct image before it could be cleared. the application was designed to only display the dialogs when the device detected that the user was currently actively engaging with the device. this was to ensure the user's attention is on the device at the time of the notification appearing. as in study , the experiment included within participant experimental phases. in the baseline phase all participants were presented with dialogs where the images that needed to be selected by user were randomly positioned. this acted as a baseline, assessing user performance before a habit could develop. in the habit phase, each individual correct image was always placed in the same location (either in the top or bottom of the dialog) for each trial. the disruption phase once again had the images appear randomly in either position. the phases were changed remotely via the server after at least days to ensure users had the opportunity to complete enough interactions for each phase. participants were not made aware of the phase change. response time was measured as the time difference between the logged time the dialogue was first presented to the logged time that a dialog was cleared. response accuracy was measured as whether the users selected the image they were asked to select in their initial response. at the start of the study, participants were directed to a website which contained participant information and a request for consent. once consent was gained, participants were directed to instructions on how to download, install and request support for the application. to ensure that participants had sufficient opportunity to install the application and gain support if required, we allowed a -day interval between initial release of the recruitment call to the commencement of the first phase of the study. once a trial (i.e. a notification dialog) was completed and logged by the server the next trial would be generated and sent to the participant's phone. as mentioned above, each withinsubject phase was administered remotely after at least days via the server and deployed by the server to each device at the next available opportunity to ensure that all phases started on the same day. the duration of the phases was chosen to ensure each user completed enough interactions per phase. participants completed an average of interactions per phase, well above the trials recommended to reach a response time plateau (parkin et al., ) needed to signal acquired automaticity. at the end of the final (third) phase, a thank you message was pushed to participants, which included instructions on how to uninstall the applications from their personal devices. the study lasted for a total duration of days. a total of participants agreed to take part in the study, contributing a total of trials. we excluded participants who did not fully complete the study or who contributed too few trials to the baseline condition ( participants) leaving a total of participants ( men, women) and trials. impossible response times below ms and above s were also removed ( trials), leaving an overall total of trials for analysis. descriptive statistics are displayed in table . on average, the remaining participants after removal interacted with a dialog notification between . and . times per day, with an overall mean of . . table : linear mixed effects regression analysis of the inverse of response times by phase. sixty-seven ( . %) entries were removed using this method, leaving trials. table shows the results of the lme analysis with the inverse transformation, while figure shows the modelled reaction times per phase untransformed. descriptive statistics for response accuracy are shown in table . like in study , a logistic mixed-effects model was run to assess the effect of phase on accuracy. this analysis again assesses the impact of the fixed effects (in this case experiment phases) on the log odds of selecting the correct item (incorrect: , correct: ), whilst controlling for individual differences in performance through random effects. table shows the results of the logistic mixed effects analysis, while figure shows the probability of correct answer per phase. the model found no statistically significant difference between the habit and either the baseline the results of study showed that, like study , participants were significantly faster at correctly answering the dialogs when the interface allowed for the formation of habits. similar to study this improvement was erased when the habit was disrupted, with participants returning to levels of performance similar to baseline performance. however, unlike study we found that accuracy did not improve when the interface changed compared to their baseline performance. these findings contradict previous research stating that habitual behaviour is less prone to errors than when a habit is not formed (graybiel, ; wood & rünger, ) . response times in study were longer than those in study . this may be because of the in the wild nature of the study. participants were interrupted with the dialog as opposed to expecting the forced choice task in a lab setting, which may have contributed to the response delay. another reason may be that both studies varied in the visual design and device context. study was conducted on a laptop, allowing the labels and pictures in the study to be clearly visible to participants. study was conducted on a much smaller screen, with participants having to read more detailed written instructions on this display. this increased focus could have led to higher selection times compared to study . nevertheless, the clear difference in response time between the phases show how in the wild measures can still be useful in quantifying increased automaticity in response to a cue, providing a useful metric for interface habit development. this work contributes insight into fundamental user behaviours, particularly on how creating an environment that allows for a habit to form can affect user task performance. the work focuses particularly on the process of formation and disruption of interface habits around option selection, such as those seen in dialog notifications on a number of devices (e.g. choosing "reply" or "archive" in an email application). we show that an interface conducive to habit formation significantly improve the speed at which people select their desired option (study & ). these findings echo those focused on other behaviours in the psychology literature which state that making a behaviour automatic can drastically improve performance (aarts & dijksterhuis, ; dezfouli & balleine, ; logan, ) . importantly, we show that the creation of interface habits can not only be supported and disrupted in a single session in the lab, but also within the context of long-term everyday use. the process of interface habit formation occurred even when interactions were dispersed throughout a block of days, at an average of interactions per day. our findings show that the improvements resulting from the formation of interface habits are drastically reduced after the interface is changed as procedural memory is no longer involved. the lab study (study ) indicated that disrupting habits has significant detrimental effects on both speed and accuracy, while the in the wild study (study ) only saw a negative impact on speed. once again, this supports previous psychological research on habits that describe the negative effects of habit disruption on speed (anderson, folk, garrison, & rogers, ; lally, van jaarsveld, potts, & wardle, ) , with only partial support for effects in accuracy (logan, ) . the discrepancy in the findings for accuracy may have arisen due to limitations of a study conducted outside controlled conditions. the real-world mobile notification context may be a contributory factor in this. dialogs were presented when performing other tasks, subsequently interrupting the user. this unexpected interruption may have thus led users to select the wrong image in error, thus inflating the number of errors made. in both studies, the habit phase led to improvements in response time. this is believed to occur due to a shift of context-response associations into procedural memory (wood & neal, ) . once in procedural memory, participants no longer need to read the labels to allow them to select the correct action and can rely solely on their automatic reaction. while it could be argued that improvements in response time occurred because the habit phase was easier, the reality is that the task was only easier due to the increase in automaticity from the development of a habitual response. if a user was not able to form habits or move associations into procedural memory, they would not see such an improvement in performance. this deterioration or inability to learn new response patterns can be seen in patients suffering from damage to brain regions associated with procedural memory (ackermann, daum, schugens, & grodd, ) but is not seen when the damage is associated with other types of memory (cohen & squire, ) . repeating the dialog behaviour using a consistent interface likely increases the strength of the stimulus-response associations within procedural memory. this learning increases the initiation and execution speed of the behaviour. (fogg & hreha, ) but also make users less likely to change to a competitor application, as the cost of transition would be increased (oulasvirta et al., ) . the research also highlights how response times can be used to measure the degree of automaticity a user has when navigating an interface, which can be used to indicate the formation of interface habits. . . adapting interfaces to account for user habits. the current research highlights the power of response time measurements as a tool for gauging the interface habit formation of users. our findings suggest that interface designers can use response times to measure the degree to which each individual user is forming an interface habit. with this type of granular data, different strategies can be employed to reduce the disruptive effects of necessary changes in the interface. for example, power users with strong interface habits may be presented with interim interfaces that can acclimatise users more gradually, reducing frustration while maintaining high performance levels. furthermore, these measures could be used to ascertain when users are mindlessly interacting with important alerts, which would signify a need to change these interfaces so as to force users to pay closer attention. performance, the nature of habits as automatic behaviours stimulated by particular cues can be exploited as a dark design pattern (greenberg, boring, vermeulen, & dostal, ) . pop-ups exemplify this unethical "bait and switch" behaviour by creating realistic looking windows and dialog boxes (cues) that are changed in their function to bring undesired results (such as opening malicious programs). this type of habit hijacking is also common in phishing scams, where malicious actors present a familiar interface (e.g. paypal website) in the hopes that users will provide important personal information (dhamija, tygar, & hearst, ) . there is a danger that unscrupulous designers may even change functionality dynamically, recording when is the best time to switch based on performance indicators of a habit being formed. (norman, ) . in safety critical circumstances such as medical surgery (machno et al., ) or transport operation (walker & strathie, ) , performing a habitual, often-practiced response in an interface without considering the consequences could be catastrophic. disrupting these habits through micro boundaries may be necessary, even if it prevents users from progressing quickly. our work explores habit formation in the context of device interaction. in particular we focus on selection tasks in a lab-based computer interaction, following with an in the wild notification dialog experiment on smartphones. the differences in our experimental results between these two studies reflect the difficulty in transferring abstract theoretical concepts from the lab to a real-world setting, as the number of confounding variables increase. that said, our findings, especially those with reference to response time, were similar across the experimental contexts. throughout the studies we have used abstract forced choice tasks as a way to simulate user option selection. this was primarily for the purposes of study control, reducing the confounds that exist if we used existing interface selection tasks. the tasks chosen were also simple and only focused on two distinct choices. this was so as to support the habit forming process over the experiments as research has shown that overly convoluted tasks could interrupt this (lally et al., ) . we also altered the tasks over two studies, to increase ecological validity. future work should investigate the role that complexity may play in the formation and disruption of interface habits as well as look to keep the tasks consistent across the studies to identify any impact this may have had on our study findings. as the dialog conditions for study could not be changed at the individual participant level, and due to some participants being delayed in setting up the application correctly on their device, participants were exposed to the baseline condition for variable amounts of time. to ensure this did not impact our findings, we removed participants that did not complete a satisfactory number of interactions during the baseline phase from the analysis. for the remaining participants, a similar number of interactions were collected across all three phases, ensuring a consistent set of responses for each condition in the analysis. our research examined the impact of creating and disrupting interface habits on interaction efficiency and effectiveness as measured by performance and accuracy. our results indicate that people can develop option selection habits both in concentrated bouts of interaction in the lab, and during more dispersed dialog interactions in the wild. the development of a habit improved task performance speed and increased user accuracy, although accuracy benefits did not occur in the wild. we show that disrupting this habit development has serious consequences for user performance, leading people to be slower when selecting options. we also show that response times can be used effectively to differentiate between users who are and are not forming an interface habit, allowing for different actions to be targeted at each group. when developing interfaces, designers must be aware of this tendency to form habits in interaction and, through design, look to support habitual behaviours. this work was funded by the hw wilson foundation, the nui travelling scholarship and science foundation ireland adapt centre /rc/ ). the datasets and analyses used for the current research are available in the open science framework repository at https://osf.io/w n e/. habits as knowledge structures: automaticity in goaldirected behavior impaired procedural learning after damage to the left supplementary motor area (sma) mechanisms of habitual approach: failure to suppress irrelevant responses evoked by previously rewardassociated stimuli analyzing reaction times random effects structure for confirmatory hypothesis 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this, then habit: exploring context-aware implementation intentions on smartphones the neural correlates of motor skill automaticity r: a language and environment forstatistical computing. vienna, austria: r foundation for statistical computing don't kick the habit: the role of dependency in habit formation apps designing the user interface a standardized set of pictures: norms for name agreement, image agreement, familiarity, and visual complexity beyond self-tracking and reminders: designing smartphone apps that support habit formation understanding the use of contextual cues: design implications for medication adherence technologies that support remembering user interface design: generative user engineering principles modeling habitual and addictive smartphone behavior habit, attitude, and planned behaviour: is habit an empty construct or an interesting case of goal-directed automaticity? reflections on past behavior: a self-report index of habit strength big data and ergonomics methods: a new paradigm for tackling strategic transport safety risks a new look at habits and the habit-goal interface habits in everyday life: thought, emotion, and action key: cord- -ywb cq authors: sarkar, indranil; garg, ravendra; van drunen littel-van den hurk, sylvia title: selection of adjuvants for vaccines targeting specific pathogens date: - - journal: expert rev vaccines doi: . / . . sha: doc_id: cord_uid: ywb cq introduction: adjuvants form an integral component in most of the inactivated and subunit vaccine formulations. careful and proper selection of adjuvants helps in promoting appropriate immune responses against target pathogens at both innate and adaptive levels such that protective immunity can be elicited. areas covered: herein, we describe the recent progress in our understanding of the mode of action of adjuvants that are licensed for use in human vaccines or in clinical or pre-clinical stages at both innate and adaptive levels. different pathogens have distinct characteristics, which require the host to mount an appropriate immune response against them. adjuvants can be selected to elicit a tailor-made immune response to specific pathogens based on their unique properties. identification of biomarkers of adjuvanticity for several candidate vaccines using omics-based technologies can unravel the mechanism of action of modern and experimental adjuvants. expert opinion: adjuvant technology has been revolutionized over the last two decades. in-depth understanding of the role of adjuvants in activating the innate immune system, combined with systems vaccinology approaches, have led to the development of next-generation, novel adjuvants that can be used in vaccines against challenging pathogens and in specific target populations. development of vaccines against infectious diseases is one of the most remarkable accomplishments in the history of mankind [ ] . smallpox has been eradicated from the world, and other diseases like diphtheria, poliomyelitis, pertussis, measles, and neonatal tetanus are significantly controlled by vaccination [ , ] . while live attenuated vaccines are immunogenic, there is a chance of live virus-induced disease progression in populations with underdeveloped or compromised immune systems [ ] . in contrast, inactivated virus vaccines are safe, but unsuitable when natural infection by the pathogen itself fails to induce any long-term immunity. recombinant subunit vaccines are considered as one of the most attractive modern vaccine types due to their high safety profiles. however, subunit vaccines are not inherently immunogenic [ , ] . to overcome this limitation, adjuvants are incorporated in subunit vaccines to enhance immunogenicity of the vaccine antigen. adjuvants facilitate the development of vaccines targeting pathogens against which live attenuated or inactivated vaccines are ineffective or undesirable. identification and selection of new adjuvants is thus critical, but also challenging, for successful subunit vaccine development. the fact that only a few adjuvants were approved for use in human vaccines till a few years ago can be at least partially attributed to the dearth of knowledge of the mechanism of action of adjuvants. however, presently six adjuvants (alum, as , mf , as , as and cpg odn) are approved for use in human vaccines. this was possible because structural characterization of several adjuvants and identification of various pattern recognition receptors (prrs) and co-stimulatory ligand receptors have enabled us to better understand the mode of action of adjuvants at a molecular level. understanding the mode of action of adjuvants is critical in designing vaccines that elicit pathogen-specific effector and long-term memory responses and in assessing the adjuvant safety at developmental and regulatory stages. the possible mechanisms of action by which adjuvants exert their adjuvanticity are discussed in the subsequent sections and represented schematically in figure . adjuvants as a delivery system in subunit vaccines [such as liposomes, immune stimulating complexes (iscoms) and nanoparticles] are considered effective in stimulating protective immunity [ ] . such adjuvants prevent rapid degradation of proteins and peptides in vivo, thereby enhancing the dose effectiveness of the vaccine antigen. liposomes are used in vaccine formulations against influenza, chlamydia, malaria, and tuberculosis (tb). coadministration of antigen with cationic liposomes leads to the induction of stronger antigen-specific immune responses than neutral or anionic liposomes [ ] . liposomes are effective vaccine delivery systems and act as carriers in adjuvants such as as , a liposome-based formulation consisting of monophosphoryl lipid a (mpla) and qs- [ ] . improved saponin-based tensoactive adjuvants (iscom, iscomatrix, and matrix-m tm ) are particulate antigen delivery systems with powerful immunostimulating activity [ ] . in iscoms, saponin, cholesterol and phospholipid form cage-like structures ( - nm in diameter). the adjuvant iscomatrix has a structurally similar structure but without the incorporated antigen (the antigen can be formulated with iscomatrix to prepare an iscomatrix vaccine) [ ] . both antigen delivery and immunostimulatory properties are present in iscomatrix [ ] . within the first few hours of injection, the antigen-iscomatrix complex traffics into draining lymph nodes (dlns) where antigen delivery takes place for uptake by the resident dendritic cells (dcs) and other antigen presenting cells (apcs) [ ] . iscoms are currently used in the development of influenza vaccines and iscomatrix in hepatitis c virus (hcv), influenza and candidate cancer vaccines in humans. the matrix-m tm adjuvant is being evaluated in vaccines against influenza, herpes simplex virus (hsv) type and malaria [ ] . nanoparticles are polymeric colloidal carriers of antigens, which enable site-directed delivery and prolonged release of antigen and facilitate alternative modes of vaccine administration (such as inhalation, optical or topical delivery). examples of polymeric nanoparticles are poly(lactic-co-glycolic acid) (plga), poly(lactic acid) (pla), poly(glycolic acid) (pga), poly(hydroxybutyrate) (phb) and chitosan [ ] aluminum salts are also used as delivery systems. crystals of alum bind to and alter the lipids of the dc plasma membrane to trigger cell activation that facilitates delivery of antigen, without alum itself being internalized by the dcs [ ] . aluminum salts are used as adjuvants in human vaccines against diphtheria, tetanus, pertussis, rabies, anthrax, and hepatitis a and b [ ] . in vitro studies revealed that the oil-inwater emulsion adjuvant, mf increases both phagocytosis and pinocytosis indirectly to promote better antigen uptake by apcs. instead of directly targeting dcs for antigen uptake, mf acts upstream by promoting influx of dc precursor cells and augmenting their differentiation into dcs [ ] . the safety of mf was demonstrated in various clinical studies with antigens from hepatitis b virus (hbv), hcv, cytomegalovirus (cmv), hsv and human immunodeficiency virus (hiv) [ ] . similar to mf , as does not directly activate dcs in vitro. intramuscular injection of as in mice promotes influx of monocytes, dcs, and granulocytes into the dlns. as is article highlights • identification of new prrs and their agonists are expected to lead to the identification of more adjuvants; in particular, prr agonists in combination adjuvants hold great promise. • systems vaccinology will provide a better understanding of the mode of action of adjuvants and allow identification of unique biomarkers of adjuvanticity. • there are many pathogens for which host-pathogen interactions have not been characterized in detail. such knowledge on hostpathogen interaction combined with the mechanism of action of adjuvants will lead to the use of specific adjuvants in vaccines against distinct pathogens. figure . schematic representation to highlight the possible mechanism of action by which adjuvants exert their adjuvanticity. adjuvants can serve as a depot that mediates recruitment of apcs or act as a delivery system to facilitate uptake of antigen by the apcs. adjuvants may activate innate immune responses by signaling through cell surface clrs (such as dectin- , dectin- , mincle), cytosolic nlrs, cell surface tlrs, endosomal tlrs or cytosolic rig-i and mda . signaling via prrs may lead to the activation of several transcription factors, which results in the production of pro-inflammatory cytokines, chemokines and type i ifns. secretion of chemokines due to adjuvants may also result in the recruitment and infiltration of more immune cells. adjuvants can activate c-gas that participates in the stingmediated irf -type i ifn pathway. adjuvants can enhance the expression of mhc and co-stimulatory molecules to mediate efficient presentation of antigen to naïve cd + t cells. depending upon the class of adjuvant, cellular (th ) and/or humoral (th ) immune responses may be induced. adjuvants also play important roles in gc reaction, affinity maturation and long-lived memory responses as a part of humoral immunity. also responsible for enhanced antigen uptake, by monocytes in particular, and antigen presentation in the dlns; this process is mediated by the presence of α-tocopherol in as [ , ] . the safety of squalene-based adjuvants (such as mf and as ) has been demonstrated by toxicological studies in animal models as well as in phase i-iii studies in humans [ ] [ ] [ ] depot effect refers to slow and prolonged antigen release at the site of injection providing continuous stimulation of the immune system. this facilitates enhanced antigen uptake by the apcs, which correlates to induction of high antibody titers. adjuvanticity of alum was originally thought to be due to depot effect; however, according to recent evidence, a depot effect is not the only mechanism by which it exerts its adjuvant activity [ ] . in a mouse model, alum was found to rapidly induce an inflammatory environment (via induction of inflammatory chemokines) that in turn triggers neutrophil recruitment and swarming at the injection site. furthermore, alum induces neutrophil death, resulting in the release of extracellular dna strands (neutrophil extracellular traps or nets) that play a significant role in the adjuvant action of alum [ ] . oil-in -water emulsions such as emulsigen®, water-in-oil emulsions such as cationic adjuvant formulation (caf) (a cationic liposome consisting of a combination of dimethyldioctadecylammonium/α,α'-trehalose , '-dibehenate or dda/tdb), as well as biodegradable micro-and nano-particles exhibit adjuvant activity via a depot effect in mice [ , ] . cationic liposomes exhibit long depot effects at the site of injection and strong electrostatic interactions with apcs. in contrast, adjuvants such as mf or iscoms do not require depot formation to exert their adjuvant activities; rather, antigen and adjuvant are cleared rapidly from the site of administration. a biodistribution study of as in mice conducted by radiolabelling each component of as revealed that all constituents of as infiltrated into the dlns within min of injection [ ] and that - % of each constituent of as was cleared from the injection site days post injection, suggesting dissociation of as [ ] . similarly, intramuscular injection of an as -adjuvanted vaccine in mice indicated rapid clearance of antigen and qs- from the injection site and into the dlns. differential biodistribution dynamics and pharmacokinetics of antigen and qs- suggested that the as and the vaccine antigen were not physically associated with each other [ ] . similarly, in matrix adjuvant formulations (consisting of nanoparticles comprised of saponin, cholesterol, and phospholipid), a physical association between matrix and antigen is not required for potent immune stimulation [ ] . in contrast, electrostatic interaction of antigen with adjuvant is required for optimal adjuvant activity of caf . similarly, optimal adjuvanticity of virosomes is achieved when the antigen of interest is associated with the virosome either through encapsulation or attachment to the bilayer via hydrophobic interaction [ ] . the success of the yellow fever vaccine yf- d, a live attenuated virus vaccine, can be attributed to its ability to activate multiple tlrs including tlr , , and , on or in dcs in mice [ ] . yf- d also activates human monocyte-derived dcs and plasmacytoid dcs (pdcs) [ ] . tlr -tlr complexes are activated by the lipopeptide analog pam csk , while tlr -tlr complexes are activated by the macrophage activating lipopeptide- (malp- ) from mycoplasma [ ] . poly(i:c) is a ligand for endosomal tlr , cytosolic retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated protein (mda ). poly(i:c) and its two derivatives, polyi:c u (ampligen) and poly(ic:lc) (hiltonol) are used in clinical trials against both tumors and infectious diseases such as hiv [ ] . tlr is targeted by monophosphoryl lipid (mpl)a. as (containing mpl) is approved for use in hbv (fendrix) and human papilloma virus (hpv) vaccines (cervarix) [ , ] . as (containing mpl) is also used in a malaria vaccine (rts,s) and the varicella zoster virus vaccine (hz/su) that have been found to be efficacious in phase iii trials [ ] . tlr recognizes bacterial flagellin. tlr signaling in cd + cd b + dcs plays an important role in intestinal iga production and th differentiation, and leads to myd -dependent, strong nuclear factor (nf)-κb activation and th- type immune responses in mice [ , ] . in contrast, flagellin acts as a th -polarizing factor for cd + t cells from human neonates and adults [ ] . tlr and tlr recognizing single-stranded rna (ssrna) molecules are targeted by small-molecule immune potentiator (smip)-based adjuvants such as imiquimod and resiquimod used in hpv virus-like particle (vlp) vaccines [ ] . tlr signaling induces b cell-mediated production of immunoglobulins (ig), interleukin (il)- and tnf-α, and natural killer (nk) cellmediated production of ifn-γ, while tlr signaling induces t cell proliferation, production of ifn-γ, il- , and il- , and memory t cell activation [ ] . tlr recognizes unmethylated cpg motif-containing microbial dna or immunostimulatory sequences (iss). tlr agonists are used in hbv vaccines to promote higher levels of protective antibodies. consequently, fewer immunizations and lower antigen doses are needed. in mice, tlr signaling leads to th type pro-inflammatory responses (il- , il- , il- , il- , tnf-α and ifn-γ), upregulation of major histocompatibility complex (mhc) and costimulatory molecules, and increased cd + t cell responses, while tlr -mediated b cell activation is responsible for induction of humoral immunity and antibody class switching [ ] . intracellular nlrs such as nod and nod receptors recognize diaminopimelatic acid (dap)-containing muropeptide from gram-negative bacteria, while nod detects the muramyl dipeptide (mdp) component present in all bacterial peptidoglycans. the adjuvanticity of the mucosal adjuvant cholera toxin (ct) is mediated through the nod receptor [ ] . adjuvants that are inducers of damage-associated molecular patterns (damps) trigger innate immune responses in vivo by damaging the host cells, thereby resulting in the release of damp factors (ex. rna, dna) for subsequent activation of the innate immune receptors [ ] . the cytosolic receptor nlrp is recognized by adjuvants such as quil-a and chitosan, as well as atp, mdp, uric acid crystals and silica. these compounds generate damp signals, such as reactive oxygen species (ros) or induce potassium efflux to activate nlrp . alum's adjuvanticity in mice is also attributed to the activation of nlrp / nacht (via swelling and rupture of the phagolysosome, release of cathepsin b into cytosol, subsequent activation of caspase and release of il- β), leucine-rich repeat (lrr), and pyrin-paad-dapin domain (pyd) domains-containing protein (nalp ) inflammasome, release of uric acid or activation of the stimulator of interferon (ifn) genes (sting)-ifn regulatory factor (irf) pathway due to the release of dna [ , ] . however, validation of these hypotheses for humans is warranted (ex. a direct role of il- β in adjuvanticity of alum in humans is debatable) [ ] . for instance, it is generally concluded that nlrp inflammasome and caspase are sometimes, but not always, required for induction of th cellassociated antibody responses in response to aluminum salts in vivo [ ] activation of caspase- is nlrp -dependent in vitro. however, no such role of nlrp is observed for qs- in vivo. qs- when formulated in liposomes activates human dcs by promoting cholesterol-dependent endocytosis with subsequent lysosomal destabilization and syk activation similar to alum [ ] . in mice, endocine, a lipid adjuvant, causes cellular damage to generate damps such as lactate dehydrogenase (ldh), dna and rna [ ] . chitosan induces release of mitochondrial dna into the cytoplasm to activate the nlrp inflammasome in mice [ ] . mf (but not aluminum hydroxide or calcium phosphate adjuvants) induces release of extracellular atp from the muscle in mice that acts as a danger signal [ ] . other damp adjuvants such as hydroxypropyl-βcyclodextrin (bcd) induce local cellular stress and death resulting in the release of the host cellular dna that serves as a damp to induce th -type immune responses [ ] . cytosolic dsdnas are sensed by several other prrs such as absent in melanoma (aim ), as well as by the protein cyclic guanosine monophosphate-adenosine monophosphate (cgamp) synthase (cgas), which simultaneously leads to the activation of sting-dependent tbk -irf -ifn- pathways and rela-tnf-α pathways [ ] . sting can bind cyclic dinucleotides (cdn), cyclic di-gmp (cdg) and cyclic di-amp (cda). in mice, cdg appears to be a safer mucosal adjuvant than cholera toxin [ ] and to promote protective immunity against h n influenza, staphylococcus, streptococcus and klebsiella infections. in mice, chitosan triggers release of intracellular dna that results in the engagement of the cgas-sting pathway in dcs to induce type ifn production and isgs, thereby promoting robust th immunity. this leads to the upregulation of costimulatory immune markers and the subsequent activation of dcs, as well as induction of igg c and cell-mediated immunity (cmi) [ ] . carbohydrate-based adjuvants include glucans, fructans, mannans, chitin/chitosan and other carbohydrate compounds derived from mycobacterium spp. (including lipoarabinomannan, muramyldipeptide/mdp, trehalose- - -dimycolate/tdm), as well as lps and saponin compounds (including qs- , a saponin in an oil-in-water emulsion) [ ] . in human monocyte-derived dcs (modc), qs- (also a component of as ) is endocytosed via the action of membrane cholesterol, with subsequent lysosomal destabilization and syk activation to promote a pro-inflammatory transcription program. in addition, cathepsin b (a lysosomal cysteine protease) is required for activation of modcs in vitro and also required for adjuvant activity of qs- in vivo [ ] . in another study, ln-resident cd b + cd + macrophages played a key role in the adjuvant activity of qs- . upon intramuscular injection in mice, qs- leads to rapid induction of local innate immune responses in the dlns and co-localises with ln-resident macrophages that are crucial for innate and effector responses to antigens formulated with qs- (via caspase / and inflammasome activation) [ ] . the primary mechanism of action of carbohydrate-based adjuvants involves interaction with prrs such as tlrs, nod and c-type lectin receptors (clrs) dectin- , dectin- and mincle on monocytes and apcs. this interaction activates nf-κb to induce inflammatory chemokine and cytokine responses [ ] . carbohydrate adjuvants also activate complement pathways to generate complement components acting as opsonins and chemokines. other important mechanisms of action of carbohydrate-based adjuvants include chemotaxis of lymphocytes, inflammasome activation (e.g. zymosan and mannans), and pore-formation, facilitating antigen entry into apcs (via interaction with cholesterol in the plasma membrane, e.g. qs- ) [ ]. adjuvants induce a series of signal transduction pathways to exert their action at both innate and adaptive levels. intramuscular injection of mpl or aso in mice is responsible for nf-κb activation in the muscles and local dlns [ ] . synthetic derivatives of mpl induce activation of tlr and selectively activate the p mapk pathway, which is strongly associated with optimal induction of ifn-γ-induced protein (ip- ), tnf-α and il- in mice [ ] . injection of as adjuvanted vaccine promotes release of ifn-γ by ln-resident nk cells and cd + t cells. pathway enrichment analysis of differentially expressed genes in injection site-dlns revealed that the ifn-signaling pathway was most enriched at and h, while the il- -driven anti-inflammatory pathway was also triggered by as at h post-injection. increased levels of ifnγ were detected early in the serum and dlns of humans and macaques vaccinated with as -adjuvanted vaccine, respectively [ ] . cellular and molecular synergy between mpl and qs- used in as in combination was responsible for this early ifn-γ response, which in turn, enhances the immunogenicity of as -adjuvanted vaccines [ ] . il- as an adjuvant activates janus kinase (jak)-signal transducer and activator of transcription (stat), phosphoinositide -kinase (pi k) and mapk pathways, thereby promoting b-cell and t-cell differentiation via sustained activation of stat and th differentiation through irf [ ] . subtle chemical alterations to mpla were made to develop a designer smip-based tlr -agonist known as sla, which induces trif signaling to produce th biased cytokines and chemokines like ifn and ip- , and less il- β. sla in oil-in-water emulsion (sla-se) was produced capitalizing on the knowledge that a combination of ifn and caspase-dependent inflammasome signaling leads to powerful adjuvant action [ ] . activation of the nf-κb pathway, as well as the p and jnk mapk pathways, programs dcs to produce il- p and to induce th responses. the extracellular signal-regulated kinase (erk)-c-fos mapk pathways favor th -type responses, while erk-retinaldehyde dehydrogenase (raldh) enzymes or β-catenin program dcs to induce t regulatory (treg) responses. similarly, complete freund's adjuvant (cfa) induces transcription of mhc-ii and b cell activation markers via the lyn-syk-pi k, the calcineurin-nuclear factor of activated t-cells (nfat) and the ras-mek-erk signaling pathways [ ] . saponin adjuvanticity relies on myd -mediated and il- receptor-signaling pathways [ ] . chitosan engages cgas-sting pathways to induce igg c and th responses in mice [ ] . intact myd signaling in each of the three types of apcs (dcs, macrophages and b cells) is essential for robust activity of tlr ligand-based vaccine adjuvants (porb, a tlr ligand and cpg, a tlr ligand) such as induction of in vivo cytokine responses, germinal center (gc) formation and antibody production [ ] . based on microarray analysis, mosca et al. demonstrated that three potent human vaccine adjuvants, mf , cpg odn, and alum, modulate a common set of genes ['adjuvant core response genes'] encoding cytokines, chemokines, innate immune receptors, ifn-induced proteins and adhesion molecules in mouse muscles [ ] . the establishment of a local immunocompetent environment due to such nonpathogenic inflammatory responses is associated with vaccine adjuvanticity. when compared to cpg odn and alum, mf was found to be the stronger inducer of adjuvant core response genes, which was reflected in enhanced and more rapid influx of mhc-ii + and cd b + cells into the injection site and more efficient transport of antigen to the dlns [ ] . both alum and mf induced chemokines involved in cellular influx such as ccl , ccl , ccl , and cxcl- [ ] . the presence of α-tocopherol in as promotes induction of leukocyte-recruiting chemokines (ccl , ccl and ccl ), neutrophil-mobilising cytokine (granulocyte colonystimulating factor (csf )) and pro-inflammatory cytokine/ chemokines (il- and cxcl- ), in mice which is in agreement with increased recruitment of granulocytes and antigenloaded monocytes into the dlns [ ] . aluminum adjuvants facilitate recruitment and differentiation of inflammatory monocytes (f / int cd b + lyg − ly c + ) into inflammatory dcs, thereby enhancing both humoral and cellular immunity [ ] . many of the above results with alum and mf have been confirmed in non-human primates [ ] . for example, vaccination with hiv vaccine adjuvanted with alum or mf triggered recruitment of monocytes, dcs, and neutrophils in the muscle [ ] . subcutaneous administration of iscomatrix in sheep induces a rapid and transient production of cytokines (il- , il- , ifn-γ) and influx of innate cells such as nk cells, nkt cells, neutrophils, migratory dcs (cd + cd − ) and cd α + dcs to the dlns [ ] . a combination adjuvant consisting of poly(i:c), a host defense peptide and pcep when delivered intranasally transiently induces production of chemokines and cytokines in murine respiratory tissues, which promotes infiltration and activation of dcs, macrophages, and neutrophils to generate improved mucosal and systemic immune responses [ ] . (a) improvement of the quality of antibody responses: innate immune responses play a profound role in regulating the magnitude, quality and persistence of antibody responses. the magnitude of the antibody response is critical in conferring protection against diphtheria, hepatitis a, lyme disease, tetanus, yellow fever, polio, rabies, and pneumococcal infections [ ] , while for rsv and meningococcal infections, the magnitude and quality of the antibody and cell-mediated response are important. adjuvant systems such as as are used in malaria (rts,s), herpes zoster (hz/su), tb and hiv vaccines, while as is used in several influenza vaccines such as trivalent inactivated h n influenza, h n prepandemic influenza, and candidate h n and h n pandemic influenza vaccines. as is used in licensed hpv- / and hbv vaccines. such adjuvant systems are known to augment antigen-specific t cell and antibody responses [ ] . in a murine study, the er stress-related pathway was found to potentially contribute to the adjuvanticity of as in vivo [ ] . furthermore, the expression of the er stress sensor kinase ire α by myeloid cells was involved in adjuvant activity of as . the er stress-related pathway was required for as mediated induction of il- , robust tfh responses, and antigenspecific antibody affinity maturation. il- plays a crucial role in differentiation of tfh cells as well as in the expression of il- by tfh cells and in production of antibodies [ ] . alum-adjuvanted vaccines act on il- producing gr- + cells to facilitate optimal priming, clonal expansion and antibody production by antigen-specific b cells in mice [ ] . in b cells, tlr ligands as adjuvants induce upregulation of surface markers involved in antigen uptake (mhc-i and mhc-ii) and surface markers involved in cross-talk with the t cells (cd , cd , and cd ), which ultimately leads to increased antigenspecific antibody production [ ] . however, when as is used as an adjuvant in the same hbv and hpv vaccine, instead of aluminum salts, higher levels of antibodies are induced in humans, indicating the added benefit of mpl (a tlr agonist) in humans [ ] . emulsigen®, an oil-in-water adjuvant, similar to mf and as , boosts innate responses and increases the number of cd + t cells required for robust antibody responses [ ] . mf supports induction of t follicular helper (tfh) cells and gc responses to vaccination by an unknown mechanism [ ] . in humans, mf and as promote early inflammation similar to results from animal studies [ ] . type ifns responses are induced in children as early as day postimmunization with mf -adjuvanted influenza vaccine, but in children immunized with non-adjuvanted vaccine, type i ifn responses are weaker and delayed. such early innate immune responses are reflected in induction of antigenspecific tfh cells days post-vaccination, and enhanced antibody responses in humans immunized with mf -adjuvanted influenza vaccine [ ] . (b) induction of gc reactions to promote memory b cell development: immunological memory is a distinctive hallmark of the adaptive immune system that contributes to protective immunity against infectious diseases. the gc reaction is central to memory development. induction of certain key molecules such as cd , inducible t-cell costimulator (icos), il- , programmed death-ligand (pd- ), cd , irf , and b-cell lymphoma protein (bcl- ) play a critical role in regulation of gc differentiation, affinity maturation and long-lived memory responses [ ] . tlrs expressed on gc b cells, follicular dcs (fdcs) and t cells have a profound effect on induction of antibody responses. nanoparticles resembling virions in size and containing tlr ligands, mpl and r , in combination with h n hemagglutinin mediate increased persistence of gcs, which significantly influence the differentiation of memory b cells critical for long-lived antibody responses in mice [ ] . a subset of cd + t cells, icos + cxcr + cxcr + t cells, was associated with protective antibody responses conferred by a trivalent split-virus influenza vaccine and efficiently induced memory b cells to differentiate into plasma cells [ ] . novel adjuvants may enhance b-cell activation in gcs and bonemarrow plasma cell survival. for example, the heat-labile enterotoxin (lt) of escherichia coli, ltk , when administered parenterally to neonatal mice, facilitates maturation of follicular dcs and generation of gcs [ ] . . . induction of cellular immunity: effector th /th / th and memory t cell responses signaling via tlr , tlr , tlr , tlr , and tlr promotes th biased immunity, while signaling via tlr /tlr , tlr /tlr and tlr promotes th -biased immunity. cd c + cd b − cd α + dcs localized in the marginal zones of lns induce th responses, as well as exhibit cross-presentation of antigens in vivo and ex vivo in mice [ ] . in humans, bdca + (cd c + ) and bdca + (cd + ) dcs (equivalent to murine cd α − and cd α + , respectively) are involved in cross-presentation of extracellular antigens [ ] . as a result of tlr -mediated enhanced mhc-i expression and type i ifn production, poly(i:c) promotes antigen cross-presentation to cd + t cells and antigen-specific ctls. in contrast, alum promotes th responses (strong antigen-specific igg and ige) and does not induce cd + t cell immunity, and even inhibits th immune responses in mice [ ] . however, when alum is present in combination with mpla, th responses can be generated as is found for aso [ ] . it is unclear whether such an aluminum salt-induced th bias exists in humans [ ] . rather, poor t cell responses are induced by aluminum salts in humans, possibly due to poor stimulation of the innate immune system [ ] . squalene-based oil emulsion is a potent inducer of both th -and th -mediated immunity and is well tolerated [ ] . adjuvants such as qs- , mf or cfa preferentially induce th -biased or a mixed th / th and th /th immune response. experimental cafs combined with immunostimulators such as tdb in tb vaccines stimulate both cellular and humoral immune responses, as well as promote efficient polyfunctional memory t cells, and th -and th -biased immune responses in mice [ ] . a sting-activating adjuvant, cdn, when formulated in a subunit vaccine and delivered intranasally, promoted a robust th immune response that correlated with long-lasting enhanced protection against mycobacterium tuberculosis in mice. adjuvanted vaccines promoting th responses may protect against intracellular pathogens by recruiting protective t cells earlier during infection [ ] . in neonates, cd + t cells are polarized towards th responses and reduced th responses. however, novel adjuvants such as ic and caf can induce adult-like th responses in newborn mice [ ] . cdg as a mucosal adjuvant induces th and th immune responses in mice [ ] . caf predominantly induces cd + t cell responses, while caf (consisting of dda, tdb and poly(i:c)) induces both cd + and cd + t cell responses [ ] . replacing as in an rts,s/ as candidate malaria vaccine with as improved antibody and cd + t cell responses, as well as protective efficacy as demonstrated by a randomized, double-blind, phase a trial [ ] . activated murine sub-capsular macrophages produce il- , which promotes generation of cd + t cells, while activated apcs also produce il- or il- , promoting generation of tfh cells, which in turn favors production of high-avidity antibodies by b cells in both mice and humans [ ] . intramuscular injection of mice with as induces early ifn-γ production by ln-resident nk cells, which is mediated by synergistic action of il- and il- in promoting ifn-γ responses. early ifn-γ production by nk cells is a prerequisite for optimal activation of dcs and induction of antigen-specific cd + t responses to as -adjuvanted antigens [ , ] . such ifn-γ responses were also observed in lns of macaques when they were injected with as [ ] . elevated levels of ifn-γ in the serum at day post-immunization and an increase in the number of cytokineproducing antigen-specific cd + t cells was also observed in humans immunized with rts,s malaria vaccine [ ] . the mechanisms of action of different adjuvants are summarized in table . the adjuvants that are licensed for use in human vaccines are listed in table , while the adjuvants in clinical trials are listed in table . . selection of adjuvants based on their mechanism of action against distinct types of pathogens mucosal surfaces are an attractive target for pathogens whose port of entry are gastrointestinal (e.g. polio virus, escherichia, salmonella, shigella, vibrio and helicobacter), respiratory (influenza virus, m. tuberculosis or mtb, adenovirus, coronavirus, rhinovirus and rsv) or urogenital tract (hsv, hpv, hiv- , chlamydia and neisseria) [ ] . mucosal adjuvants can be categorized as toxin-based (lt and ct), immunostimulatory (mpl, cpg, and qs ) and delivery system (emulsigen® and iscoms). two commonly used oral toxin-based adjuvants are [ ] . both are potent, but also toxic, when used as mucosal adjuvants. protective efficacy was attained when intranasal vaccines containing mutant lt adjuvants were used against hsv, bordetella pertussis and streptococcus pneumoniae [ ] . natural infection with rsv induces poor antibody responses with impaired effector functions, and perturbs localization and persistence of effector and memory t cells [ ] . induction of a potent, local mucosal immune response is required to prevent infection and a high systemic antibody response is also required to interrupt disease progression. nanoemulsions are safe for intranasal delivery and induce th and th responses in mice with no significant inflammation [ ] . sastry et al. tested multiple adjuvants such as sigma adjuvant system (sas, an oil-in-water adjuvant), carbopol, alum, adjuplex, poly(i:c), poly(ic:lc), mpla, addavax and montanide isa, and found that the adjuvantmediated increase in rsv-specific neutralizing antibody responses was context-dependent (i.e. whether pre-existing immunity was present or not) and species-specific (i.e. mice vs. calves) [ ] . formulation of the rsv fusion (f) protein with a combination adjuvant, triadj, elicited protective, mucosal and systemic, immune responses against rsv in mice and cotton rats [ ] . the mucosal epithelial barrier limits the bioavailability of vaccine antigens for sampling by apcs. adjuvants such as polyethyleneimine and chitosan are used as penetration enhancers and immunostimulants to augment binding to the mucosal surfaces and activate innate immunity in a mouse model [ ] . chitosan polymeric nanoparticles stimulate the nasal-associated lymphoid tissues (nalt) to produce mucosal secretory iga, igg, tnf-α, il- , and ifn-γ. pro-inflammatory cytokines and chemokines also act as mucosal adjuvants. proinflammatory cytokines such as il- , il- , il- , il- , and il- induce mucosal cd + ctls and antigen-specific iga. similarly, chemokines such as ccl (or mcp- ) enhance mucosal iga and ctl responses [ ] . neutralizing antibodies may protect against some acute self-limiting mucosal pathogens, but for highly invasive pathogens causing chronic infections (such as hiv, hcv, herpesviruses, and mycobacteria), mucosal innate and adaptive immune responses including cd + , th , and cd + ctls, as well as secretory iga and igg neutralizing antibodies at the port of pathogen entry, are required for effective and optimal protection [ ] . mucosal adjuvant-containing vaccines elicit both local and systemic immune responses, effective at local as well as distant sites [ ] . to control enteropathogens, orally administered vaccines must overcome challenges of antigen degradation and immune tolerance [ ] . biodegradable micro-or nanoparticles are required that are resistant to low ph and can target antigen to m cells. u-omp , a bacterial protease inhibitor from brucella abortus, is an oral adjuvant suitable for subunit vaccine formulation, which can inhibit stomach and gut proteases and delay antigen digestion at the lysosome to enhance antigen presentation and recruitment of immune cells to gastrointestinal mucosa [ ] . intranasal immunization of mice with poly-i:c u (ampligen) in an h n influenza vaccine promoted increased levels of protective, mucosal iga and systemic igg [ ] . however, only a few mucosal vaccines are licensed for humans, primarily due to a dearth of safe and effective mucosal adjuvants [ ] . although during the last few decades there has been a constant development of new and effective mucosal adjuvants, most of them are toxic. for example, ltk when intranasally injected can trigger transient facial nerve paralysis or bell's palsy [ ] . thus, there is an urgent need to address safety issues of mucosal adjuvants. in a phase iii trial, poly-i:c u (ampligen) was demonstrated to be safe [ ] . a randomized phase i/ii trial was conducted to determine the safety and efficacy of ampligen in patients with stage ii-iv human epidermal growth factor receptor (her )-positive breast cancer. the result from this trial will give important insight into the application of ampligen in therapeutic cancer vaccines [ ] . pathogenic fungi and protozoan parasites have complex life cycles and switch among several different forms during their life. histoplasma capsulatum grows as a mold in the soil at low temperature, but upon inhalation into the lungs, it switches to yeast form and causes histoplasmosis. interaction of infected macrophages with cd + and cd + t cells leads to increased production of th cytokines, il- , ifn-γ, and tnf-α, that are critically important in generating protective immunity against h. capsulatum infection in mice. leukotrienes, lipid mediators derived from arachidonic acid metabolism, are found to be potent adjuvants against such fungal infections [ ] . malaria vaccine development is impeded by the complex life cycle of plasmodium spp., the intracellular stage in its life cycle, large physical size, surface antigenic diversity and enormous genetic and genomic plasticity [ ] . the parasite replicates intracellularly (and thus is partially protected from immune recognition) and also sequesters any innate immune ligand away from prrs in the sporozoite and gametocyte stages of their life cycle. a malaria vaccine needs to establish humoral immunity to prevent merozoites from entering the erythrocytes and the liver or destroy the merozoites through opsonization and cmi. rts,s/as (mosquirix) is a malaria candidate vaccine targeted against the infectious sporozoite stage and designed to enhance both antigen-specific humoral and cellular immunity. th effector cells are essential to target asexual blood stages, while eventual control and/or clearance of the parasites requires antibody-mediated responses [ ] . mpl and qs- , the two components used in as have important functions. mpl is a tlr agonist that induces production of ifn-γ by t cells and antibody isotype switching to igg a/c in mice, while qs- induces neutralizing antibodies and cytotoxic t cell responses [ ] . as requires synergistic activities of both mpl and qs- for optimal adjuvant activity. as in combination with plasmodium antigens induces rapid and transient innate immune responses in the injection site and dlns, activates immune cells (including apcs), as well as generates -fold higher antibody titers when compared to natural exposure [ ] . however, in a large phase iii trial in children and young infants in seven sub-saharan african countries, although rts,s/as prevented a considerable number of cases of clinical malaria in infants and young children over - years, the vaccine efficacy declined with subsequent follow-ups in the infants, and did not provide significant protection against severe malaria [ ] . nonetheless, rts,s/as plays a significant role in the control of malaria in areas of high transmission when used in conjunction with other effective preventive measures (rts,s clinical trials partnership). poly(i:c) and its derivatives are of great importance for vaccines that need to induce a th /ctl immune response against various viruses and pathogens including p. falciparum [ ] . pam csk was used in a malaria vaccine containing p. falciparum circumsporozoite protein b cell epitopes and universal t cell epitopes, which resulted in the induction of high titers of antigenspecific igg , igg and igg in immunized volunteers [ ] . herpesviruses are large viruses with a complex genome. primary infection with varicella zoster virus (vzv) causes varicella (chickenpox) and may go into latent phase in human cranial and dorsal root ganglia. aging or immune dampening results in decline of vzv-specific cmi, which may induce reactivation of the virus and cause shingles. hence, cmi is necessary to prevent reactivation of the latent virus. the vzv vaccine hz/su (shingrix) composed of the vzv glycoprotein e subunit (ge) antigen and as b was recently approved for the prevention of herpes zoster in adults aged years or older [ ] . as was selected as the adjuvant for the vzv vaccine, because compared to other adjuvant systems, as induced higher numbers of ifn-γ secreting cd + t cells, and thus improved t cell as well as antibody responses, with acceptable clinical safety profiles [ ] . hpv effectively evades innate immunity by inhibiting the ifn receptor signaling pathways and activation of isgs via the e and e proteins. hpv also downregulates tlr and does not induce any danger signal to alert the immune system [ ] . this prolongs the duration of infection and delays the onset of adaptive immunity. thus, an effective cmi is required to clear and control hpv infection. effective vaccine-induced immunity against hpv should consist of cmi to the early proteins, e and e , and neutralizing antibodies against the virus coat protein l . two currently approved hpv vaccines, cervarix (a bivalent hpv / vaccine, gsk) and gardasil (a quadrivalent hpv / / / vaccine, merck) are highly protective against hpv , , and [ ] . both are li vlps; however, cervarix is as adjuvanted, while gardasil is aahs (amorphous aluminum hydroxyphosphate sulfate)-adjuvanted. vlps strongly activate the stromal dcs in the injection site that migrate to the dlns, or may directly bind to the surface of apcs or other immune cells and migrate to the lns, where they prime naïve b cells [ ] . according to a recent study in girls aged - years, two doses of cervarix elicited superior hpv- / antibody responses compared to two or three doses of gardasil. the differences in immunogenicity between the two vaccines may be due to the different types of adjuvants used. as enhances humoral immune responses and cmi by triggering local and transient cytokine responses that promote enhanced activation and presentation ability of apcs [ ] . significantly higher antibody titers are induced in mice immunized with hpv- l vlps adsorbed onto aahs as compared to vlps adsorbed onto aluminum hydroxide along with induction of an improved l -specific ifn-γ secreting t cell response [ ] . mtb causing tb is an intracellular pathogen that has the ability to survive within the hostile environment of the alveolar macrophages after being phagocytosed and to multiply unchecked. cd + t cells, cd + t cells, ctls, th cells, nk cells, and activated macrophages are critical in controlling mtb infections. bacillus calmette-guérin (bcg) vaccine fails to protect adults from pulmonary tb and prevent transmission of mtb in adolescents and adults [ ] . thus, there is an urgent need for improved vaccines against tb. one of the potential vaccine strategies against mtb is to eliminate or control latent infection and prevent reactivation or progression to clinical tb in latently infected patients. this may be accomplished by incorporating adjuvants that are capable of inducing both cd + and cd + t cell responses in both immunocompetent and immunocompromised individuals. mechanisms of antibody-mediated protection against tb include opsonization, complement activation, and fc receptor engagement. current research is focused on adjuvants that act on innate lymphoid cells (ilcs), nk cells and non-classical t cells such as cd , mr , hla-e and γδ t cells present in large numbers in the circulation and mucosa [ ] . although the immune correlates of protection from tb disease are not validated yet, vaccines currently in clinical development predominantly focus on generating cd + and cd + th -type immune responses. adjuvants such as mineral salts, saponin, emulsigen®, micro-or nanoparticles, toxin derivatives, cationic lipids, cpg dna, adjuvant systems and cytokines have been tested in subunit vaccine preparations, either alone or in combination with bcg in a prime-boost strategy [ ] . the strongest th -inducing adjuvants for tb are unmethylated mycobacterial dna and cpg odn, which promote ctl activation and ifn-γ production [ ] . tlr / and tlr / ligands are presented on the surface of mtb (triacylated and diacylated forms of mycobacterial p lipoprotein) or secreted by the bacterium, while nlrs such as nod are responsible for intracellular recognition of mycobacteria [ ] . novel adjuvants, including dda, tdb, ic , poly(i:c), gelatin, cpg odn, mpla, glycopyranosyl lipid adjuvant (gla) in combination with squalene (se) known as gla-se, mf , caf , and as b are also being clinically tested. dda promotes generating humoral, cell-mediated and ifn-γ responses against mtb, while as and mf induce strong th immunity against mtb. all these adjuvanted subunit vaccines induce protective immunity and enhance bcg-primed immunity in animal models [ ] . in a randomized, double-blind, phase b trial, a candidate tuberculosis vaccine, m /as e , demonstrated a clinically acceptable safety profile and conferred % protection against active pulmonary tuberculosis in adults with latent mtb infection [ ] . nanoparticle-based vaccines are critical for the induction of protective th -type immune responses to intracellular pathogens. the liposomal caf adjuvant promoted th and long-lasting memory t cell response in human tb vaccination trials. caf -adjuvanted tb vaccine stimulates the clr, and mincle, and triggers the syk/card signaling cascade to activate the th signaling pathway [ ] . one of the biggest challenges in vaccine development is the fact that the immunological mechanisms that govern vaccine safety and efficacy are still largely unknown. in recent years, systems vaccinology has emerged as an interdisciplinary approach that relies on high-throughput omics-based techniques to study vaccine-induced changes in the entire genome, set of transcripts, proteins, and metabolites in various tissues. a systems vaccinology approach has been used to elucidate immune responses to vaccines against yellow fever [ ] , influenza [ ] , malaria [ ] , smallpox [ ] and hiv [ ] . in addition, a systems vaccinology approach identified molecular and cellular immune signatures of a vaccine against bordetella pertussis [ ] . ip- was identified as an early innate immune signature that correlated with antibody responses to an ebola vaccine (rvsv-zebov) [ ] . computational analysis of the transcriptomic profile in human peripheral blood mononuclear cells (pbmcs) induced by yellow fever vaccine yf- d identified two molecular signatures: eukaryotic translation initiation factor alpha kinase (eif ak ) and tnfrsf , encoding the receptor for the b-cell growth factor blys-baff [ ] . eif ak correlated with the magnitude of the cd + t cell responses, while tnfrsf correlated with the magnitude of neutralizing antibody responses. other genes such as calreticulin, c-jun, and glucocorticoid receptor were also induced by yf- d, and this induction correlated with cd + t cell responses [ ] . in another study with young healthy adults, intramuscularly administered tiv induced higher antibody levels and plasmablasts when compared to intranasally delivered live attenuated influenza vaccine (laiv) with induction of distinct transcriptional signatures such as enhanced expression of type ifn genes in laiv recipients, but not in tiv recipients [ ] . based on a systems vaccinology approach, tlr agonists as adjuvants were found to potently enhance the immunogenicity of influenza vaccine, resulting in an improved antibody response in humans [ ] . the longevity of the immunoglobulin response post vaccination could be predicted from the ability of the adjuvanted vaccine to induce proliferation of antigen-specific il- + icos + cxcr − cd + t cells in the peripheral blood. systems vaccinology also identified two biomarkers (junb and ptx ) of mf and the skeletal muscle tissue cells (in addition to apcs) as direct target of mf for its adjuvant action in mice [ ] . caproni et al. investigated molecular signatures induced by different tlr-dependent (cpg odn, resiquimod and pam csk ) and tlr-independent (mf and alum) adjuvants in influenza subunit vaccines to establish the innate immune correlates of adjuvanticity by using dna microarrays in a mouse model [ ] . two adjuvants, mf and pam csk increased overall antibody and hai titers, and induced active infiltration of cd b + cells, especially neutrophils, to the injection site. this suggests early induction of cd b + cells due to an emulsion-based adjuvant to be predictive of subsequent robust humoral immunity. systems vaccinology has also been applied to identify novel mechanisms of induction of th responses by an adjuvant. for instance, the th -promoting adjuvant activity of cysteine protease allergen is dependent on the production of ros by dcs. as a result of induction of ros, oxidized lipids are induced that in turn promote epithelial cell-mediated production of thymic stromal lymphopoietin (tslp), resulting in the recruitment of il- + basophils to the lns for induction of th -type immune responses in mice [ ] . genes associated with memory b cell formation and productive antibody responses such as bcl , bcl a, tank, plcg , and cd are induced when mice are immunized with ovalbumin (ova) adjuvanted with tlr and tlr agonists [ ] . in a study with the candidate malaria vaccine rts,s/as b in human subjects, enhanced expression of genes involved in immunoproteasome formation, psme in particular, was found to be responsible for conferring protection from parasitemia. induction of the immunoproteasome enhances mhc antigen presentation, which in turn, indirectly enhances antibody responses and directly augments cd + t cell development and production of ifn-γ, tnf-α, il- , and cd l. the above immune signatures may contribute to the protective efficacy of the candidate malaria vaccine [ , ] . a comparative systems analysis of four vaccine adjuvants, gla-se, ic , caf , and alum, in mice revealed distinct molecular signatures. gla-se induced massive changes in the transcriptomic profile in the whole blood and dlns that correlated with increased cellular influx (such as cd c + gr + mdcs) in the dlns, in contrast to limited transcriptomic changes induced by other adjuvants. co-expression analysis of differentially expressed genes in whole blood revealed that caf and gla-se (but not ic ) induced transcriptional signatures related to innate immune responses. the analysis also revealed modules enriched for genes associated with tfh and gc-mediated b cell responses; for example, gla-se induced nfatc , nfatc , and il r; caf induced batf and ic induced pou af . a systemic analysis of protective immune responses to three rts,s vaccinations with a subsequent controlled human malaria challenge of the vaccine recipients with plasmodium-infected mosquitoes was carried out. molecular signatures of b cell and plasma cells in human pbmcs were found to be positively correlated to protection, while the nk cell signatures correlated negatively with protection, indicating multiple mechanisms of protective immunity against p. falciparum [ ] . in a study by burny et al., different adjuvants [as b , as e , as , as , and alum/al(oh) ] induced common innate pathways and were responsible for improved adaptive responses when used with a model antigen (hbv surface antigen or hbsag) in humans. as b , as e , and as induced comparable innate profiles and so did as and alum. furthermore, the ability to activate innate immunity (ifn-signaling pathway, in particular) was linked to enhanced adaptive responses elicited by as -and as -adjuvanted vaccine. early changes in immune markers, such as crp, il- , ifn-γ, and ip- , correlated with the magnitude of the adaptive responses [ ] . systems vaccinology also identifies signatures of vaccine safety and efficacy. non-specific adverse side effects observed for vaccines that fail in human clinical trials are frequently associated with over-stimulation of certain components of the innate immune system. systems vaccinology can be applied to screen adjuvants to help design protective and safe vaccines [ ] . correlates of protection have been established for a number of licensed vaccines as reviewed by tomaras et al. [ ] . however, attempts to identify correlates of protection are still ongoing for tb, while the commonly assumed immune correlates often fail to correctly predict an individual's risk of developing malaria [ ] . for hiv, complex immune correlates of protection characterized by multiple types of immune responses are found to be involved in controlling hiv- transmission [ ] . for vaccines for which the immune correlates of protection are unknown, systems vaccinology approaches can be used to identify signatures induced rapidly after vaccination that will help to predict the later immune outcome. a systems vaccinology approach can also help in identifying vaccine non-responders as well as vaccine high-and low-responders [ ] . innate and adaptive immune responses are profoundly influenced by any significant changes in metabolic activity. inflammation triggered by vaccine adjuvants results in a shift in energy supply leading to metabolic acidosis and impaired oxygen supply, which in turn results in phenotypic shifts. these phenotypic shifts heavily affect the metabolic state of an individual. lipid metabolism plays an important role in inflammation. liquid chromatography-mass spectrometry (lc-ms) is employed to identify and quantify cell-or tissue-specific metabolites [ ] . metabolite immune-correlates such as nucleotides, amino acids, lipids, fatty acids, and anti-oxidants may represent inflammatory mediators and/or biomarkers that profoundly influence several inflammatory processes such as cellular infiltration, activation of signaling pathways and oxidative stress [ ] . thus, a comprehensive understanding of the molecular signatures induced by adjuvants early after vaccination will help to predict the later adaptive immune responses in humans. furthermore, such knowledge will also improve or help in (re-)designing next-generation adjuvants and drive the development of next-generation vaccines with the concerted effort of vaccinologists, clinicians, systems biologists, statisticians, as well as industrial and regulatory authorities. the relationship between adjuvants, innate pathways/ receptors activated, immune responses triggered, and the type of pathogens ideal for such adjuvant-mediated immune responses are summarized in table . in this review article, we have summarized the mechanism of action of different classes of adjuvants. we also discussed why this knowledge is important in context of distinct disease targets and how this knowledge can be utilized to improve the development of adjuvanted vaccines against challenging pathogens. we also briefly highlighted the important role of the new-age systems vaccinology approaches in better understanding an adjuvant's mode of action, and identification of unique cellular and molecular biomarkers of adjuvanticity. it is important to note here that mouse models offer flexibility and accessibility to study intricate facets of the mechanism of action of adjuvants and responses to immunization with adjuvanted vaccines. while the results from animal studies often overlap with the results from human studies, there are several dissimilarities as well. for instance, mf -adjuvanted vaccine in mice induces both cell-mediated and humoral immune responses, which is not observed in humans at any age, rather they tend to develop a th -th response. even in humans, immune responses need to be investigated not only in the serum but also in the dlns and other lymphoid organs; not only at the priming site but also in the distant effector sites such that a holistic and reliable assessment of mechanism of action of adjuvant and/or vaccine can be made encompassing all possible immune parameters. even the immunological correlates that have recently been identified by gene profiling or systems vaccinology for different adjuvants/vaccines are defined more at the population level and much less at the individual level [ ] . all these considerations must be taken into account while designing effective and safe vaccines/adjuvants. recent advancements have allowed researchers to conclude that clinical-grade adjuvants have distinct immunological profiles and signatures, which can be used to target different pathogens. based on pathogen-specific immune response requirements (i.e. th , th or th responses, or mixed th / th or th /th responses, etc.), next-generation adjuvants can be rationally developed and incorporated into human vaccines. currently, all approved human adjuvants mostly induce only antibody responses. however, recent adjuvant research has led to the development of novel adjuvants capable of inducing cmi (especially required for malaria, tb and hiv), as well as antibody responses. new immunostimulatory adjuvants or immunomodulatory compounds are under investigation to induce cmi and high antibody titers. novel combination adjuvants are being tested in candidate human vaccines with promising results that have strong implications for use in vaccines against challenging infectious pathogens and different target populations. this is potentially due to activation of multiple innate immune sensing signal transduction pathways by combination adjuvants. novel adjuvants are required that can target emerging new pathogens or reemerging old pathogens. such pathogens often have a more complex host-pathogen interaction, which needs better understanding and further characterization. among these new-generation adjuvants, several are proprietary, which may make it difficult to purchase them and conduct independent parallel trials. factors such as genetic background, preexposure to pathogens or vaccine antigens, age, nutritional and immunological status of vaccine recipients, all dictate the final effectiveness of adjuvanted vaccines. nevertheless, with the aid of structural, systems and reverse vaccinology, epitope prediction and other technological advancements, adjuvant technology is now gradually progressing towards a more personalized approach. there has been an exponential growth in the field of adjuvant research. while alum was historically used as the only licensed adjuvant for more than years, six new adjuvants were approved in the last years. the next five years will see substantial progress in obtaining licensure for more varied types of adjuvants. understanding innate immunity and the role of prr agonists as adjuvants in stimulating the innate immune system has revolutionized adjuvant technology. systems biology has immense contribution in the development of effective and potent adjuvants, and will continue to do so in the coming years. correlates of adjuvanticity or immune signatures, and biomarkers of adjuvant safety and protective efficacy will further streamline adjuvant research. tailor-made adjuvants will find their use against distinct pathogens and in specific target populations. better characterization of adjuvants by new omics-based technologies will facilitate licensing of new adjuvants. since there is a pressing need for developing vaccines against a multitude of very virulent/emerging pathogens, we need to develop subunit vaccines and not live vaccines, and hence adjuvant selection is critical. the mucosal surface is the preferred route of entry for most pathogens. therefore, mucosal immunization is considered to be most effective in preventing mucosally transmitted infections. however, the major hurdle in the development of mucosal vaccines is the lack of safe and effective mucosal adjuvants due to toxicity issues. there is specific need for standardized, more comprehensive and pertinent methodologies for safety evaluation to enable development of safe mucosal adjuvants [ ] . mucosal adjuvants are also required to promote bioavailability of vaccine antigen. another requirement is the development of mucosal adjuvants with an optimal targeting ability so as to reduce undesirable adverse side effects. since efficacy and toxicity of most mucosal adjuvants appear to be intrinsically linked, a risk-benefit ratio needs to be ascertained for these adjuvants. attention must also be directed to studying antigen-adjuvant interactions instead of irrational mixing of an adjuvant in a vaccine formulation [ ] . oil-in-water emulsions are very promising adjuvants and characterization/analysis of components added in emulsion preparations in more detail will facilitate improvement of such adjuvants [ ] . papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers vaccine-preventable disease table working group. historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states modes of action for mucosal vaccine adjuvants vaccine adjuvants: from to and beyond. vaccines (basel) an overview of adjuvant formulations and delivery systems liposomal vaccine delivery systems liposomes as vaccine delivery systems: a review of the recent advances iscom-based vaccines: the second decade iscomatrix adjuvant for antigen delivery iscomatrix: a novel adjuvant for use in prophylactic and therapeutic vaccines against infectious diseases meta-analysis on randomized controlled trials of vaccines with qs- or iscomatrix adjuvant: safety and tolerability vaccine delivery using nanoparticles alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity vaccine adjuvants: mode of action vaccines with the mf adjuvant do not stimulate antibody responses against squalene from discovery to licensure, the adjuvant system story correlates of adjuvanticity: a review on adjuvants in licensed vaccines design and evaluation of a safe and potent adjuvant for human vaccines novel respiratory syncytial virus-like particle vaccine composed of the postfusion and prefusion conformations of the f glycoprotein adjuvants: classification, modus operandi, and licensing neutrophil swarming and extracellular trap formation play a significant role in alum adjuvant activity comparison of multiple adjuvants on the stability and immunogenicity of a clade c hiv- gp trimer non-clinical safety and biodistribution of as -adjuvanted inactivated pandemic influenza vaccines enhancement of adaptive immunity by the human vaccine adjuvant as depends on activated dendritic cells iscom technology-based matrix m adjuvant: success in future vaccines relies on formulation working together: interactions between vaccine antigens and adjuvants yellow fever vaccine yf- d activates multiple dendritic cell subsets via tlr , , , and to stimulate polyvalent immunity identification of novel synthetic toll-like receptor agonists by high throughput screening immune adjuvant effect of molecularly-defined toll-like receptor ligands. vaccines (basel) • in-depth review of the use of tlr-ligands as adjuvants and discussion of the mechanism of action of such ligands efficacy of a prophylactic adjuvanted bivalent l virus-like-particle vaccine against infection with human papillomavirus types and in young women: an interim analysis of a phase iii double-blind, randomised controlled trial new hepatitis b vaccine formulated with an improved adjuvant system rectal and vaginal immunization of mice with human papillomavirus l virus-like particles microbiota influences vaccine and mucosal adjuvant efficacy towards an understanding of the adjuvant action of aluminium rna is an adjuvanticity mediator for the lipid-based mucosal adjuvant the vaccine adjuvant chitosan promotes cellular immunity via dna sensor cgas-stingdependent induction of type i interferons viral evasion of dna-stimulated innate immune responses the mucosal adjuvant cyclic di-gmp enhances antigen uptake and selectively activates pinocytosis-efficient cells in vivo carbohydrate-based immune adjuvants lysosome-dependent activation of human dendritic cells by the vaccine adjuvant qs- central role of cd (+) lymph node resident macrophages in the adjuvanticity of the qs- component of as adjuvants in the driver's seat: how magnitude, type, fine specificity and longevity of immune responses are driven by distinct classes of immune potentiators. vaccines (basel) excellent insight into the correlates of adjuvanticity of wellknown and specialized adjuvants, roles on induction of adaptive immune responses as well as targeted and rational use of adjuvants in vaccines as , an aluminum salt-and tlr agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity selective activation of the p mapk pathway by synthetic monophosphoryl lipid a cellular and molecular synergy in as -adjuvanted vaccines results in an early ifngamma response promoting vaccine immunogenicity il- signaling in immunity a structure-function approach to optimizing tlr ligands for human vaccines molecular characterization of in vivo adjuvant activity in ferrets vaccinated against influenza virus mode of action of adjuvants: implications for vaccine safety and design toll-like receptor ligand-based vaccine adjuvants require intact myd signaling in antigen-presenting cells for germinal center formation and antibody production molecular and cellular signatures of human vaccine adjuvants identification of molecular and cellular biomarkers induced in mouse muscle upon injection with mf oil-in-water emulsion the adjuvants aluminum hydroxide and mf induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells adjuvant system as containing alpha-tocopherol modulates innate immune response and leads to improved adaptive immunity alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells induction of lymphocyte recruitment in the absence of a detectable immune response littel-van den hurk s. formulation of the respiratory syncytial virus fusion protein with a polymer-based combination adjuvant promotes transient and local innate immune responses and leads to improved adaptive immunity immunological mechanisms of vaccination different adjuvants induce common innate pathways that are associated with enhanced adaptive responses against a model antigen in humans activation of the endoplasmic reticulum stress sensor ire alpha by the vaccine adjuvant as contributes to its immunostimulatory properties promotion of b cell immune responses via an alum-induced myeloid cell population preclinical evaluation of bacterially produced rsv-g protein vaccine: strong protection against rsv challenge in cotton rat model towards an evidence based approach for the development of adjuvanted vaccines highlights the role of immunepotentiators in activation of the innate immune system, which is implicated in the development of next-generation adjuvants as well as clinical adjuvanted-vaccines immunity to viruses: learning from successful human vaccines induction of icos +cxcr +cxcr + th cells correlates with antibody responses to influenza vaccination the adjuvant lt-k can restore delayed maturation of follicular dendritic cells and poor persistence of both protein-and polysaccharide-specific antibody-secreting cells in neonatal mice adjuvants enhancing cross-presentation by dendritic cells: the key to more effective vaccines? front immunol sting-activating adjuvants elicit a th immune response and protect against mycobacterium tuberculosis infection a liposome-based mycobacterial vaccine induces potent adult and neonatal multifunctional t cells through the exquisite targeting of dendritic cells intranasal c-di-gmpadjuvanted plant-derived h influenza vaccine induces multifunctional th cd + cells and strong mucosal and systemic antibody responses in mice randomized, double-blind, phase a trial of falciparum malaria vaccines rts,s/ as b and rts,s/as a in malaria-naive adults: safety, efficacy, and immunologic associates of protection mechanisms of action of adjuvants synthetic cationic peptide idr- provides protection against bacterial infections through chemokine induction and enhanced leukocyte recruitment (tlr ) interacts with flagellin and profilin through disparate mechanisms cpg oligonucleotides as cancer vaccine adjuvants. vaccines (basel) flagellin as a vaccine adjuvant the development of mucosal vaccines for both mucosal and systemic immune induction and the roles played by adjuvants discusses the role of mucosal adjuvants in stimulating both mucosal and systemic immunity against pathogens infecting via mucosal surfaces u-omp from brucella abortus is a useful adjuvant for vaccine formulations against salmonella infection in mice choice and design of adjuvants for parenteral and mucosal vaccines. vaccines (basel) vaccine development for respiratory syncytial virus adjuvants and the vaccine response to the ds-cav -stabilized fusion glycoprotein of respiratory syncytial virus van den hurk s. induction of mucosal immunity and protection by intranasal immunization with a respiratory syncytial virus subunit vaccine formulation mucosal adjuvants: opportunities and challenges what role does the route of immunization play in the generation of protective immunity against mucosal pathogens? intranasal immunization with h n vaccine plus poly i:poly c u, a toll-like receptor agonist, protects mice against homologous and heterologous virus challenge tlr agonists and proinflammatory antitumor activities leukotrienes are potent adjuvant during fungal infection: effects on memory t cells antigenic diversity and immune evasion by malaria parasites immune mechanisms in malaria: new insights in vaccine development induction of cross-reactive cytotoxic t-lymphocyte responses specific for hiv- gp using saponin adjuvant (qs- ) supplemented subunit vaccine formulations the future of the rts,s/as malaria vaccine: an alternative development plan vaccine adjuvant uses of poly-ic and derivatives recommendations of the advisory committee on immunization practices for use of herpes zoster vaccines cell-mediated immune responses to a varicella-zoster virus glycoprotein e vaccine using both a tlr agonist and qs in mice hpv -immune response to infection and vaccination efficacy of human papillomavirus l protein vaccines (cervarix and gardasil) in reducing the risk of cervical intraepithelial neoplasia: a meta-analysis immunobiology of hpv and hpv vaccines comparative immunogenicity and safety of human papillomavirus (hpv)- / as -adjuvanted vaccine and hpv- / / / vaccine administered according to -and -dose schedules in girls aged - years: results to month from a randomized trial adjuvants in tuberculosis vaccine development tb vaccine development and the end tb strategy: importance and current status nod and toll-like receptors are nonredundant recognition systems of mycobacterium tuberculosis multi-stage subunit vaccines against mycobacterium tuberculosis: an alternative to the bcg vaccine or a bcg-prime boost? phase b controlled trial of m /as e vaccine to prevent tuberculosis systems biology approach predicts immunogenicity of the yellow fever vaccine in humans systems biology of vaccination for seasonal influenza in humans expression of genes associated with immunoproteasome processing of major histocompatibility complex peptides is indicative of protection with adjuvanted rts, s malaria vaccine integrated analysis of genetic and proteomic data identifies biomarkers associated with adverse events following smallpox vaccination molecular signatures of a tlr agonist-adjuvanted hiv- vaccine candidate in humans molecular and cellular signatures underlying superior immunity against bordetella pertussis upon pulmonary vaccination systems vaccinology identifies an early innate immune signature as a correlate of antibody responses to the ebola vaccine rvsv-zebov predictive markers of safety and immunogenicity of adjuvanted vaccines mf and pam csk boost adaptive responses to influenza subunit vaccine through an ifn type i-independent mechanism of action the t helper type response to cysteine proteases requires dendritic cell-basophil cooperation via ros-mediated signaling key roles of adjuvants in modern vaccines systems analysis of protective immune responses to rts,s malaria vaccination in humans complex immune correlates of protection in hiv- vaccine efficacy trials identification of immune signatures predictive of clinical protection from malaria systems vaccinology: enabling rational vaccine design with systems biological approaches new approaches to understanding the immune response to vaccination and infection vaccine adjuvants: putting innate immunity to work the importance of adjuvant formulation in the development of a tuberculosis vaccine recent advances of vaccine adjuvants for infectious diseases the authors like to thank all the current and previous members from the laboratory as well as the animal care facility at vido-intervac, university of saskatchewan, canada for their contribution. this is vido-intervac manuscript no . publication of this manuscript is supported by grant mop from the canadian institutes of health research (cihr). the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. key: cord- -ipa wz authors: poland, g. a.; ovsyannikova, i. g.; kennedy, r. b. title: personalized vaccinology: a review date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ipa wz abstract at the current time, the field of vaccinology remains empirical in many respects. vaccine development, vaccine immunogenicity, and vaccine efficacy have, for the most part, historically been driven by an empiric “isolate-inactivate-inject” paradigm. in turn, a population-level public health paradigm of “the same dose for everyone for every disease” model has been the normative thinking in regard to prevention of vaccine-preventable infectious diseases. in addition, up until recently, no vaccines had been designed specifically to overcome the immunosenescence of aging, consistent with a post-wwii mentality of developing vaccines and vaccine programs for children. it is now recognized that the current lack of knowledge concerning how immune responses to vaccines are generated is a critical barrier to understanding poor vaccine responses in the elderly and in immunoimmaturity, discovery of new correlates of vaccine immunogenicity (vaccine response biomarkers), and a directed approach to new vaccine development. the new fields of vaccinomics and adversomics provide models that permit global profiling of the innate, humoral, and cellular immune responses integrated at a systems biology level. this has advanced the science beyond that of reductionist scientific approaches by revealing novel interactions between and within the immune system and other biological systems (beyond transcriptional level), which are critical to developing “downstream” adaptive humoral and cellular responses to infectious pathogens and vaccines. others have applied systems level approaches to the study of antibody responses (a.k.a. “systems serology”), [ ] high-dimensional cell subset immunophenotyping through cytof, [ , ] and vaccine induced metabolic changes [ ]. in turn, this knowledge is being utilized to better understand the following: identifying who is at risk for which infections; the level of risk that exists regarding poor immunogenicity and/or serious adverse events; and the type or dose of vaccine needed to fully protect an individual. in toto, such approaches allow for a personalized approach to the practice of vaccinology, analogous to the substantial inroads that individualized medicine is playing in other fields of human health and medicine. herein we briefly review the field of vaccinomics, adversomics, and personalized vaccinology. vaccines have been one of the most effective public health strategies in preventing infectious diseases. a decade ago, we described the idea of vaccinomics and adversomics, based on the immune response network theory [ , ] , which utilizes immunogenetics/imunogenomics and systems biology approaches to understand the basis for inter-individual variations in vaccineinduced immune responses in humans, as well as the basis for adverse side effects from vaccines [ ] . vaccinomics and adversomics explore the influence of genetic and non-genetic regulation on the heterogeneity of vaccine-induced immune responses at both the personal and population levels [ ] . in particular, vaccinomics and adversomics utilize high-throughput, high-dimensional systems biology approaches, which aim to predict variations in protective and maladaptive innate and adaptive immune responses to vaccines [ ] [ ] [ ] [ ] , ] . in this regard, the basis of personalized (and predictive) vaccinology is the assessment of an individual's genetic background, sex, as well as other factors that may impact vaccine immunogenicity, efficacy, and safety [ ] [ ] [ ] [ ] . we and others have widely published on the applicability of the tools and concepts of vaccinomics, including immunogenetics and immunogenomics, to the knowledge-based directed development of new and improved vaccine candidates [ ] [ ] [ ] [ ] . the application of these concepts is likely to allow for explanation, quantification, and prediction of vaccine-induced protective immune responses-including the http://dx.doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. development of predictive immune signatures in response to vaccines. indeed, we have previously published what we believe is the first draft of a mathematical model and predictive equation describing the non-random events that lead to a pre-determined immune response [ ] : b i x iþe y = measure of immune response b o = intercept b i = coefficient for the ith variable x i and indicates the amount of change in y for a unit change in x i Ε = random deviations from the model we recognize that such an equation, given the current state of the science, is incomplete and cannot yet predict immune responses. but we present it as an early directional attempt to quantify such an equation. such an approach begins to move us into a st-century model of directed vaccine development and an advanced understanding of how, and by what mechanisms, vaccines and vaccine adjuvants trigger both useful and maladaptive innate and adaptive immune responses. we believe that vaccinomics and adversomics represent approaches counter to the standard methods of vaccine development until recently. historically, vaccine development has been empirical, despite many emerging and re-emerging complex, hyper-variable pathogens-many with elaborate immune escape mechanisms. in addition, vaccine coverage rates continue to suffer as society is risk-averse toward vaccines and demands levels of safety that may not be achievable. finally, the ''one-size-fits-all" approach to the practice of vaccinology ignores the complexity and diversity of the human immune system and host genome. thus, the promise of vaccinomics and related paradigms is to identify specific immune response profiles, immunosignatures, and biomarkers that predict vaccine safety and/or efficacy, and which may lead to new vaccine candidates. vaccinomics provides the opportunity to examine not only immune response genes likely to be involved in vaccine response, but also the possibility of identifying the influence of new (uncharacterized) genes on vaccine-induced immunity. in turn, the identification and directed study of such genetic variants allows recognition, often at the molecular level, of the effects of differential binding, processing, and expression/presentation of antigenic viral peptides used in vaccine development, identification of the differential range of presented peptides (genetic restriction), altered secretion patterns (cytokines) in response to vaccines or vaccine adjuvants, altered transcription of important genes (signaling molecules) and gene products, altered binding of virus/antigens by membrane-based receptors (tlrs, other), differential receptor function, expression, and affinities, and the impact of epigenetics on vaccine-induced immune responses. we have utilized this knowledge in our own laboratory to create a research-oriented paradigm of ''discover-validate-characterize-apply," which may be used in new candidate vaccine development ( fig. ) [ ] . in this paradigm, we have been able to utilize vaccinomics approaches to discover genetic variants that are significantly associated with subsequent downstream immune responses, validate that such variants are indeed associated, then seek to characterize the mechanism whereby such effects occur and, finally, apply this knowledge-often in functional studies that confirm the effect on immunity. such knowledge can be exploited in developing immune strategies to enhance or circumvent genetic restrictions, for example, in triggering vaccine-associated immune responses, by ''reverse engineering" around a given genetic or other obstacle to generating protective immune responses. there are a growing number of studies reporting unbiased genome-wide assessments of genetic variation and its influence on adaptive (humoral and cellular) vaccine-induced immune responses across multiple viral and bacterial vaccines. for example, candidate and gwas immunogenetic and phamacogenetic studies have identified polymorphisms in hla, kir, mica, and btn genes associated with immune responses to pathogens causing disease in humans, such as hepatitis c [ ] , mycobacterium leprae [ , ] , human immunodeficiency virus [ ] , and measles [ ] [ ] [ ] . similar studies have identified novel genes impacting immune responses to vaccines, including hepatitis b, rubella, influenza a, smallpox, anthrax, and mumps [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our gene association studies of measles-mumps-rubella (mmr) vaccines have demonstrated that inter-individual variations in measles vaccine virus-induced humoral and cellular responses are significantly associated with polymorphisms in immune response genes and, together with hla alleles, explain % of the inter-individual variability in humoral response [ , [ ] [ ] [ ] . these findings, which illustrated the importance of key hla alleles in the adaptive humoral immune response to measles vaccine, led to the identification of naturally processed and presented measles-derived peptides isolated from specific hla polymorphisms associated with vaccine non-and hyper-response [ , ] . these peptides containing specific components (adjuvants and biodegradable nanoparticles) are now being utilized in a reverse-engineering strategy to develop peptide-based candidate measles vaccines. likewise, homan et al. have attributed diminished protection to differential hla presentation of t and b cell epitopes between vaccine and wild type strains of mumps virus [ ] . this diminished efficacy could theoretically be overcome by incorporating defined critical immunogenic peptides into an improved vaccine. tlr genes represent an important link between the innate and the adaptive immune system [ , ] . as an example, we have demonstrated that measles vaccine-induced humoral responses are significantly associated with coding polymorphisms in the tlr (rs ) and tlr (rs ) genes [ ] . for the rubella vaccine and tlr gene, a tlr gene snp rs was associated with rubella-specific gm-csf production [ ] . our recent mumps vaccine study has identified and replicated tlr snps associated with a % decrease in antibody titer, and a tlr snp associated with a % increase in t cell response (unpublished data). these data strongly suggest that robust tlr activation by measles, mumps, and rubella viruses is crucial for optimal vaccine response. supporting these findings is a study demonstrating that an inactivated mumps vaccine containing a protollin-based tlr / adjuvant is highly immunogenic in a mouse model; it led to superior total igg levels, higher neutralizing antibody titers, greater mucosal iga production, and enhanced th /th cytokine secretion [ ] . one potential application of this finding is to identify the specific and critical interactions between tlrs (and other genes) and virus, leading to advances in our knowledge of the precise mechanisms driving immunity to mmr vaccine. significant sex differences in humoral and cellular immune responses to vaccines are apparent [ , ] . additionally, local and systemic adverse rates are generally higher in females versus males. protective antibody responses are significantly higher in females than males after vaccination against influenza, yellow fever, measles, mumps, rubella, hepatitis a and b, herpes simplex (hsv) , rabies, smallpox, and dengue viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . sex-based differences in humoral immune responses are observed through various age groups [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , suggesting that sex steroid hormones are not the singular mediators of sex differences in humoral immune responses to vaccines [ , ] . this suggests that genetic, or other, factors may be an important driver of sex-related differences in humoral immune response [ ] . despite significant evidence of immune response differences between the sexes, for the most part, vaccine studies have not examined and analyzed immune response outcomes by sex [ , ] . in fact, little information is known about potential mechanisms for sex-based effects, which should be a priority for vaccine research studies. discovery of specific factors involved in sex-based differences in immune response may allow the identification of new correlates of vaccine immunogenicity. in a cohort of older (ages - ) and younger (ages - ) previously vaccinated individuals, the seasonal trivalent influenza vaccine induced > . -fold higher a/h n -specific hai antibody titers in women than men across both age groups [ ] . similarly, a study of standard seasonal influenza vaccine and high-dose influenza vaccine responses in a sex-balanced cohort of elderly subjects (ages - ) demonstrated significantly higher rates of seroconversion in females than in males [ ] ; however, no significant differences in antibody measures were found between males and females after seasonal influenza vaccination in another cohort of older adults (ages - ) [ ] . a study by furman et al. examining gene expression, serum cytokines/ chemokines, cell subsets, and phosphorylation events found several serum markers (lept, il- ra, crp, gm-csf, and il- ) to be more highly expressed in females than males after influenza vaccine [ ] . this same report used a systems biology approach to identify a gene cluster involved in lipid biosynthesis that is regulated by testosterone and significantly correlated with poor humoral responses following influenza vaccination in men [ ] . these data suggest that this gene cluster (e.g., genes involved in lipid metabolism) could be an important driver of sex-related differences in humoral immune response. this collective knowledge could substantially assist future personalized vaccine development efforts through the generation of new knowledge and the identification of targets and biomarkers that predict vaccine responses in specific populations (e.g., females vs. males; young vs. old; obese vs. lean). further research is needed to clarify the effects of sex on immune response. identification of molecular immune signatures of sex differences in innate and adaptive immune responses to vaccines may provide evidence necessary for additional efforts in designing personalized vaccination and vaccinomics approaches (i.e., in which males and females might be vaccinated differently using different doses or different vaccines) to provide equal protection while reducing side effects [ , , ] . a significant global public health issue is the aging of the population. as individuals age, immunosenescence develops, leading to poorer immune responses to vaccines. immunosenescence is an age-related dysregulation of the immune system due to ageassociated changes in innate and adaptive immune system components, which leads to impaired immunity and protection following immunization or infection [ ] [ ] [ ] . published data reveal that innate and adaptive immunity is decreased with age, but the systems-level mechanisms for these findings are unclear [ , ] , particularly in regard to influenza and other viral vaccine responses where the morbidity, mortality, and associated healthcare costs are greater in older individuals [ ] . major signs of innate immune dysfunction commonly observed in the elderly include, but are not limited to, altered cytokine secretion; decreased nk cell activity; reduced tlr expression; and a chronic inflammatory state (elevated levels of il- b, mcp- , tnf-a, and serum il- ) known as ''inflamm-aging" [ , [ ] [ ] [ ] . age-related humoral immune dysfunction, for example, might be overcome through optimal stimulation of innate and/or th cell-specific genes, which may be different in males and females. for example, adjuvanted zoster subunit vaccine (hz/su) reduced the risks of herpes zoster, and postherpetic neuralgia in immunocompetent persons years of age and older [ ] . this hz/su vaccine contains varicella zoster virus glycoprotein e and a novel as b adjuvant system aimed to improve and preserve with age zoster-specific cd + t cell responses [ ] . a tlr agonist gla-se (glucopyranosyl lipid adjuvant formulated in a stable emulsion) has been shown to enhance th responses to influenza vaccine in older adults [ ] , suggesting a potential mechanism for targeting innate receptor agonists (e.g., tlrs) that enhance innate immune responses against influenza. given the substantially diminished efficacy of influenza and other vaccines with age and the importance of developing improved vaccines [ ] , data from vaccinomics studies could be used to inform directed and rational development of next-generation influenza vaccines-potentially circumventing immunosenescence-related factors. systems biology approaches provide a unique opportunity to identify biomarkers likely to be involved in immune responses to vaccination [ ] [ ] [ ] [ ] , , ] . fourati et al. applied a systems vaccinology approach to examine gene signatures and molecular pathways of age-related hyporesponse to hepatitis b vaccine (hbv) in naïve older adults [ ] . they observed the b cell signaling pathway (and higher memory b cell frequencies) and inflammatory pathway (and increased frequencies of activated pro-inflammatory innate cells) were strongly correlated with higher and low antibody responses to hbv, respectively. this signature, including serum cytokine profiling and flow cytometric correlates of response, predicted the antibody response to hbv with up to % accuracy [ ] . this study demonstrates that a systems biology approach can be used to predict age-related immune response to vaccination. obesity is another major public global health concern. in the us, % of adults and nearly % of children and adolescents are now overweight or obese [ ] . weight gains across all countries have been demonstrated to be associated with increasing socioeconomic status. obesity has been shown to be a predictor of impaired immunogenicity (e.g., decreased antibody response) to hepatitis b, tetanus toxoid, rabies, and influenza vaccines [ ] [ ] [ ] [ ] , and as such can be considered a marker, or state, of immunosuppression at its extremes. these data suggest that obesity is correlated with poorer vaccine-induced immune responses in humans, and further research is required to understand the immune mechanisms that are altered in obesity. as individuals age, their circulating leptin levels rise with a concomitant reduction in leptin signaling; this results in leptin resistance, which is a finding associated with obesity [ ] . leptin resistance has been shown to adversely affect the immune response in obese subjects, including responses to influenza virus [ , ] . for example, obese individuals demonstrate decreased activation of influenza-specific cd + t cells compared to healthyweight persons, including decreased production of ifn-c and granzyme b, suggesting that influenza vaccination may not be as effective in the obese population as in healthy-weight individuals [ ] . given only moderate seroprotection of influenza and other vaccines in obese older adults [ ] , and the importance of developing improved influenza vaccines [ ] , systems biology studies designed to identify the mechanisms for improved immune response are needed. in fact, data from vaccine studies could be used to inform directed and rational development of personalized vaccines that optimally stimulate innate and adaptive immune responses in males and females and overcome immune deficiencies induced by obesity [ ] . careful vaccine studies comparing lean and obese persons could provide foundational data used to improve vaccine-induced protection in the obese, a subpopulation with an elevated risk for serious vaccine-preventable illnesses and suboptimal vaccine-induced protective responses [ ] . adversomics utilizes tools-much like those used in vaccinomics-to identify, characterize, and predict adverse, or maladaptive, immune responses to vaccines [ , , ] . the promise of adversomics would be to develop or identify either predictors or immune signatures of maladaptive immune responses that lead to harm rather than benefit, and to better understand the generation and mechanisms of such maladaptive immune responses. we have asked the question, as have other scientists, ''does it make sense in the st century to give the same vaccine, dose, and at the same frequency to everyone, regardless of age, weight, gender, race, genotype, and medical condition?" for example, we give adult males and females the same dose, and the same number of doses of vaccines, ignoring the findings that females nearly always have superior humoral immune responses to males for all vaccines studied, and yet experience significantly more side effects-more adverse events, of greater duration, and of higher intensity [ , , ] . while the field is young in implementation, research has already revealed associations between specific genes or snps and adverse immune outcomes. for example, associations between cytokine gene expression and fever after smallpox vaccine have been identified [ ] . other studies have demonstrated correlations between smallpox vaccine-induced fevers and il a and il snps [ ] . other smallpox vaccine-induced adverse events such as fever, rash, and enlarged lymph nodes have been significantly associated with mthfr, irf , and il snps haplotypes [ ] . while smallpox vaccine is not used in the general population, such studies stand as examples of the usefulness of vaccinomic approaches. finally, other recent studies have identified generic fever gene networks (tnfa) after vaccine administration [ ] , and relationships between mmr vaccine administration and snps in ifi l, cd , scn a, a, and tmem (ano ) genes [ ] . despite the tremendous success of vaccines, vaccinologists face several current challenges, including difficulty in developing vaccines for hypervariable viruses (hiv, rhinovirus, hepatitis c virus, coronavirus) and complex pathogens (malaria, mycobacterium tuberculosis); newly emerging pathogens, such as zika virus (zikv); complications imposed by aging and immunosenescent populations; inadequate understanding of the neonatal and newborn immune systems; increasingly immune deficient or immunocompromised populations due to hiv, cancer, or medications; sexbased differences in vaccine response and adverse-event rates; enhanced scrutiny of vaccine safety; and as noted global increases in age and weight. in addition, vocal and active anti-vaccine groups whose messages are not easily countered by facts or scientific studies have materially and detrimentally affected vaccine coverage rates [ ] [ ] [ ] . vaccinomic approaches can be utilized to better understand these issues; this information can then be used to inform new approaches, new understandings, and new vaccine candidates. just as new technologies have created exciting new opportunities in personalized medicine, they have brought with them novel challenges in addition to those mentioned above. in order for the full potential of personalized vaccines to be achieved, we must overcome additional challenges, such as the need for the following: larger genotype:phenotype datasets (often in the many thousands to ten thousands) integrating increasingly diverse high-throughput, highdimensional data types biomarkers that can reliably distinguish which product patients receive based on the likelihood of their response or an adverse side effect vaccines with different mechanisms of action may require a move away from humoral correlates of protection for licensure; in this regard, correlates of protection based on cellular immune outcomes are likely to play an important role in future vaccines more sophisticated biostatistical and bioinformatics approaches that can identify patterns and causative networks within terabyte levels of extremely high dimensional data types from the economic side: methods of technology transfer and funding mechanisms to move novel vaccines developed through vaccinomic approaches into low and middle-income countries who often most need specific vaccines (malaria, others) we have seen the shift from ''vaccinology . ," which is the empirical ''isolate-inactivate-inject" paradigm, to ''vaccinology . "-the use of recombinant technology and novel adjuvants. however, even this paradigm is limited by our incomplete mechanistic understanding of adjuvants and innate immunity. as we adopt approaches such as those listed above, we envision a movement of the field into an era of ''vaccinology . ," during which we expect to see the use of vaccinomics and systems-level approaches to develop new vaccines; innovative vaccine-antigen packaging methods; and adjuvant development targeted at the innate response pathways best suited for a given pathogen. a common reaction to this paradigm of personalized vaccinology is questioning cost and economics. at one level, such considerations are simply ''too soon" in the development of the science to effectively answer. however, like progress being made in individualized medicine, it is likely that being able to provide the right vaccine to the right patient-for the right reasons and at the right dose-will lead to improved medical outcomes and reduced costs at the population level. personalized vaccinology is the goal of applying the concept of personalized medicine to vaccines. rapid strides in omics technologies and foundational work applying systems biology, computational immunology and reverse vaccinology have facilitated modern approaches to vaccine design and development enabling us to create vaccine formulations for new and re-emerging pathogens. egg-based influenza vaccines take > months to create. the recent licensure of cell culture-based influenza vaccines demonstrate that rapid, scalable processes can now be implemented in order to create vaccine against emerging influenza strains (e.g., h n , h n , h n , h n , h n ) within weeks [ ] and can be safely administered to individuals with egg allergies [ ] . the ebola outbreak in liberia, sierra leone, and guinea in provides an example of the need to rapidly develop vaccine candidates [ ] . dna vaccines, virus-like particle vaccines, and replicating/nonreplicating viral vector vaccines have all been created and tested. among the most promising are a replication-competent, recombinant vesicular stomatitis virus vector expressing the glycoprotein of ebola zaire (rvsv-zebov), [ ] a variety of adenovirusvectored vaccines expressing ebola glycoprotein, [ , ] a modified vaccinia virus ankara-based vaccine encoding the ebola zaire glycoprotein (mva-bn-filo), [ , ] and dna-based vaccinesone expressing glycoproteins from both zaire and sudan, and the other expressing the marburg glycoprotein [ ] . although the rvsv-based vaccine elicits high titers of neutralizing ab, it is contraindicated in children and those with compromised immune systems. viral vector vaccines present the problem of developing robust immunity to the vector as well as the target immunogen, limiting their usefulness to a single vaccination. the availability of vaccines in multiple vector backbones opens up the possibilities for prime-boost vaccination strategies for ebola, similar to those that have been applied to hiv, malaria, and tuberculosis [ ] [ ] [ ] [ ] . in this regard, a prime-boost regimen using the mva-based vaccine as the booster vaccination has shown considerable promise [ ] . another example of modern vaccine development being applied to a new pathogen can be seen with the response to zika virus. a purified, formalin-inactivated vaccine (zikv piv) has been developed by the walter reed army institute of research (wrair) [ ] and is being evaluated in several clinical trials (nct , nct , nct ), while other inactivated vaccines are in preclinical development [ ] . two variants of a plasmid dna vaccine containing the prm-env proteins have been developed by niaid and one of the formulations is currently in a phase i clinical trial (nct ) [ ] . inovio pharmaceuticals developed their own plasmid dna vaccine (also expressing prm-env), which is currently in two clinical trials (nct , nct ). rna-based vaccines [ ] and a variety of subunit and viral vector-based vaccines are also in development [ , , ] . dna and rna-based vaccines can be rapidly made at minimal costs compared to other formulations and are fairly stable, without the cold-chain requirements of live virus-based vaccines. subunit vaccines are typically safer than whole virus-based products, which represents an active area of investigation not only for pathogens with no existing vaccines, but also for improving on established vaccines. our group and others have identified pathogen-derived epitopes as preliminary steps in the development of safe, stable, and effective peptide-and protein-based vaccines for smallpox, influenza, measles, tuberculosis, staphylococcus, and myriad other viral and bacterial pathogens [ , [ ] [ ] [ ] [ ] [ ] . parallel efforts by different groups to create new vaccines result in a spectrum of potential products that can be uniquely tailored to specific population groups. live viral vaccines rapidly inducing robust immunity can be used in healthy individuals where time is of the essence (e.g., in outbreak scenarios), while inactivated or subunit vaccines can be used in vulnerable populations such as pregnant women or those with immunocompromising conditions, or in young children where the presence of maternal antibody interferes with whole virus vaccines. vaccines based on different viral vector backbones can be combined into effective primeboost regimens. vaccines with specific adjuvants may be most appropriate for the elderly in order to overcome immunosenescence, or in the very young in order to compensate for immune system immaturity. we, along with increasing numbers of other scientists, believe that personalized vaccinology will revolutionize the practice of vaccinology to the benefit of human health. as part of the development of this field of science, vaccinomics and adversomics will allow us to develop molecular immune signatures of adaptive and maladaptive immune responses to vaccines, develop early biomarkers of vaccine response in vaccine trials, identify who should get what vaccine and at what dose, and increase safety and public confidence in vaccines by reducing the likelihood of serious adverse events related to vaccines. in many ways, however, personalized vaccinology is most challenged by the difficulty in moving the field away from the post-wwii population-level paradigm of ''one dose of every vaccine for everyone," toward an individualized or personalized approach based on the unique factors relevant to a given individual. in his book, the structure of scientific revolutions [ ] , thomas kuhn recognized that ''we wrongly believe scientific progress is a process of linear accretion of knowledge, that science is predicated on the belief that the scientific community understands what the world is like, and that we suppress or resist 'fundamental novelties' because they are seen as subversive to our firmly held beliefs of what the world is like." later in his book, he suggests that ''new advances always have and always will reveal that science and medicine includes bodies of belief incompatible with beliefs we hold today, and that advancements come when we reject a time-honored scientific theory in favor of another incompatible with it." these cognitive biases have, in our opinion, been manifest in our discussions with scientific colleagues as we developed this field of science. schopenhaur, the german philosopher, suggested that new discoveries are at first ridiculed, then opposed, and finally accepted as self-evident. vaccinomics and adversomics appear to be moving from the ridiculed and opposed steps, and into the not-yet quite self-evident phase of the continuum. part of the challenge is that often the concept of personalized vaccinology suggests to the reader that a unique vaccine will be developed for each individual. while that is one tactic being used in the cancer-vaccine field, it is neither necessary nor practical for the prevention of infectious diseases. rather, the personalized vaccinology approach would suggest the development of specific vaccines based on factors that relate to overcoming the potential for poor immunogenicity and the potential for adverse events. an excellent example is influenza vaccines. a mere decade or so ago, only a trivalent injectable influenza vaccine was available. quadrivalent vaccines were unavailable. for with one exception, everyone received the same vaccine and dose, regardless of age, weight, immunosuppression state, etc. at the current time in the us, multiple influenza vaccines are available so that the right vaccine, for the right patient, can be given at the right time. for example, laiv (live attenuated influenza vaccine) can be used in younger subjects or the needle-phobic. high-dose or mf -adjuvanted vaccines can be chosen for the elderly. recombinant vaccines can be chosen for those with egg allergy, and so on. this is the approach that should be taken with all vaccines. in some cases it may mean merely adjusting the dose based on weight, gender, or age. in other cases it may mean utilizing an adjuvanted or non-adjuvanted vaccine based on immune status. other examples include the recently licensed mf adjuvanted influenza vaccine (fluad Ò ), which has demonstrably higher immunogenicity and efficacy than its nonadjuvanted counterparts, [ ] [ ] [ ] or the highly effective as adjuvanted zoster glycoprotein e vaccine, which does not contain live virus and may be more broadly suitable for administration to older individuals [ , ] . thus, the movement toward a new paradigm of vaccine practice, based on a personalized approach, is occurring in the st century based on new scientific knowledge, market demand, safety considerations, immunogenicity concerns, public health trends (age, obesity, other), and the simultaneous pull of individualized medicine in other medical arenas. the net result is likely to be higher vaccine coverage rates, increased public confidence in vaccines, improved immunogenicity and adverse event rates, and a reduction or elimination in the morbidity and mortality related to vaccine-preventable diseases. as a result, we anticipate a new era of personalized ''predictive vaccinology," whereby we abandon a ''one size and dose fits all vaccine approach" in order to design and develop new vaccines, and acquire the ability to make the following predictions for each individual: whether to give a vaccine based on likelihood of response (and perhaps need); the likelihood of a significant adverse event to a vaccine; and the number of doses likely to be needed to induce a protective response to a vaccine [ ] . current vaccine development is largely empirical. vaccines are tested by trial and error, are mass produced, and given to the entire population using the same antigen dose, route of administration, number of vaccinations, and at the same age. in contrast, the new vaccine-development paradigm begins with the ''discovery" of new knowledge by integrating unbiased, comprehensive analysis of the genome, transcriptome, proteome, metabolome, microbiome, and immunome-along with the assessment of multiple measures of immune function-in order to under-stand and evaluate perturbations of the immune system. findings are then ''validated" in replication cohorts or additional model systems. the new knowledge is then ''applied" to the creation of new vaccine formulations that can undergo additional testing to start a new round of ''discovery," or can move into clinical trials in order to develop vaccine products engineered to elicit (or avoid) specific effects on the immune system. each product is tailored to specific subgroups such that robust, protective immunity can be elicited in the old and young, lean and obese, or male and female, while avoiding inappropriate immune responses due to genetics, metabolism, race, gender, malnutrition, immunosuppression, and other host factors or underlying conditions. r ai , and contract no. hhsn c (n ai ). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. the authors would like to acknowledge the contributions of the centers for disease control and prevention (cdc), which provides financial support to the world health organization initiative for vaccine research (u ck ). dr. poland is the chair of a safety evaluation committee for novel investigational vaccine trials being conducted by merck research laboratories. dr. poland offers consultative advice on vaccine development to merck & co. inc., avianax, dynavax, novartis vaccines and therapeutics, emergent biosolutions, adjuvance, seqirus, and protein sciences. drs. poland and ovsyannikova hold three patents related to vaccinia and measles peptide research. dr. kennedy has received funding from merck research laboratories to study waning immunity to mumps vaccine. these activities have been reviewed by the mayo clinic conflict of 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. /bs.ircmb. . . sha: doc_id: cord_uid: u x jaw in mammals, cytosolic detection of nucleic acids is critical in initiating innate antiviral responses against invading pathogens (like bacteria, viruses, fungi and parasites). these programs are mediated by multiple cytosolic and endosomal sensors and adaptor molecules (c-gas/sting axis and tlr /myd axis, respectively) and lead to the production of type i interferons (ifns), pro-inflammatory cytokines, and chemokines. while the identity and role of multiple pattern recognition receptors (prrs) have been elucidated, such immune surveillance systems must be tightly regulated to limit collateral damage and prevent aberrant responses to self- and non-self-nucleic acids. in this review, we discuss recent advances in our understanding of how cytosolic sensing of dna is controlled during inflammatory immune responses. the innate immune system, which is conserved across virtually all multi-cellular organisms on the planet (from sponges to insects, plants, and vertebrates), includes a diverse set of receptors capable of detecting molecular patterns like sugars, lipids, polymers, and nucleic acids that are principal in prompting protective responses (broz and monack, ; kieser and kagan, ; kumar et al., a) . in vertebrates, cytosolic detection of nucleic acids (derived from microbes like viruses, intracellular bacteria, fungi and parasites) is critical in initiating innate (characterized by production of type i ifns) and adaptive (characterized by t and b cell responses) immune responses (wu and chen, ) . recent efforts have focused on identifying relevant immune surveillance sensors and components of downstream signaling-toll-like receptors (tlrs) and their cognate ligands, cytosolic sensing of rna (primary mediated by the rig-i/ips- axis), cytosolic sensing of dna (primary mediated by the cgas/sting axis), and the inflammasome pathway (primary mediated by nod-like receptors; nlrs) (broz and monack, ; kieser and kagan, ; kumar et al., a ). yet, while the identities of immune surveillance systems have been revealed, inflammatory programs mediated by these sensors must be tightly regulated to prevent aberrant and inappropriate responses to selfderived ligands (like self rna and dna) that may be released from damaged cells, senescent cells, apoptotic cells, or during fertilization (barber, ; de oliveira mann and kranzusch, ) . indeed, rig-i, the cytosolic receptor for rna distinguishes self from non-self rna through interaction with a -triphosphate that is unique to viral rna (wu and chen, ) . though exceptions to this requirement have been reported, the ability of rig-i to distinguish self from non-self in this way ensures that anomalous immune responses to cellular rna do not occur. responses to dna are far more agnostic. cgas, in collaboration with sting, does not distinguish between cellular and foreign dna (barber, ; crowl et al., ) . indeed, ht-dna (herring testes-dna), as well as isd (interferon-stimulated dna) oligonucleotides, is known to act as potent stimulators of cgas/ sting in multiple mammalian cell types. it has also been suggested that chronic cgas/sting activation induced by self dna may be responsible for induction of aberrant inflammatory diseases like systemic lupus erythematosus (sle), aicardi-goutières syndrome (ags), and polyarthritis (barber, ; crowl et al., ) . while apoptotic cells represent a possible source of dna, the existence of dnases in cytoplasmic (e.g., dnase-iii, also known as trex ) and lysosomal compartments (e.g., dnase-ii) can clear potential ligands and ensure that inappropriate responses are not initiated (barber, ; crowl et al., ) . in the event of mitochondrial dna (mtdna) release into the cytoplasm (which occurs following mitochondrial damage), intracellular caspase activation controls the aberrant immune response (mcarthur et al., ; rongvaux et al., ; white et al., ) . similarly, cgas/sting and the necessary cofactors, and cellular dna are compartmentalized such that sensing of self-dna is avoided; the receptor in the cytosol and the ligand (dna) in the nucleus (barber, ) . however, recent publications have illustrated that cell cycle progression in the context of dna-damage may lead to the formation of micronuclei which elicit cgas/sting-mediated dna sensing (harding et al., ; mackenzie et al., ) . in addition, cellular dna can serve as a cgas/sting ligand following cellular senescence (defined as the senescence-associated secretory phenotype; sasp) (gluck et al., ; yang et al., ) . induction of sasp factors, like inflammatory cytokines and chemokines, may then reinforce senescent cells via autocrine and paracrine routes. similarly, during fertilization, sperm cell-derived dna can be found in oocyte cytoplasm may serve to activate cytoplasmic nucleic acid sensing (nas) pathway to induce inflammatory responses. thus, there are at least two contexts unrelated to infection where cytosolic responses must be repressed: during mitosis when chromosomal dna naturally exists in cytoplasm, and during fertilization when sperm-derived dna enters oocyte cytoplasm. such control can be achieved through the downregulation of molecules necessary for prompting responses to nucleic acids, and/or the upregulation of a negative regulators that act as molecular safeguards. in this review, we provide an update on cellular machinery that negative controls cytosolic sensing of dna and discuss therapeutic opportunities for the germ-cell specific nlr family member, nlrp , a recently identified inhibitor of dna-sensing during fertilization. in contrast to the identification of molecular machinery responsible for tlr and rna-sensing rig-like receptor (rlr) pathways, identification of a universally accepted cytosolic dna sensor and its related signaling pathway involved a more untidy trajectory (unterholzner, ) . since the existence of a cytosolic sensor for dna was first postulated (one that could elicit tlr-independent production of ifn to transfected double-stranded dna; dsdna) (ishii et al., ; stetson and medzhitov, ) , a great deal of effort has gone into revealing its identity. first, dnadependent activator of irf (dai; also referred to as dlm- /zbp ) was shown to participate in the activation of irf downstream of dsdna sensing (takaoka et al., ) . evidence suggests that following cellular exposure to synthetic dsdna or dna virus infection, dai can form a signaling complex together with tbk and irf to initiate production of type i ifns. however, reports also suggested that cells from dai-deficient mice did not exhibit impaired responses to synthetic b-form dsdna and dna genomes derived from bacteria (ishii et al., ) . ifi (ifn-γ-inducible protein ), a pyhin family member protein, was also shown to be involved in the recognition of synthetic dsdna and dna genomes of nuclearly replicating viruses (e.g., hsv- , kshv, hcmv, and ebv) (unterholzner et al., ) . in addition, ifi was also shown to participate in the dna damage response through promoting apoptosis and senescence, suggesting that such processes may mediate inflammatory diseases (unterholzner, ) . related to ifi is yet another putative cytosolic dna sensor, absent in melanoma (aim ), which, through an inflammasome dependent pathway, induces production and secretion of il- β and il- rather than type-i ifns after dna exposure (burckstummer et al., ; fernandes-alnemri et al., ; hornung et al., ) . more recently, aim -like receptors (alrs), consisting of members possessing a pyrin signaling domain and a dna-binding hin domain, have been shown to not contribute to type i ifn production downstream of dna sensing, nor to autoimmune diseases like as ags which are associated with responses to self-dna (gray et al., ) . the establishment of human ifi -deficient cells further illustrated competent type-i ifn responses to hcmv infection. nevertheless, ifi may participate in the recognition of cytosolic dna in a cell type and/or species-specific manner. an additional putative dna sensor, the ddx helicase, was identified through rnai screening and shown to be involved in dna recognition in immunocompetent cells rather than epithelial cells (zhang et al., ) . shortly after its discovery, ddx mediated signaling was shown to go through the bacterial-derived second messenger cyclic dinucleotide (cdn) molecules cyclic di-amp and cyclic di-gmp (parvatiyar et al., ) . solved crystal structures confirmed that the binding regions for these ligands overlapped, suggesting that ddx can recognize multiple ligands via the dead domain (omura et al., ) . of note, both ifi and ddx have been proposed to act upstream of sting (see below) through physical interactions. yet, their collaborative role in modulating cytosolic sensing of dna requires further investigation using genetically engineered mice. additionally, several dna damage inducible host factors like the catalytic subunit of dna-dependent protein kinase (dna-pkcs), and its binding co-factors ku / , or mre (meiotic recombination ) have also been implicated in cytosolic sensing through direct interactions with dna ligands (ferguson et al., ; kondo et al., ) . importantly, while dna-pkcs appear to have critical roles in dna-mediated immune responses, dna-pkcs deficient cells exhibit normal expression of many isgs upon stimulation with dna and dna virus infection. nonetheless, these observations reinforce the notion that dna damage response induces type i ifn production (brzostek-racine et al., ) . use of dna damage induced agents like , -dimethylbenz-α-anthracene (dmba) has helped shed light on the underlying events initiate the dna damage-induced immune response via cytosolic dna sensing pathway and implicates nucleosome leakage in eliciting cgas/sting-dependent signal activation via recognition of self-dna (barber, ) . finally, lrrfip , cytosolic protein, has been shown to recognize both dsdna and dsrna derived from pathogens, and may control the production of type-i ifns through the transcriptional co-activator β-catenin rather than irf -potentially acting as an amplifier of cytosolic nas (yang et al., ) . collectively, evidence indicates that while the long list of candidate cytosolic dna sensors may have redundant functions, they vary in ligand specificity as well as cell type and tissue distribution. between and , prior to the identification of several candidate dna sensors described the above, barber and colleagues reported that sting (stimulator of ifn genes, also referred as mita, mpys, or eris, and encoded by tmem ), an endoplasmic reticulum (er) localized protein consisting of multiple transmembrane regions, acts as an essential molecule for cytosolic sensing of dna (barber, ; ishikawa and barber, ; ishikawa et al., ) . several dna species appear to trigger sting-dependent signaling via tbk /irf and ikk/nf-κb axis in a length dependent manner and sting has also been attributed with a role in responses to plasmid-based vaccines and induction of long-term immunity. and though evidence suggests that sting may interact directly with dsdna, the physiological relevance of these observations remains to be explored (abe et al., ) . nevertheless, upon ligand stimulation, sting translocates from the er to the perinuclear-golgi region, forms a signaling complex with tbk and leads to phosphorylation and activation of irf . in addition, sting signaling appears to self-regulate by inducing protein degradation through a ubiquitin-mediated proteasome pathway and termination of signal transduction. a subsequent study indicated that sting dimers bind cyclic dinucleotides (cdns), suggesting that sting acts as the direct innate immune sensor for cdns (burdette et al., ) . other reports indicated that the type-i ifn and pro-inflammatory cytokine inducing flavonoid compounds, , -dimethylxanthenone- -acetic acid (dmxaa, also known as vadimezan or asa and initially identified as a potent tumor vasculature disrupting agent in mice), and -carboxymethyl- -acridanone (cma) may trigger sting dependent signal activation through direct interactions (prantner et al., ; roberts et al., ) . however, it was also known that these compounds displayed such sting activating properties in mice but not human cells. consistent with the unfortunate failure of these drugs in human clinical trials, these observations may be explained by structural differences in the dmxaa-binding sites across the human and mouse proteins (gao et al., ) . while the identification of sting undoubtedly contributed to our understanding of the underlying molecular events that control cytosolic dna-mediated immune responses, it was universally accepted that a sensor upstream of sting remained to be discovered. indeed, the breakthrough came in when chen and colleagues uncovered a role for cyclic gmp-amp (cgamp), and the catalytic enzyme (cyclic gmp-amp synthase (cgas), encoded by mb d and c orf ) in response to several cytosolic dna ligands (sun et al., ; wu et al., ) . cgamp is a cdns that consist of varying phosphodiester linkages (a -phosphodiester linkage and a canonical -phosphodiester linkage; the cgamp isomer is known as -cgamp) and cgas undergoes conformational rearrangement following direct binding to dna which leads to the synthesis of cgamp from cellular stores of atp and gtp. cgamp produced by cgas upon dna stimulation in turn acts as a cognate sting ligand to activate signaling and downstream production of type i ifn (barber, ) -it is interesting to note that similarly to other prrs (tlr, rlr and the inflammasome pathway) cgas activation of the sting pathway does not require direct interaction the sensor and adaptor that mediates signal transduction. importantly, the cgas/sting pathway plays a crucial role in the induction of autoimmune and inflammatory diseases (see section ), highlighting the need to properly control the dna sensing pathway for the maintenance of cellular homeostasis and immune response. taken together, while the functional and physiological relevance of several candidate dna sensors (e.g., ddx , ifi , and dna-pkcs) needs to be fully investigated, cgas and sting have emerged as bona fide players in cytosolic sensing of dna (fig. ). intracellular post-translational modifications (ptm), including phosphorylation, ubiquitination, and ubiquitin-like modifications like sumoylation and isgylation, glutamylation, acetylation or methylation, are critical in controlling cellular responses. these modulators of pathway activity participate in signal transduction by tuning enzymatic states, subcellular localization, protein stability and degradation, as well as protein-protein interactions. not surprisingly, recent evidence implicates several ptms in the cgas/sting pathway (summarized in fig. ). trim and trim , both e ubiquitin ligases, can positively regulate sting-dependent signal activation through conjugation of k -linked poly-ubiquitination of sting (trim at position k , and trim at k , k , and k (tsuchida et al., ; zhang et al., ) . similarly, k -linked poly-ubiquitination of sting at four lysine residues (k , k , k , and k ), mediated by e ubiquitin ligase complexes of amfr (autocrine motility factor receptor)-gp /insig (insulin-induced gene ), also leads to positive regulation of sting function (wang et al., ) . more recently, mul (mitochondrial e ubiquitin protein ligase ) was also shown to be responsible for k -linked poly-ubiquitination of sting at k , leading to specific enhancement of irf -dependent signaling but not nf-κb (ni et al., ) . conversely, two distinct ubiquitin ligases, rnf (ring finger protein ) and trim α (tripartite motif containing α), lead to termination of signal activation through k -linked polyubiquitination of sting at k and k which target it to subsequent degradation (wang et al., a; zhong et al., ). epstein-barr virus (ebv), a dna virus, coopts this regulatory property of the dna sensing pathway to evade host innate immune response. ebv induces expression of trim which in turn induces k -linked poly-ubiquitination of sting, and attenuation of sting-mediated antiviral response (xing et al., ) . proper regulation of signal transduction requires the poly-ubiquitin conjugation system to be reversible. indeed, inactive rhomboid protein (irhom ) contributes to stabilize sting protein by recruiting the deubiquitinating enzyme eif s (eukaryotic translation initiation factor subunit ) and de-conjugating rnf -mediated k -linked poly-ubiquitination (luo et al., ) . in addition, it appears that irhom may regulate the translocation of sting in response to dna through recruitment of er translocon-associated protein trapβ, which was previously identified as a sting-interacting protein (ishikawa and barber, ) . conversely, the ubiquitin specific protease (usp ) can counteract k -linked poly-ubiquitination and rnf -mediated k -linked poly-ubiquitination of sting, thereby facilitating sting-mediated signaling (zhang et al., ) . similarly, rnf catalyzes k -linked poly-ubiquitination of sting at the same conjugating site as rnf (k ), thereby preventing rnf -mediated poly-ubiquitination of sting, which positively regulates sting function (qin et al., ) . another ubiquitin specific protease, usp , also participates in this process, suggesting that collaboration between two distinct usps may catalyze de-ubiquitination of sting for signal activation. moreover, usp has also been shown to act as a de-conjugating enzyme of k -linked and k -linked poly-ubiquitination of sting, though the role of k -linked poly-ubiquitination of sting remains to be defined (sun et al., ) . nevertheless, de-ubiquitinated sting fails to form signaling complexes with tbk , thereby suppressing signal transduction, supporting a repressive role for usp . while multiple ubiquitin ligases have been implicated in directly regulating sting protein, much less is known about their modulatory role on cgas function. a recent report illustrated that the ubiquitin ligase rnf acts as a positive regulator of cgas via k -linked poly-ubiquitination at both k and k and may contribute to the enzymatic activity of cgas and promoting synthesis of cgamp (wang et al., a) . it was also reported that rnf expression is elevated in pbmcs of sle patients, suggesting that it could play a role in auto-inflammatory disorders. though an initial report described a role for trim in targeting sting for ubiquitination, it may also conjugate mono-ubiquitin to cgas at k (seo et al., ) . thus, negative regulation of cgas function by a ubiquitin ligase remains to be identified. in short, while the role of multiple lysine (k)-residues in sting protein has been implicated in regulating its activity, it remains unclear how ubiquitination of sting and cgas is regulated to orchestrate productive and appropriate immune responses. additionally, the biological significance and machinery regulating non-degradative poly-ubiquitin linkage (like k and k ) of sting remains to be fully resolved. recent studies have also implicated sumoylation in modulating signaling through the cgas/sting axis. the first report indicated that trim , an e ubiquitin ligase, may promote stabilization of cgas and sting by targeting them for sumoylation (hu et al., ) . in contrast, senp , a sumo-specific protease, was shown to mark these proteins for degradation via proteasomal and chaperone-mediated autophagy pathways. though cgas k and k (which are conserved between mouse and human), and sting k (corresponding to k in murine sting), were shown to regulate this process, a conflicting report suggested that sumoylation at k , k , and k residues of cgas suppressed functions including dna-binding, conformational rearrangement, and enzymatic activity-and that senp may reverse this suppression through catalyzing de-sumoylation of cgas (cui et al., ) . other ptms, including glutamylation, mediated by tubulin tyrosine ligaselike enzymes (ttlls), have also been shown to be involved in modulating cgas-mediated immune responses (xia et al., ) . poly-glutamylation and mono-glutamylation of cgas, mediated by ttll and ttll , respectively, negatively regulate cgas-mediated dna binding and enzymatic activity. conversely, the intracellular carboxypeptidases ccp and ccp counteract mono-glutamylation and poly-glutamylation of cgas, suggesting that cgas function is tightly regulated through cellular glutamylation and de-glutamylation. in addition to glutamylation, recent work also implicates palmitoylation, the covalent attachment of fatty acids (like palmitic acid) to cysteine (c) residues of a substrate proteins, in modulating sting (specifically, at positions c and c ) mediated signaling and type i ifn production downstream of dna sensing (mukai et al., ) . perhaps most recognized and best studied ptm is the phosphorylation of substrate proteins, and sting is no exception. recent studies have shown that sting possesses a number of serine (s) residues in the c-terminal region that can act as potential phosphorylation sites, though the primary phosphorylation site is serine- (s ) (konno et al., ; tanaka and chen, ) . while tbk , whose primary target is irf , was suggested to be involved in the phosphorylation of sting, in vitro experiments hinted otherwise (konno et al., ) . in addition, sting-phosphorylation and downstream degradation appear normal in tbk -deficient mouse embryonic fibroblasts stimulated with sting agonists, suggesting that if tbk does indeed target sting, its role is redundant (abe and barber, ) . subsequent studies reported that the autophagy-related serine/threonine protein kinases ulk and ulk as well as the ribosomal protein s kinase (s k ) are involved the phosphorylation of sting (konno et al., ; wang et al., ) . while multiple candidate sting kinases are being explored, only one has been implicated in directly controlling cgas function, and its identity came out of studies on hsv-host interactions. induced by hsv- , akt, also known as protein kinase b (pkb), phosphorylates cgas at s (s in murine cgas), thereby suppressing the synthesis of cgamp and attenuating cgas/sting-mediated antiviral responses (seo et al., ) . thus, hsv infection has coopted cellular machinery to negatively regulate cgas function and evade immune responses. taken together, while the ptms that control cgas/sting function remain to be fully defined, understanding the underlying network that controls these processes may contribute to the establishment of potent therapeutic approaches for tuning cgas/sting-mediated signaling (see section ). while cytosolic sensing of nucleic acids and induction of innate immune responses is critical for eliminating invading pathogens, inappropriate responses to dna from necrotic or apoptotic cells can result in the development of autoimmune diseases like sle, ags (characterized by high levels of anti-nuclear antibodies (ana) and high levels of circulating proinflammatory cytokines). to limit such aberrant inflammatory responses, vertebrates have evolved mechanisms to tightly control the availability of self dna and regulate inflammatory responses through cgas/sting dependent signaling (fig. ) . for example, mice lacking dnase ii, which acts in macrophages to degrade chromosomal dna derived from apoptotic cells, exhibit embryonic lethality due to anemia induced by the accumulation of undigested dna (kawane et al., ) . rescue of these mice through deletion of the type i ifn receptor, ifnar highlights the pathogenic potential of ifn, though aged mice develop tnfα-mediated severe polyarthritis (kawane et al., ) . additionally, mice lacking the three-prime repair exonuclease , trex (encoded by dnase iii), which degrades nicked dsdna as well as single-stranded dna, exhibit a significantly shortened lifespan due to constitutive activation of pro-inflammatory genes across multiple organs (yang et al., ) . similarly, using genetically engineered mice, dnase i has also been implicated in the development of sle (napirei et al., ) , though its role in nucleic acid sensing remains to be elucidated. importantly, the dramatic phenotypes observed in both dnase ii and trex deficient mice can be reversed through abrogation of cgas or sting gene expression, suggesting that the cgas/sting axis can initiate auto-inflammatory diseases (ahn et al., ; gall et al., ; gao et al., ; gray et al., ) . adding credibility to these studies, mutations in trex have also been implicated in ags and sle patients. furthermore, a recent report suggests that mutations in sting (specifically, n s, v m, and v l) may cause vascular and pulmonary syndrome (vaps), an auto-inflammatory disease characterized by abnormal inflammation across multiple tissues, including skin, vasculature, and lung (liu et al., ) -in vitro interrogation of these mutations revealed that they result in a gain-of-function phenotype and likely contribute to constitutive production type i ifn ( jeremiah et al., ) . furthermore, mutations in rnaseh , which degrades rna/dna hybrids, have also been implicated in the development of ags (crow et al., ) , and mutant mice possessing mutated human allele (g s) die perinatally (pokatayev et al., ) . collectively, these studies and observations highlight the importance of tightly regulating dna sensing and point to intracellular dnases like dnase ii and trex critical gatekeepers in controlling cgas/sting access to self-dna. strong selection pressure, coupled with high mutation rates and short generation times, has led to intricate strategies employed by viruses to counter host immune surveillance and establishment of infection. while the subject is more comprehensively covered by others, here we focus on a few examples from recent reports that highlight the roles of ptms and protein stability in regulating cytosolic sensing of dna (fig. ) . for example, the hepatitis b virus (hbv) polymerase suppresses cytosolic sensing of dna by interfering with k -linked poly-ubiquitination of sting (liu et al., ) . though the clinical significance of this observation remains to be resolved-given that hepatocytes lack functional sting dependent signaling (thomsen et al., ) -the data highlight the role of polyubiquitination in regulating sting function. indeed, other viruses have evolved machinery to modulate k -linked poly-ubiquitination of sting. the human coronavirus (hcov)-nl , severe acute respiratory syndrome (sars) cov, porcine epidemic diarrhea virus (pedv) papain-like protease, hepatitis c virus non-structural protein b (ns b), as well as the tax protein of human t lymphotropic virus type- (htlv- ), all suppress sting through either direct physical interactions or regulation of the polyubiquitination process ding et al., ; nitta et al., ; sun et al., ; wang et al., b; yang et al., ) . other examples include the human papillomavirus (hpv ) derived e protein and human adenovirus type- (had ) derived e a protein, both of which suppress cytosolic sensing of dna through direct interactions with sting (lau et al., ) . an lxcxe motif encoded in both proteins, and conserved among many dna tumor viruses, mediates disruption of sting-dependent signaling, suggesting a possible functional link between oncogenesis and antagonization of sting function. another oncogenic dna virus, kaposi sarcoma-associated herpesvirus (khsv) encodes a viral interferon regulatory factor (virf ) gene capable of preventing dna sensing via direct association with sting (ma et al., ) . additionally, though details remain to be resolved, the murine gammaherpesvirus (mhv ) encoded de-ubiquitination (dub) enzyme orf was shown to antagonize cytosolic sensing of dna (sun et al., ) . in addition to targeting sting (described above), the hcv employs the ns / a protease to target the mitochondrial adaptor ips- for cleavage, thereby short-circuiting the signaling cascade (chan and gack, ) . in a rather exquisite example of host restriction, dengue virus (denv), a mosquitoborne flavivirus which infects hundreds of millions of people annually, encodes the ns b/ns protease which cleaves human but not murine sting (aguirre et al., ) . importantly, as tbk mediated signaling is not suppressed by ns b/ns in murine cells, mice produce higher levels of type i ifns and effectively restrict denv replication. though far fewer examples exist of viral targeting of cgas, recent reports suggest that capsid proteins of hiv- and hiv- may dampen cgas function by recruiting cellular cpsf and cyclophilin-a (lahaye et al., ; rasaiyaah et al., ) . what is clear is that understanding virus-mediated immune evasion strategies can provide critical insights into regulatory functions that control cellular responses to dna in the cytosol. the nucleotide-binding domain leucine-rich repeat-containing receptor (nlr) family contains five subfamilies that are subclassified according to the composition of domains in their n-termini-the nlrc family contains a caspase activation and recruitment domain (card), and the nlrp family contains a pyrin domain (pyd). nlrs are known for their role in triggering caspases to cleave pro-il- β and il- into mature proinflammatory cytokines in response to various pathogen associated molecular patterns (pamps) and danger associated molecular patterns (damps) (saxena and yeretssian, ; zhong et al., ) . however, recent advances have revealed that some members of the nlr family may also regulate tissue homeostasis and modulate innate immune signaling pathways (kufer and sansonetti, ; saxena and yeretssian, ; zhong et al., ) . several nlrps (including nlrp , , , , , and ) are expressed primarily in mammalian oocytes, and nlrp , nlrp , and nlrp have been shown to have important roles in reproduction and development (mcdaniel and wu, ; murdoch et al., ; tong et al., ; westerveld et al., ; zhang et al., ) . additionally, nlrc and nlrc , as well as several nlrps, have been demonstrated to function as negative regulators of prrs (table ) . for example, nlrp suppresses negative regulator of tlr pathway cui et al. ( ) both tlr and tnfα-mediated nf-κb activation at the level of ikk complex formation (bruey et al., ; fontalba et al., ) . similarly, nlrp inhibits rig-i signaling by recruiting dtx , an e ubiquitin ligase which marks tbk for degradation (cui et al., ) . nlrp and nlrp suppress nf-κb signaling and are involved in the maintenance of intestinal homeostasis and tumorigenesis (allen et al., ; anand et al., ; lupfer and kanneganti, ; zaki et al., )-more recent reports also point to a role for nlrp in anti-viral immunity in mouse intestines (wang et al., b) . nlrx , the only mitochondrially localized member of this family, modulates sensing of dsrna and virus-induced ros production (guo et al., ; ma et al., ; moore et al., ; qin et al., a; tattoli et al., ) . in collaboration with iqgap , nlrc suppresses dna sensing lymphoid cells, myeloid cells, and epithelial cells through interactions with sting (tocker et al., ; zhang et al., ) . in addition, nlrc can suppress tlr signaling by modulating ikk activation (cui et al., ) , though nlrc deficiency does not affect this signaling pathway (kumar et al., b) . finally, the primate specific nlrp suppresses tlr-mediated activation of nf-κb by targeting traf for degradation in myeloid cells and b-cells (ellwanger et al., ; qin et al., b; wu et al., ) . together, these emerging properties call into question whether the established role for this family in activating proinflammatory responses may in fact represent an exception rather than a rule. as highlighted above, inappropriate activation of the nas pathway can result in highly destructive immune responses. yet, molecular dampeners, structural determinants and topological barriers described above as well as novel checks and balances yet to be discovered tightly regulate the sting/cgas axis and defend against such circumstances. one instance when distinguishing between self and non-self dna is particularly important is fertilization, when sperm cell derived dna can be found in oocyte cytoplasm. yet, while sting/cgas and rest of the nas pathway (which are all expressed in oocytes) are afforded access to this foreign dna, a nucleic acid sensing response is not triggered. in a recent publication, abe and colleagues reasoned that germ cells must therefore possess a robust mechanism to negatively regulate cytosolic nucleic acid sensing (abe et al., ) . through a combination of data-mining and experimental approaches, the group identified candidate genes that may serve as putative regulators of nas in oocytes. among these, four (trim , tgif lx, c orf , zpld ) significantly suppressed cgas-mediated signaling, and one, nlrp (nacht, lrr and pyd domains-containing ; a component of the inflammasome family of proteins), shut off nas virtually all together. what followed were a series of observations that delineate a role for nlrp as a rheostat for nucleic acid sensing, highlight the importance of controlling innate immune responses to foreign and endogenous ligands, and suggest that tight regulation of these processes is critical in maintaining proper immunologic homeostasis in germline. specifically, the authors demonstrate that nlrp associates with both sting and mavs and prevents downstream signal transduction (fig. ) . in turn, these adaptor degradation of lrr activation nlrp fig. nlrp -mediated inhibition of cytosolic nucleic acid sensing. upon activation of sting dependent signaling, conformational rearrangement from inhibitory-form to an active-form of nlrp is induced. targeting of tbk for k -linked poly-ubiquitination is then initiated and results in proteasome dependent degradation of tbk and termination of sting dependent as well as rig-i dependent signaling. similarly, nlrp is targeted for degradation to avoid persistent immunosuppression. abbreviations: nlrp , nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein ; pyd, pyrin domain; nacht, naip (neuronal apoptosis inhibitory protein), ciita (mhc class ii transcription activator), het-e (incompatibility locus protein from podospora anserine) and tp (telomerase-associated protein), lrr, leucine-rich repeat; ub, ubiquitin; p, phosphorylation. molecules induce the proteasomal degradation of nlrp , a feedback loop that may be critical in preventing persistent immunosuppression and proper induction of innate immune responses under appropriate conditions (for example, post fertilization of oocytes). importantly, several human diseases, including crohn's disease, celiac disease, blau syndrome, rheumatoid arthritis, type- and - diabetes, sle, cryopyrin-associated periodic syndromes (caps), inflammatory bowel diseases, colitis as well as colon cancer, have been shown to be associated with mutations in nlrps (saxena and yeretssian, ; zhong et al., ) . other nlrps (like nlrp , nlrp , and nlrp ) have been associated with miscarriage and infertility (docherty et al., ; huang et al., ) . in line with these observations ectopic expression of a k x nlrp allele (rs ; coding for a nonsense mutation that introduces an early stop codon in nlrp ) results in reduced suppression of tbk -mediated signaling (abe et al., ) . with an allele frequency of . % in the human population, and a minor allele frequency of % in east asian and ad mixed american populations, infertility associated with homozygosity of this gene may affect in , individuals. as an immunological rheostat, nlrp safeguards against inappropriate cytosolic responses to nucleic acids and the acquisition of such a function may have been a prerequisite to sexual reproduction. indeed, homologs of the inflammasome family of proteins have been found widely across life-arguing in favor of their involvement in immunity and broader cellular processes, as is observed with other expanded gene families. the past decade has seen rapid advancement in our understanding of how cells sense cytosolic dna, including the identification of receptors and molecular machinery that participate in this process as well as recognition of multiple diseases associated with this pathway. recent findings highlight the importance of controlling innate immune responses to dna and suggest that tight regulation of these processes is critical in maintaining proper immunologic homeostasis across multiple organs. thus, understanding the underlying network of regulators that control signaling through the cgas/sting axis will undoubtedly contribute to the development of potent therapeutic agents against auto-immune and inflammatory diseases, as well as certain cancers. cytosolic-dna-mediated, sting-dependent proinflammatory gene induction necessitates canonical nf-kappab activation through tbk sting recognition of cytoplasmic dna instigates cellular defense germ-cell-specific inflammasome component nlrp negatively regulates cytosolic nucleic acid sensing to promote fertilization denv inhibits type i ifn production in infected cells by cleaving human sting sting manifests self dna-dependent inflammatory disease nlrp suppresses colon inflammation and tumorigenesis through the negative regulation of noncanonical nf-kappab signaling nlrp negatively regulates innate immunity and host defence against bacterial pathogens sting-dependent cytosolic dna sensing pathways sting: infection, inflammation and cancer the birds, the bees, and innate immunity newly described pattern recognition receptors team up against intracellular pathogens pan /nalp /pypaf , an inducible inflammatory mediator that regulates nf-kappab and caspase- activation in macrophages the dna damage response induces ifn an orthogonal proteomic-genomic screen identifies aim as a cytoplasmic dna sensor for the inflammasome sting is a direct innate immune sensor of cyclic di-gmp viral evasion of intracellular dna and rna sensing role of nlrp and nlrp in the maintenance of intestinal homeostasis sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex mutations in genes encoding ribonuclease h subunits cause aicardi-goutières syndrome and mimic congenital viral brain infection intracellular nucleic acid detection in autoimmunity nlrc negatively regulates the nf-kappab and type i interferon signaling pathways nlrp negatively regulates type i interferon signaling by targeting the kinase tbk for degradation via the ubiquitin ligase dtx senp potentiates cgas activation by relieving sumo-mediated inhibition of cytosolic dna sensing cgas conducts micronuclei dna surveillance hepatitis c virus ns b blocks the interaction of sting and tbk to evade host innate immunity mutations in nlrp are associated with reproductive wastage and multilocus imprinting disorders in humans the nlr family pyrin domain-containing protein contributes to the regulation of inflammatory signaling dna-pk is a dna sensor for irf- -dependent innate immunity aim activates the inflammasome and cell death in response to cytoplasmic dna nlrp , an inhibitor of the nf-kappab pathway, is transcriptionally activated by nf-kappab and exhibits a nonfunctional allelic variant autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease structure-function analysis of sting activation by c[g( , )pa( , ) p] and targeting by antiviral dmxaa activation of cyclic gmp-amp synthase by self-dna causes autoimmune diseases innate immune sensing of cytosolic chromatin fragments through cgas promotes senescence cutting edge: cgas is required for lethal autoimmune disease in the trex -deficient mouse model of aicardi-goutières syndrome the aim -like receptors are dispensable for the interferon response to intracellular dna nlrx sequesters sting to negatively regulate the interferon response, thereby facilitating the replication of hiv- and dna viruses mitotic progression following dna damage enables pattern recognition within micronuclei aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc sumoylation promotes the stability of the dna sensor cgas and the adaptor sting to regulate the kinetics of response to dna virus a genetic association study of nlrp and nlrp genes in idiopathic recurrent miscarriage a toll-like receptor-independent antiviral response induced by double-stranded b-form dna tank-binding kinase- delineates innate and adaptive immune responses to dna vaccines sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity inherited sting-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations requirement of dnase ii for definitive erythropoiesis in the mouse fetal liver chronic polyarthritis caused by mammalian dna that escapes from degradation in macrophages multi-receptor detection of individual bacterial products by the innate immune system dna damage sensor mre recognizes cytosolic double-stranded dna and induces type i interferon by regulating sting trafficking cyclic dinucleotides trigger ulk (atg ) phosphorylation of sting to prevent sustained innate immune signaling nlr functions beyond pathogen recognition pathogen recognition by the innate immune system nlrc deficiency does not influence cytokine induction by virus and bacteria infections the capsids of hiv- and hiv- determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway activated sting in a vascular and pulmonary syndrome hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dna-sensing pathways irhom is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting unsolved mysteries in nlr biology modulation of the cgas-sting dna sensing pathway by gammaherpesviruses nlrx negatively modulates type i ifn to facilitate kshv reactivation from latency cgas surveillance of micronuclei links genome instability to innate immunity bak/bax macropores facilitate mitochondrial herniation and mtdna efflux during apoptosis identification of oocyte-selective nlrp genes in rhesus macaque monkeys (macaca mulatta) nlrx is a regulator of mitochondrial antiviral immunity activation of sting requires palmitoylation at the golgi mutations in nalp cause recurrent hydatidiform moles and reproductive wastage in humans features of systemic lupus erythematosus in dnase -deficient mice ubiquitination of sting at lysine controls irf activation hepatitis c virus ns b protein targets sting and abrogates rig-imediated type i interferon-dependent innate immunity structural and functional analysis of ddx : a bispecific immune receptor for dna and cyclic dinucleotide the helicase ddx recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response rnase h catalytic core aicardi-goutières syndrome-related mutant invokes cgas-sting innate immune-sensing pathway in mice -dimethylxanthenone- -acetic acid (dmxaa) activates stimulator of interferon gene (sting)-dependent innate immune pathways and is regulated by mitochondrial membrane potential rnf temporally regulates virus-triggered type i interferon induction by two distinct mechanisms nlrx mediates mavs degradation to attenuate hepatitis c virusinduced innate immune response through pcbp nlrp disrupts mavs signalosome to inhibit type i interferon signaling and virus-induced apoptosis hiv- evades innate immune recognition through specific cofactor recruitment the chemotherapeutic agent dmxaa potently and specifically activates the tbk -irf- signaling axis apoptotic caspases prevent the induction of type i interferons by mitochondrial dna nod-like receptors: master regulators of inflammation and cancer akt kinase-mediated checkpoint of cgas dna sensing pathway trim -mediated monoubiquitination of cgas for cytosolic dna sensing recognition of cytosolic dna activates an irf -dependent innate immune response coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway evasion of innate cytosolic dna sensing by a gammaherpesvirus facilitates establishment of latent infection usp negatively regulates antiviral responses by deubiquitinating sting dai (dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response sting specifies irf phosphorylation by tbk in the cytosolic dna signaling pathway nlrx is a mitochondrial nod-like receptor that amplifies nf-kappab and jnk pathways by inducing reactive oxygen species production lack of immunological dna sensing in hepatocytes facilitates hepatitis b virus infection the scaffolding protein iqgap interacts with nlrc and inhibits type i ifn production mater, a maternal effect gene required for early embryonic development in mice the ubiquitin ligase trim regulates innate immune responses to intracellular double-stranded dna the interferon response to intracellular dna: why so many receptors? ifi is an innate immune sensor for intracellular dna the e ubiquitin ligase amfr and insig bridge the activation of tbk kinase by modifying the adaptor sting trim alpha is a negative-feedback regulator of the intracellular dna and dna virus-triggered response by targeting sting nlrp regulates intestinal antiviral innate immunity s k-sting interaction regulates cytosolic dna-mediated activation of the transcription factor irf the e ubiquitin ligase rnf facilitates the cgas-mediated innate immune response htlv- tax impairs k -linked ubiquitination of sting to evade host innate immunity mutations in the testis-specific nalp gene in men suffering from spermatogenic failure apoptotic caspases suppress mtdna-induced sting-mediated type i ifn production innate immune sensing and signaling of cytosolic nucleic acids cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna nlrp attenuates toll-like receptor signalling by targeting traf for degradation via the ubiquitin ligase rnf a glutamylation of the dna sensor cgas regulates its binding and synthase activity in antiviral immunity trim promotes dna virus infections by inhibiting innate immune response trex exonuclease degrades ssdna to prevent chronic checkpoint activation and autoimmune disease the cytosolic nucleic acid sensor lrrfip mediates the production of type i interferon via a beta-catenin-dependent pathway proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease cgas is essential for cellular senescence the nod-like receptor nlrp attenuates colon inflammation and tumorigenesis expression analysis of the nlrp gene family suggests a role in human preimplantation development the helicase ddx senses intracellular dna mediated by the adaptor sting in dendritic cells trim protein modulates type i interferon induction and cellular antiviral response by targeting mita/sting protein for k -linked ubiquitination nlrc , a member of the nlr family of proteins, is a negative regulator of innate immune signaling induced by the dna sensor sting usp recruits usp to promote innate antiviral response through deubiquitinating sting/mita the ubiquitin ligase rnf regulates antiviral responses by mediating degradation of the adaptor protein mita functions of nod-like receptors in human diseases we apologize to authors whose work could not be cited due to space constraints and narrow focus of the review. s.d.s. acknowledges funding from nih r (gm - ) and nih u (ca - ). key: cord- - ikkl hk authors: wilson, christopher; jumbert, maria gabrielsen title: the new informatics of pandemic response: humanitarian technology, efficiency, and the subtle retreat of national agency date: - - journal: nan doi: . /s - - - sha: doc_id: cord_uid: ikkl hk digital communication technologies play an increasingly prominent role in humanitarian operations and in response to international pandemics specifically. a burgeoning body of scholarship on the topic displays high expectations for such tools to increase the efficiency of pandemic response. this article reviews empirical uses of communications technology in humanitarian and pandemic response, and the ebola response in particular, in order to propose a three-part conceptual model for the new informatics of pandemic response. this model distinguishes between the use of digital communication tools for diagnostic, risk communication, and coordination activities and highlights how the influx of novel actors and tendencies towards digital and operational convergence risks focusing humanitarian action and decision-making outside national authorities’ spheres of influence in pandemic response. this risk exacerbates a fundamental tension between the humanitarian promise of new technologies and the fundamental norm that international humanitarian response should complement and give primacy to the role of national authorities when possible. the article closes with recommendations for ensuring the inclusion of roles and agency for national authorities in technology-supported communication processes for pandemic response. recent decades have seen a dramatic rise in global pandemics. from the sars pandemic in , to avian influenza in , h n in , ebola in , and the appearance of the zika virus in latin america in , these developments are inextricably bound up in modern socio-technical developments and processes of globalization. advances in global air travel, agricultural technology, urbanization, and pollution all facilitate the appearance and spread of contagious diseases (see wolfe ; ramalingam ) . simultaneously, new media and technologies have also come to play a profound role in the way that global pandemics are identified, traced, understood, managed, treated, and perceived. digital communication technologies play an increasingly significant role in different aspects of global pandemic response, presenting novel opportunities to mitigate risks and enhance response efficiency. in doing so, they also confound traditional domains of information and communication practices in pandemic response (mager ) and introduce a novel collection of international and transnational actors to areas that have traditionally been the purview of national authorities. the capacity of digital communications tools to process, systematize, and make sense of large amounts of data has attracted the attention of practitioners, policy makers, and scholars alike (brownstein et al. ; tusiime and byrne ; wesolowski et al. ; zwitter and hadfield ; meier ; holeman et al. ) , and has raised significant expectations regarding their use in pandemic response in particular (odugleh-kolev ) . these expectations are countered by an emerging critical scholarship concerned with the novel risks that accompany humanitarian technologies (sandvik et al. (sandvik et al. , comes ) , how communication technologies impact power relationships between national and international actors in humanitarian crises (burns ; letouzé et al. ; mcdonald ) , and the ways in which new technologies are both constituted by, and contribute to reshaping social practices in a given society (amicelle et al. ) . with the exception of work by ihlen and levenshus' ( ) on technology in crisis and risk management and roberts and elbe's ( : ) on syndromic surveillance systems, however, the role of digital communication tools in pandemic response has received scant critical attention. this article contributes to filling that gap by conceptualizing the broad variety of ways in which digital communication technologies are brought to bear in global pandemic response. a three-part conceptual framework for informatics is constructed on the basis of empirical examples from recent pandemic responses and contemporary policy debates, drawing on the ebola response in particular. this model highlights how the application of new communication technologies in pandemic response is often accompanied by an influx of novel actors and convergence of previously distinct activities within single technological platforms or institutional operations. applying a critical reading of knowledge politics to these dynamics emphasizes the potential of new technologies to complicate global pandemic response, and the associated risk of relocating decision-making and agency outside of national authorities' spheres of influence. this article is organized in five sections. following this introduction, a second section describes the literature upon which the conceptual framework for pandemic informatics is developed. the third section presents that framework and describes each of its three components in detail. the fourth section notes two underlying dynamics that are consistently cited in conjunction with the use of digital communications in pandemic response: the introduction of novel actors and convergence of activities in single technological platforms and across organizational entities. the final section concludes by noting the significant challenges that this poses for effective coordination of pandemic response between national and international actors, and elaborates on the tendency of technologically driven informatics to decrease national authorities' sphere of influence in pandemic response. reflecting on seminal and watershed humanitarian policy for humanitarian coordination, the article concludes by suggesting that the roles and authority of national actors be explicitly designed in workflows for internationally coordinated pandemic response, and identifies preliminary measures through which this might be pursued. this conceptual exercise draws on descriptions of digital technology deployment in several contexts and from a variety of sources. in addition to academic research, this includes liberal reference to the so called "grey literature" produced by humanitarian implementing agencies, think tanks and volunteer groups, as well as descriptions of humanitarian communication in popular press and in online media maintained by civil society organizations and other commentators. while such sources are not subject to significant quality or review standards, and often lack the detail found in other types of literature, they tend to describe a much wider variety of activities, and do so without the significant time lag that accompanies peer reviewed research. often, activities and initiatives described in organizational reports, popular media, or ngo blogposts represent novel combinations of activities only anticipated in scholarly work and add a depth and richness to the scope of activity conceptualized in this article. the current analysis is grounded in response to the ebola pandemic, as arguably the most globally active and thoroughly documented example of pandemic response. accordingly, second section uses examples of digital technology innovation from the ebola response to construct a preliminary framework, with an emphasis on the incentives and objectives for using technological tools and strategies. the third section will then present each component of that preliminary framework in detail, using examples from other pandemics and from response policy more generally to validate and refine the framework. a great deal has been written about how communication technologies were used in the global response to west african ebola, including academic articles (odugleh-kolev ; tulenko ; sacks et al. ; sandvik et al. ; harman and wenham ) , assessments of multinational and non-governmental response (acaps ; adams et al. ; dubois et al. ; smith ) , and case studies documenting specific instances of technology use or country communication processes (nethope global broadband and innovations alliance ; acaps ; levine et al. ) . the most comprehensive overview is provided by fast and waugaman's ( ) -page report for usaid, "fighting ebola with information," which provides a starting point for this article's conceptual framework. after briefly describing the types of activities covered in fast and waugaman's report, an argument is made for re-categorizing and assessing those activities according to the objectives they pursue, which provides an initial basis for the framework developed in this article. fast and waugaman provide a thorough account of how information and technology were mobilized in the west african ebola response, based on case analysis, literature review, and over interviews. their analysis includes a typology of technology tools commonly utilized in the response ( - ) and nine in-depth case studies of specific information flows that relied on digital tools to facilitate response ( - ). these case studies include descriptions of activities, technological tools, actors involved, outcomes, challenges, and objectives and provide a useful starting point for drawing conclusions about the breadth of activities utilizing digital communications tools. fast and waugaman organize and present their case studies by virtue of the differences the integration of digital technologies enabled, such as increasing the diversity of information flows (e.g. "up" for data collection, as well as horizontally among peer groups, and back "down" through feedback loops) among a greater plurality of actors (e.g., frontline health workers, citizens, governments, and "remote" responders) ( ). the organizing principle here is a spatial understanding of coordination across pandemic response. connecting and coordinating actors that would otherwise not have exchanged information is certainly one of technology's most prominent contributions. a close read of these case studies, however, reveals a number of additional functions and objectives and allows for a more nuanced assessment of their impact. the liberian sms-based initiative, mhero, for example, was structured not only to connect frontline workers and governments, but also explicitly "aimed to strengthen the government's health information system […] and to provide critical information to support health workers on the frontlines of the crisis" ( ). the ebola community action platform (ecap) did not aim only to connect response organizations and extension workers, but more accurate information on the state of contagion and response was quickly leveraged towards public health messaging via billboards, radio, posters, handouts, and "person-to-person drama activities at the community level" ( ). these examples are notable not only for the breadth of actors they engage, but for the deliberate piggybacking of novel objectives and efficiencies, and the same dynamic is discernable across all of fast and waugaman's nine case studies. this matters. the way in which objectives are articulated and claimed has consequences for how responsibilities are allocated and priorities made in humanitarian response (burns ; mcdonald ) . in much the same way as the power asymmetries implied by humanitarian technology are often eclipsed by a presumption of technology's democratizing potential to re-distribute power (sandvik et al. : ) , fast and waugaman's preliminary mapping of digital information flows posits connectivity as an immediate and obvious benefit to pandemic response, without assessing the ways in which it asserts and reinforces power relationships. as the sociologists would have it, however, technological tools and political instruments "are less inert intermediaries than partly autonomous actants that contribute to orientating actors' behaviours" (amicelle et al. ) . novel connectivity in pandemic response inevitably structures relationships of power and responsibility in a messy field, asserting and assigning influence and roles in humanitarian response. critical scholars have described these dynamics as "knowledge politics" (burns ). the degree to which actors are able to influence these knowledge political assertions and assignments, we term informatics discretion, relies significantly on their participation, capacity and expertise in the types of information and communication modalities at issue (elwood ) . reconceptualizing digital information flows in pandemic response according to their objectives allows for a close reading of the ways in which this occurs and the implications it has for pandemic response coordination. a careful review of fast and waugaman's nine case studies suggests at least three broad categories of objectives that manifest themselves consistently across different modes of connectivity. digital communications in fast and waugaman's case studies are leveraged to determine the way in which ebola was spreading and the nature of risks posed by the pandemic, in order to coordinate activity among different types of response actors, including national authorities, international humanitarian aid workers and front-line health care providers, and in order to communicate with the general public regarding health risks and appropriate behavior to mitigate those risks. in order to test and refine these broad categories, they were assessed in the context of the broader literature on west african ebola response, and in the broader context of pandemic response and humanitarian policy, including grey literature and popular media, as described above. convenience sampling and citation tracing were used to identify relevant academic literature, while scans of the websites of civil society groups identified in academic research were used to identify sources in grey literature and popular press. when distinct objectives or efficiencies were described in the context of digital information flows, these were grouped according to the above categories. those categories were then refined into a three-part conceptual model of informatics in pandemic response, which is described in the following section. the previous section's review of objectives and efficiencies driving the use of digital communication technologies in ebola response suggested three broad categories: (a) diagnostic efforts, through which the characteristics and spread of infectious diseases are assessed in order to inform treatment and response; (b) risk communication practices, through which communities and individuals are informed about pandemic risks in an effort to mitigate those risks and curb contagion; and (c) coordination processes, through which different actors involved in pandemic response are allocated roles and responsibilities, in an attempt to maximize the efficiency of their work and avoid superfluous or parallel efforts. this section provides detailed description of these categories, validated and refined through reference to other contexts. it should be noted that some of these categories recall established fields of study. risk communication and coordination activities are relatively well delineated in scholarly work and policy documents (see plough and krimsky ; akl et al. , respectively) , while our understanding of diagnostic efforts combines a variety of activities occurring across the spectrum of response, from health surveillance, to the identification of the pandemic and clarification of symptoms, to contact tracing and contagion modeling. it should be emphasized that our objective here is not to recount or re-conceptualize established fields of practice, but rather to sketch the different mechanisms and patterns through which technology and information are deliberately employed, and to question the consequences this has for governance and power relationships in pandemic response more generally. this first component groups diagnostic activities related to collecting, systematizing, and processing information about a disease outbreak, and mapping its spread and associated needs, which we term diagnostics. this constitutes diagnosis at a societal level, and should not be confused with the medical diagnosis of individuals. the analysis of information has been central to understanding, anticipating and responding to infectious diseases at least since john snow became the "father of modern epidemiology" by drawing dots around a map of london water pumps during the cholera epidemic in (hempel ) . in the humanitarian context, collecting information is central to the implementation of an efficient response, including situational information, needs assessment, and operational information (king ; van de walle et al. ). this corresponds with humanitarian practice which has traditionally assumed the "information imperative" to be central to the humanitarian imperative-that is, the need to collect as much relevant information as possible, to enhance evidence for decision-making basis and improve efforts to assist people suffering in crisis contexts (bui et al. ; darcy and hofmann ; miller et al. ; saab et al. ) . scholars have explored a number of ways in which technology improves humanitarian diagnostics, including the use of mobile phone network data for human mobility mapping and contact tracing (tatem et al. ; aslam et al. ; wesolowski et al. ; gittelman et al. ; bharti et al. ; bengtsson et al. ) , and big data or social media scraping for contagion modeling (brownstein et al. ; chunara et al. ) . such techniques allow humanitarian actors to extract information, diagnosing needs and epidemiological trends without direct contact with affected populations. other applications of digital media for diagnostic activities introduce completely different actors. novel initiatives in response to the ebola pandemic in - include hackathons organized in western capitals to map resources for west african response, and western non-governmental organizations (ngos) that provide communication-based outbreak models to multinational humanitarian organizations (sangokoya ; moore ; dittus et al. ) or build geographic information system (gis) maps for national authorities (timo lüge ). early work is even underway to develop artificial intelligence responses to combat the spread of infectious diseases, by using multiple sources of publically available data to algorithmically predict the appearance and spread of disease (barron ). such innovative approaches are compelling and have attracted an understandable amount of interest and optimism, not in the least due to perceived gains in efficiency. novel collaborations and web-based health surveillance systems have regularly been quicker than the world health organization (who) to publish reports on epidemic outbreaks (anema et al. (anema et al. : (anema et al. - , and jennifer gardy, a senior scientist at the british columbia centre for disease control, has argued that epidemics are always essentially a race between the spread of info and spread of virus, and that technology has given scientists a critical edge (edmunds ) . whether or not such approaches are inherently more efficient, they are noteworthy for their inclusion of novel actors. this is perhaps most apparent in web-based initiatives such as wiki systems, which are used to facilitate collaborative pandemic modeling among geographically disparate scientists (kno.e.sis ), or the humanitarian data exchange, which through the leadership of a traditional humanitarian agency, was used to coordinate data sharing between a diverse group of actors, including local ngos and remote volunteer mapping communities (verhulst ; fast and waugaman : ) . though not solely dependent on digital media, a similar dynamic is visible in the proliferation of global health networks, such as goarn (global outbreak alert and response network) and promed (program for monitoring emerging diseases), which combine the efforts of national and international civil society and regulatory bodies, to surveil health risks internationally through a mix of traditional and digital media, and which to some degree eclipse the traditional roles of international and national health regulatory bodies (ramalingam : - ) . the second component we identify is risk communication, which we understand as the processes of communicating the risks associated with a pandemic outbreak, primarily to relevant and potentially affected publics. it is a widely recognized field of practice defined by the american national academy of sciences as: an interactive process of exchange of information and opinion among individuals, groups, and institutions. it involves multiple messages about the nature of risk and other messages, not strictly about risk, that express concerns, opinions, or reactions to risk messages or to legal and institutional arrangements for risk management. (cited in covello et al. : - ) . the interactive character referenced in that definition is the subject of some debate. in their handbook on global health communication, editors waisbord and obregon argue that the field of global health communication is characterized by a theoretical split, which places a preference for behavior change and unidirectional approaches against critical theories of participatory engagement (waisbord and obregon : - ). civil society and practitioner rhetoric tends to link participatory communicative models with technological advances, framed in normative terms that are highly critical of traditional, less participatory approaches in the humanitarian and development sectors (chao ) . this normative perspective is also assumed by several scholars (kaiser ; abraham ; maxwell et al. ; gillman ; Özdamar and ertem ; madianou et al. ) , and participatory approaches to risk communication have been argued to improve outcomes and efficiency. odugleh-kolev ( ) argues that a structural and interactive understanding of risk communication is particularly important in pandemic response, where coordinated, functional, and systemic communication is implied in activities such as assessing transmission risk and mapping contagion patterns in rural communities ( ). participatory approaches to risk communication tend to incorporate diagnostic activities, since digital media technologies facilitate simultaneous broadcast and collection of humanitarian information, at marginal cost, as will be discussed below. for clarity, this analysis focuses on risk communication implemented nationally or sub-nationally, targeting communities potentially affected by pandemics, and disregard the international role of for instance social media in risk communication. in doing so, we can identify at least three ways in which digital media are used intentionally to bolster risk communication strategies. again, responses to the ebola outbreak of are illustrative: firstly, digital media was expected to expand the reach and interactivity of risk communications. the use of new media technologies, particularly mobile phones, is often expected to dramatically increase the reach of risk communication to rural and remote communities, as well as front line health providers. mobile penetration rates in developing countries were estimated to have surpassed % at the time of the ebola outbreak (wesolowski et al. ) , and a review of ebola response in nigeria cited mobile-delivered training to health workers as "vital" for promptly declaring the country "free of ebola" (west : ). digital media also allow for scaled co-creation of health communication content, such as programs to produce videos together with youth infected with ebola in sierra leone, as a means of addressing stigmatization and developing trust networks among affected populations (zuckerman ). secondly, digital media allow for feedback and interaction to be integrated into risk communications. a project called u-report, led by the united nations children's fund (unicef), combines mobile phone-based surveys with opportunities for user feedback and was used to channel infection reports and concerns regarding public health activities in liberia during the ebola outbreak (muah et al. ) . similarly, the international conglomerate ibm combined outgoing radio communications with interactive short messaging system (sms) functionality to implement interactive risk communications surrounding ebola in sierra leone (bell, ) . some studies suggest that these affordances were particularly effective when targeting health communication to specific sub-groups (ems and gonzales ) . finally, digital media are sometimes expected to obviate institutional and resource limitations on the development of media platforms and content production for public health authorities. that digital media is faster and cheaper to produce and disseminate than paper is widely recognized. in addition, participatory content creation such as the sierra leone video program described above can effectively outsource some degree of communications work, as do content creation efforts which move beyond specific communities and aim to engage "the crowd" in generating content. such approaches have been pursued in generating both platforms and content for risk communication, either through international volunteer communities or institutionalized global health networks, or initiated by third parties, as in a series of hackathons organized in new york to develop mobile apps for spreading information about ebola in affected countries (sangokoya ) . the third component we identify is coordination, which is a persistent challenge and polemic in the humanitarian sector (stephenson ; bisri ). the introduction of new actors and new technologies promise to mitigate this challenge, even as they contribute to it in novel ways. ramalingam ( ) notes that the contemporary governance system for international diseases incorporates at least five different types of actors (intergovernmental organizations, national governmental organizations, non-governmental organizations, private foundations, and public/private partnerships and consortia), whose activities are marked by competition and lack of collaboration (ibid - ). he argues that this institutional disarray, when coupled with other socio-economic developments and trends of globalization, poses a threefold governance challenge to global health: the challenge of capitalizing on the diversity of actors, the challenge of bringing traditional actors up to speed with innovative organizational and technical approaches, and balancing the incentives and responsibilities of individual countries in treating diseases that do not respect borders (ibid ). simultaneously, timely, accurate, and appropriate information is widely regarded as a cornerstone for effective humanitarian coordination (bui et al. ; miller et al. ; saab et al. ) and digital media are often expected to dramatically enhance the coordination potential of information (moss and townsend ) . the most remarkable applications of digital media to coordination informatics for pandemic response are likely the creation and activation of global health networks, mandated to surveil infectious diseases internationally and alert regarding their outbreak. these networks have at least partially filled a coordination gap for identifying and initiating responses to global pandemics (see burkle et al. ) , but coordination in the implementation of specific response remains largely lacking. volunteer and technical communities (v&tcs) represent another prominent example of the coordination challenges posed by novel actors in humanitarian response. volunteer communities are composed of thousands of individuals around the globe who are moved to contribute time and energy to humanitarian response efforts virtually and remotely, often by collecting or processing humanitarian information such as incident reports or spatial data over social media. though different communities vary significantly in their membership, degree of organization and formal relationships with humanitarian coordinating bodies, their integration into a field marked by seasoned professional field staff and conservative information management is consistently marked by cultural and institutional tensions (harvard humanitarian initiative ). officially and institutionally this is visible in the challenges that surround the development of ethical codes of conducts for digital humanitarians (meier : - ; resor ) or the development of an activation protocol through which ocha is to include the standby task force (svt, one of the most prominent v&tcs) in humanitarian response operations (burns ; gorp ). in ebola response, v&tcs were particularly prominent in establishing novel information exchange platforms, such as a skype channel for coordinating data collection and identifying data gaps (fast and waugaman : - ) , though there is some evidence that lack of integration into formal coordination mechanisms actually "contributed to gaps in awareness of existing tools and duplication of effort" (ibid ). this dynamic recalls of other instances where the introduction of digital tools complicates rather than facilitates humanitarian efforts (bui et al. ; miller et al. ; saab et al. ) , and is consonant with the more general assessment that the uncoordinated introduction of novel actors into ebola response exemplifies "wider dysfunction in the provision of global health security" (harman and wenham : ). tapia et al.'s ( ) two case studies on humanitarian coordination offer a possible explanation for the failure of information systems to lead to enhanced coordination despite explicit efforts. on the basis of a careful literature review and analysis of two humanitarian coordinating bodies, they examine "the instrumental use of it as a mechanism by which ngos collaborate" (ibid ) and identify an important distinction between it-driven coordination efforts that are conceptualized as technical or informational challenges, and those that are conceptualized as organizational or process challenges, which they describe as more formidable. tapia et al. conclude that coordination efforts with modest goals and modest demands on organizational processes are likely to be more successful and increase opportunities for successively more progressive coordination efforts (ibid ). according to this logic, it would be reasonable to expect that digital media coordination would be most successful when it did not demand changes in organizational practice (i.e., coordinating actors who are already inducted in the use of specific media and coordination efforts that do not require changes to data formats or data collection procedures). this section describes two novel tendencies revealed by the above distinctions: the influx of novel actors that accompany and drive the use of new technologies in pandemic response, and the tendency of new technologies to compound previously distinct activities and workflows within single platforms or single institutional operations. new informatics introduce a host of actors and intermediaries not traditionally included in pandemic response and coordination. the ways in which this influx interacts with traditional structures for pandemic response can be considered according to a sandwich model, in which interaction is introduced from above and from below. the bottom slice of the sandwich in this metaphor is interaction with affected populations, where novel informatics present at least two types of challenges. firstly, social media and big data introduce promising new sources of information on which to base decision-making in pandemic response, but for whose meaningful use humanitarian organizations tend to lack the institutional and technical capacity, and national authorities even more so (harvard humanitarian initiative ; odugleh-kolev ; smith ; read et al. ) . simultaneously, the participatory ethos of new technology encourages humanitarian organizations and government authorities alike to deliberately engage affected communities in the design, implementation and evaluation of humanitarian response (kaiser ; maxwell et al. ; gillman ; Özdamar and ertem ) , yet poses a number of non-trivial hurdles to meaningful engagement. here too, technical capacities tend to be weakest with national governments, necessitating partnership with international organizations to invest in participatory interventions. infrastructural requirements and issues of access and representativity can also frustrate intentions to utilize participatory technologies. in a humanitarian context, political realities can often be the most meaningful obstacle, even when all issues of capacity, infrastructure, and access are surmountable. for all these reasons, national authorities are rarely in a position to unilaterally dictate the ways in which new technologies are leveraged to interact with affected populations. informatic challenges at the top end of the sandwich arise from the (sometimes unsolicited) engagement from novel international actors. of particular note are v&tcs and digitally native civil society organizations that are small, nimble, and eager to disrupt established practice. these actors present fundamental challenges to humanitarian coordination by their very engagement. because they do not fit neatly into traditional humanitarian coordination mechanisms, yet tend to demand attention and heighten expectations, novel international actors at the top level of pandemic informatics are a powerful force for asserting knowledge politics in informatics of response. the information flows described by fast and waugaman illustrate not only information exchanges in this sense, but fundamental assertions and negotiations about what kind of information is relevant and where and by whom those decisions are made. the moments at which this happens are not regulated by traditional policies of humanitarian coordination or cluster mechanisms, but occur in the practical application of technologies to knowledge and information, what some scholars have termed moments of closure in humanitarian knowledge politics (burns ). in some instances, international informatics are efficiently structured to serve national authorities. the introduction of data and information clearing houses presents opportunities for national authorities to assert control of national agendas, for example, such as when the humanitarian data exchange enabled guinean ministries to track training of infection prevention and control training efforts in the country (fast and waugaman : ) . this dynamic appears exceptional in a chaotic response environment marked by a "myriad of actors with no clear role or leadership" (harman and wenham : ) , however. a pandemic response context where "many of the information collection systems that organizations set up during the response were not linked to national systems or national capacity" ( ) necessarily reinforces the capacity and agency of international actors, and often novel actors, at the expense of national authorities' influence over response processes. it is also worth noting informatics practices that transcend this two-level model. most clearly in opposition to the agency of national authorities is the introduction of "hidden actors", such as application developers, producers of hardware or network managers with de facto influence over humanitarian data during its collection and processing (see gillman : ). equally notable are instances in which international actors bypass engagement with the traditional response environment altogether. in some instances, this appears to occur exclusively at the international register, with little or no contact with any in-country actors. examples include international networks of scientists collaborating to improve diagnostic tools (eclipse ), or hackathons organized in foreign capitals to develop data models or applications for implementation in pandemic response (sangokoya ; gordon ; lodato and di salvo ) . such initiatives raise serious questions about opportunity cost and efficient use of resources. to summarize, digital communication technologies and information flows introduce a host of novel actors to pandemic response. traditional humanitarian actors may experience this at the top level through the novel engagement of international actors, or at the bottom level through pressure to engage with affected populations and their data. in each instance, the influx of novel actors carries a risk for decision-making authority and agency to be moved into novel fora, further outside the influence of national authorities. this may not occur in an absolute sense; indeed, the role of national governments in driving ebola response strategies has been significant (dubois et al. : ) . yet the potential for exclusion merits careful consideration, particularly given the already pronounced tendency for agency of national authorities to be limited by the militarization of humanitarian response (sandvik ) and the increasing prominence of international ngos in public health service provision (prince ) . such dynamics are particularly vulnerable in the contexts of pandemic and crisis response, and reviews of the ebola response have also noted how decision-makers' identities shape response strategies (dubois et al. : ) . the application of digital technologies has undoubtedly opened up a host of opportunities for decision-making by novel actors. this may occur in situations where national authorities enjoy a limited role, such as the network communications of global watchdog networks in which ngos, scientific communities, international health networks and government agencies contribute to multiple digital communications streams and daily webinars to coordinate disease surveillance and response (ramalingam : - ) . it may also occur in digital for where there is no direct participation by national authorities, or in situations where national authorities lack the basic technical capacities to engage in a natural coordinating role, such as when "a host of academics, private philanthropists and technology companies" lobbied telecom companies for access to call detail records in order to develop their own response strategies (sandvik et al. : ) . though there are some cases in which novel actors and information practices support a stronger role for national authorities in pandemic response and a greater capacity to exercise agency and decision-making in the design and implementation of that response (ramalingam : - ; fast and waugaman : - ) , but this appears rare and the conditions under which it occurs are unclear. the overwhelming picture is one in which technologically driven informatics exacerbate coordination challenges (kim ) , driving the enactment of knowledge politics outside formal structures of humanitarian clusters and beyond the influence of national authorities. though the boundaries between diagnostic activities, risk communication, and response coordination were perhaps never entirely clear, these areas have traditionally been institutionally and procedurally distinct. digital communications' affordances and functionalities make it increasingly possible to combine activities from these areas in single processes. this recalls theories of technological convergence, in which the increasing capacity of media tools to integrate multiple functionalities corresponds with broader shifts in markets and genres (kim ) . as described by henry jenkins (huerta and tsimring ) : our cell phones are not simply telecommunications devices; they also allow us to play games, download information from the internet and receive and send photographs or text messages. any of these functions can also be performed through other media appliances. one can listen to the dixie chicks through a dvd player, car radio, walkman, computer mp files, a web radio station or a music cable channel. fueling this technological convergence is a shift in patterns of media ownership. whereas old hollywood focused on cinema, the new media conglomerates have controlling interests across the entire entertainment industry ( ). a detailed exploration of how these dynamic maps onto humanitarian technology exceeds the scope of this article, but we feel justified in arguing that there are at least two comparable dynamics. first, we will use the term digital convergence to refer to the ways in which technology enables a concentration of diverse tasks in single platforms and workflows (the same platform conducting a range of tasks). second, the examples cited in this article consistently exemplify how this type of convergence is coincident with what we term operational convergence, whereby specific types of information and communication management tasks are distributed across novel institutional and organizational groups of actors (same tasks conducted by a range of actors). below, we briefly describe four examples that demonstrate how this can occur in pandemic response. these examples are not explored in depth, but are meant to illustrate the consistent interplay of convergence with digital media and the injection of novel actors across a variety of humanitarian settings. the first example is what ramalingam terms "watchdog and knowledge networks" ( ), understood as networks involved in the "early detection of disease, characterization of the disease, and subsequent reporting and communication directed to decision makers in governments, international bodies and other key audiences" (ibid - ). especially instructive is ramalingam's analysis of goarn, the global outbreak alert and response network. as an "operational arm" of the who and network of networks that together surveil the outbreak and spread of infectious diseases, goarn functions as a de facto coordination mechanism for international and national actors engaged in pandemic response. the scope of goarn's activity in this regard is impressive, having responded to over outbreaks in over countries between and , and the network has played a crucial role in the sars outbreak in and the avian influenza outbreaks of . notably, the efficiency and scope of goarn's activities is explicitly attributed to the fact that coordination and information exchange occur across a diverse digital infrastructure that supports text messaging, email, and web-based applications, all of which are employed in tandem to ensure the right knowledge and information get to where they are needed at the right time, and importantly, allows a two-way exchange of information across the network ( ). notably, the diverse communications between national authorities, who staff, ngos, and scientific institutions are consolidated in a coherent institutional framework and through weekly webinars. this process interoperates diagnostic and coordination, feeding them directly into the establishment of response protocols, including protocols for risk communication, which are in many instances executed by the same actors that provided diagnostic information, by virtue of their capacity for rapid, two-way communication with populations. though it is unclear the degree to which this operational convergence of such tasks diminishes the agency of national authorities in a general sense, it is reasonable to expect that the inclusion of multiple actors decreases governments' scope for top down control. this may often be for the better in terms of effective response, as is likely the case with sars in , when goarn's access to digital communications platforms facilitated the supply on non-governmental diagnostic information, which likely contributed to acknowledgement of the outbreak by the chinese government. assisted contact tracing provides a second example. contact tracing is the epidemiological practice of identifying the individuals who have come into contact with infected individuals, in order to map the spread of a disease (huerta and tsimring ) . in , a private company named odisi developed a platform for "assisted contact tracing" (act), which digitized this process through the use of integrated voice recognition software. individuals in ebola-affected communities were able to report their contacts using mobile phones and then received follow-up messages regarding care and updates on the ebola response. this digitized approach increased the efficiency of data collection by eliminating the need for human interviewers, and also allowed the integration of other types of data (paper and mobile data), which increased the platform's diagnostic capacity dramatically. though the platform was designed and implemented as a diagnostic tool, the affordances offered by digital media quickly presented other opportunities. the automated registry of exposed individuals was quickly adapted for risk communication purposes via sms follow-up messages, and the digitization of rapid analysis of data promptly positioned the act platform to play a coordinating role among parallel diagnostic initiatives. here, we see a clear digital convergence of diagnostic and risk communication activities on a mobile phone platform, at the discretion of a private company. a third example is offered by u-report, a free sms-based polling tool launched in uganda in through a civil society partnership in order to monitor the quality of human rights and governance in the country, with a focus on polls related to human rights. in early , platform users noted early signs of the outbreak that would later come to be known as "nodding sickness" and would claim over lives in the following months. u-report did not have a health mandate and did not solicit these early epidemic reports, but received them because the communication platform was already in place and integrated into the communication habits of users. in this sense, the presence of a digital media infrastructure very much conditioned the implementation of a diagnostic tool, which promptly provided a site for national coordination, as u-report collaborated with the ministry of health and the who to develop and implement a -stage communications and mobilization plan. here again we see the digital and operational convergence of diagnostic activities and risk communication activities by national authorities with the support of international organizations. notably, while national authorities are directly engaged in both sending and receiving content, they do not have direct control of the operational and financial processes that support the initiative. fourth and lastly, the humanitarian data exchange (hdx) was established by the un office of coordination for humanitarian affairs in , in an effort to improve and coordinate access to humanitarian data. the ebola crises page collected data sets from un, governmental, civil society, and private sector data sources and invited users to contribute their own data to the site. the page also featured maps and visualizations developed in collaboration with private charitable foundations and private businesses. as such, the site represents a near seamless integration of all the components of pandemic informatics. it directly served the needs of independent diagnostic efforts by providing access to quality humanitarian data; it performed a coordinating function by establishing standards and expectations for the use and production of humanitarian data, and visualizations and graphics created by the community were incorporated into the hdx gallery for download and use in independent risk communication efforts. here, we see convergence between digitally enabled coordination efforts, deeply rooted in multilateral humanitarian institutions, and the diagnostic processes that they enable. unlike the examples above, the question of who engages with the hdx and how is not significantly pre-determined. the platform is designed to be open, and to the extent that contributors are invited and approved by ocha, it is reasonable to expect that it is open to national authorities. to the extent that technological capacity still limits national authorities from engaging with the platform, convergence of diagnostic and coordination activities nonetheless consolidates what we can call informatic discretion outside their spheres of influence. what is striking about these examples is not necessarily the fact that activities related to different informatic components interact; that has to some degree perhaps always been the case. what is striking is the degree to which they do so automatically, as conditioned by digital media, and within the purview of the actor driving the technological and informatic innovation. this is almost never national authorities, due to capacity issues described above. to the extent that reliance on technology and the introduction of novel actors drive informatic discretion beyond the influence of national authorities, this phenomenon is likely to be exacerbated by instances of technological and operational convergence. this analysis has reviewed the ways in which digital technologies have been deployed in humanitarian pandemic response, and proposed a three-part conceptual model for assessing these informatics according to the objectives that are pursued. doing so revealed two consistent and interrelated consequences: -the influx of novel actors from both affected populations and from the international register engenders novel fora for asserting knowledge politics, influence, and informatic discretion in response. these fora support the increased engagement of novel actors, but are often inaccessible to national authorities due to limits on technical capacity and political position. -the efficiency of technologically driven informatics tends towards technological and operational convergence, in which multiple types of activities are collapsed into single platforms or institutional processes. convergence has the effect of further consolidating sites for informatic discretion beyond the influence of national authorities. these two dynamics are concomitant and mutually reinforcing. each is directly enabled by the introduction of novel technological tools and strategies to pandemic response, but also facilitates the other (novel actors tend to be the ones who actually combine informatics functionalities, often without warning and through their own innovations), and complicates traditional pandemic response informatics in mutually reinforcing ways. it is tempting to anticipate a certain momentum at play. it is also tempting to frame this as a coordination problem. novel actors and models for participation strain the humanitarian system precisely because humanitarian roles and responsibilities are rigidly defined, but not immutable. the dynamics of convergence would, moreover, seem to promise significant increases for efficiency and coordination were obstacles to information sharing surmounted, and gaps in institutional cultures for using technology filled. but herein lies the challenge. though technology seems consistently to imply dramatic gains in humanitarian efficiency, it consistently frustrates efficient coordination, and despite millions of dollars spent, in the case of multi-lateral, large-scale coordination efforts around information, there have only been several very public failures (tapia et al. : ) . this is likely because the challenge manifest in these informatics is more fundamental, having to do with the ways in which institutions and organizations leverage technologies to assert knowledge politics "in ways that rely upon the differential influence and authority that is granted to particular forms of knowledge or representations" (barnett ) . without addressing the differential capacities and expertise that drive a propagation of knowledge politics beyond national authorities' spheres of influence, coordination exercises are unlikely to be anything more than exercises. in this scenario, a humanitarian technological context marked by exuberant expectations and a chaotic lack of coordination is problematic. more troubling is the underlying recession of national authorities from those fora in which knowledge and influence are asserted and contested. the tendency of international humanitarian response to sideline national authorities is not new. the possible exacerbation of this dynamic by new response informatics is, however, particularly problematic because the enthusiasm surrounding novel technologies so efficiently occludes the challenges that they pose to fundamental coordination norms of humanitarian coordination. there is wide agreement that international humanitarian intervention should complement and support national authorities' response as a temporary measure until the point at which national authorities are able to assume control over national processes, facilities, and infrastructure (jahre and jensen ; harvey and harmer ; ocha inter-agency standing committee ). the details of this relationship prompt arduous contention and debate in the context of traditional humanitarian coordination mechanisms, such as the un cluster system (harvard humanitarian initiative ; sandvik et al. ; mcdonald ) , and to a modest degree, in critical commentary on the application of humanitarian technology (sandvik et al. ). until they are equally visible in the discourses and planning processes that drive remote volunteering, university hackathons, mobile network-enabled contact tracing, and participatory mapping efforts, it is hard to imagine ways in which to reassert the agency and influence of national authorities in new response informatics. so what is next? there are at least three opportunities to begin addressing this. firstly, the degree to which digitally driven informatics exacerbate challenges to the agency of national authorities in the context of humanitarian coordination should be explicitly included in critical discourses that resist techno-optimism in the humanitarian sector. this involves expanding notions of responsible humanitarian technology, innovation and data-use to include reflections on how digital informatics impact the spheres of influence of national authorities in humanitarian response. it constitutes an additional type of risk to be considered when questioning the risks associated with humanitarian experimentation (harvey and harmer ) . secondly, established mechanisms for coordinating complementary and supplementary humanitarian support to national authorities in humanitarian response, including the un cluster system, should deliberately anticipate these dynamics and work to mitigate their effects. significant work is ongoing to improve the ways in which national and international actors interact in humanitarian response (unga ; oecd ; jahre and jensen ; odugleh-kolev ) . the policies and procedures that result from these efforts should include explicit processes for accommodating the types of novel actors, platforms and instances of convergence described above. explicitly identifying roles and sites for interaction with national authorities would have the twin benefit of providing entry points for those actors according to established coordination norms, and providing opportunities for technical capacity development to national authorities. lastly, prominent humanitarian coordination bodies that have significant experience using digital communication technologies and interacting with the novel actors they introduce, should facilitate structured interaction between these actors and representatives of national authorities, in an effort to identify good practices for reinforcing national agency in digitally-driven informatics. taken to their logical conclusion, relatively low-cost investments in events and joint trainings by organizations such as ocha, could easily lead to the production of credible and welcome 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technology in the ebola response in west africa humanitarian information management and systems theoretical divides and convergence in global health communication. obregon/the handb glob heal commun using mobile technology to improve maternal health and fight ebola: a case study of mobile innovation in nigeria the viral storm: the dawn of a new pandemic age. allen lane zuckerman e ( ) new media, new civics? governing big data authors' contributions cw contributed to the design of this research, desk review, and writing of the manuscript. mgj contributed to the design of this research, desk review, and writing of the manuscript. both authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -by cujyy authors: guo, hai; yang, zhuen; huang, ran; guo, anqi title: the digitalization and public crisis responses of small and medium enterprises: implications from a covid- survey date: - - journal: front doi: . /s - - - sha: doc_id: cord_uid: by cujyy the covid- outbreak is a global crisis that has placed small and medium enterprises (smes) under huge pressure to survive, requiring them to respond effectively to the crisis. smes have adopted various digital technologies to cope with this crisis. using a data set from a survey with chinese smes, the study examines the relationship between smes’ digitalization and their public crisis responses. the empirical results show that digitalization has enabled smes to respond effectively to the public crisis by making use of their dynamic capabilities. in addition, digitalization can help improve smes’ performance. we propose a theoretical framework of digitalization and crisis responses for smes and present three avenues for future research. at the end of , a novel coronavirus disease broke out suddenly and spread rapidly to become a global pandemic. by late june , covid- had infected more than million people worldwide, including more than , people in china. this public health crisis has posed great challenges for the survival and development of firms, with small-and medium-sized enterprises (smes) suffering in particular. the covid- pandemic has been economically destructive in many ways. first, as more and more governments block cities in order to control the pandemic, the global supply chain has been significantly disrupted as both imports and exports are blocked. second, delays in the resumption of work have greatly reduced firms' production capacity while fixed costs such as salary and rent remain unchanged, leading to serious cash flow issues. third, the reduction in demand due to the outbreak has posed serious threats to service sectors such as catering, hospitality, and cultural tourism. worse, the damage caused by the covid- outbreak is expected to be longlasting and have a chilling effect on global economic growth. smes play a vital role in promoting technological innovation, improving employment, and maintaining social stability (o'regan et al. ). however, due to their shortage of resources, smes are much more vulnerable to public crises than other enterprises (barron et al. ; mayr et al. ) . the existing literature has examined the roles of production recovery, corporate social responsibility, and community participation in reducing the threat of public crises on smes (ballesteros et al. ; kearins ; neise and diez ) . in particular, firms' dynamic capabilities have been found to be the key to public crisis responses (lin and wu ; linnenluecke ; martinelli et al. ) . unfortunately, the ways in which smes should build and leverage dynamic capabilities in public crises like the covid- outbreak remain largely unclear. in this study, we argue that digitalization has the potential to help smes respond effectively to public crises by activating their dynamic capabilities (vial ) . digitalization refers to the use of digital technologies such as information, computing, communication, and connection technologies to promote organizational changes (bharadwaj et al. ; sebastian et al. ; vial ) . in the context of the covid- outbreak, much research has suggested that the adoption of digital technologies plays an important role in crisis responses. in china, the government has encouraged the use of big data, artificial intelligence (ai), cloud computing, and other digital technologies in pandemic monitoring, virus tracing, disease treatment and work resumption. for example, big data technology can provide powerful support for real-time pandemic monitoring and tracking. the adoption of online office software enables employees to work remotely in a flexible manner. based on data from an online questionnaire survey conducted with chinese smes, the present study explores the relationships among digitalization, crisis response strategies to the covid- outbreak, and the crisis response performance of smes. the survey results clearly show that digitalization can help smes employ emergency responses as well as respond strategically to public crises in the long run, thus contributing to the improvement in smes' performance. drawing on the dynamic capabilities perspective, we propose a theoretical framework of digitalization and public crisis responses and present several avenues for future research. invalidating normal practices and rules and leading to huge economic losses and even humanitarian disasters (wasileski et al. ) . firms can be seriously affected by public crises. such crises lead to instability, forcing firms to adjust their internal resources and capabilities to adapt to or operate upon the changing environment (martinelli et al. ) . as firms must respond to public crises as quickly as possible (bundy and pfarrer ; helmer and hilhorst ; williams ) , it is highly valuable to examine how firms should respond to such crises (cui et al. ; geroski et al. ; mayr et al. ; thornhill and amit ) . public crisis responses involve the posture that is taken with respect to modifying or adapting one's actions in a turbulent crisis environment (smart and vertinsky ) , which can be divided into two types: emergency responses in the short term for survival, and strategic responses in the long term for development (müller ; smart and vertinsky ) . in the short term, firms must make emergency responses to mitigate the immediate negative effects of a crisis (müller ) . firms' normal production and operation activities will be temporarily interrupted during the crisis (martinelli et al. ) , requiring them to implement immediate actions that promise immediate results, such as resuming production, improving efficiency, and reducing costs (müller ) . in addition, firms may have important social responsibilities in crisis areas (neise and diez ) . they can obtain support from the government and the community to perform better during the crisis by undertaking social responsibilities (kearins ) , such as protecting the basic rights of employees and donating to communities (ballesteros et al. ) . however, it is not sufficient to address ongoing threats (müller ) . in the long term, firms should strive to turn threats into opportunities, which requires strategic responses (ginsberg ; müller ; smart and vertinsky ; smith and sipika ) . for example, the practical value of digitalization has been widely recognized in the context of the covid- outbreak. further, the crisis has also triggered strategic changes, including changes in product lines, market width, and external relations (boeker ; kirtley and o'mahony ; romanelli and tushman ) . therefore, in the long term, firms should endeavor to pursue opportunities in the crisis environment through strategic responses (wan and yiu ; wenzel et al. ) . the key to public crisis responses is to understand the crisis environment in a timely manner, seize opportunities, and reconfigure resources to cope with the crisis (ballesteros et al. ; yang and hsieh ) . therefore, the dynamic capabilities perspective is highly relevant to crisis response research (fainshmidt et al. ; helfat ; linnenluecke ; martinelli et al. ) . as the covid- outbreak has been both unexpected and unpredictable, firms must possess dynamic capabilities to cope with it in a non-procedural, innovative, and dynamic manner. dynamic capabilities are the comprehensive capabilities to build, integrate, and reconfigure internal and external resources when coping with a rapidly changing environment (teece (teece , teece et al. ) , which are regarded as powerful tools for firms to create and sustain value in a changing environment (eisenhardt and jeffrey ; lin and wu ) . the turbulent nature of the environment becomes even more prominent in public crises. thus, crises represent a real opportunity for firms to unleash the full potential of their dynamic capabilities (linnenluecke ; martinelli et al. ; yang and hsieh ) . in the context of a crisis, dynamic capabilities can be divided into three dimensions: the capability of sensing the crisis, the capability of seizing new opportunities in the crisis (ballesteros et al. ; teece ) , and the capability of reconfiguring resources to cope with the crisis. first, firms with dynamic capabilities have the potential to sense or understand the crisis in a timely manner (ballesteros et al. ; lampel et al. ; teece ) . admittedly, no firm could predict the arrival of the covid- outbreak, but some firms may have sensed the spread of the outbreak and predicted that it would have a significant impact on their business. by assessing how the daily operations of the local community would be influenced by the potential crisis, such as the disruption of production and distribution activities, market failures, and staff shortages, firms can better perceive the crisis. without sensing and understanding a crisis, it is virtually impossible to develop the comprehensive and interlinked strategies that are required to respond to it (müller ) . second, firms with dynamic capabilities are more likely to identify and capture new opportunities in a crisis (ballesteros et al. ; danneels ; easterby-smith et al. ). a public crisis breaks social patterns and gives birth to new business opportunities. for example, the shutdowns caused by the pandemic have provided opportunities for online businesses to flourish. firms equipped with dynamic capabilities can better address valuable opportunities and are more likely to create and absorb new knowledge from the external environment, providing an impetus for changing under the crisis (ballesteros et al. ; makkonen et al. ) . for instance, during the covid- outbreak, meituan launched the "unmanned delivery" plan, which involved contact-free deliveries, by restructuring its intelligent distribution system. third, firms can integrate and reconfigure their internal and external resources to cope with a crisis (makkonen et al. ) . organizational inertia may pull firms away from observing external environment changes and adapting to them (newey and zahra ) . as a result, firms without the capability to reconfigure their resources might fail to implement either short-or long-term crisis response strategies. overall, dynamic capabilities are critical for firms' public crisis responses. then comes the question: what kind of firms are more likely to be able to dynamically respond to public crises? to answer this question, this study highlights the important role of firms' digitalization efforts, particularly under the context of the covid- outbreak. digitalization refers to the process of organizational transformation through the adoption of digital technologies (sebastian et al. ; vial ) , mainly manifested in organizations as digital artifacts, digital platforms, digital infrastructures (briel et al. ; giones and brem ; nambisan ; nambisan et al. ; yi et al. ) , as well as digital business and management models (srinivasan and venkatraman ) . digital technologies are a mix of computerized information and communication technologies (sturgeon ) and can be classified into seven types: social, mobile, big data, cloud guo et al. frontiers of business research in china ( ) (yoo ). thus, digitalization or digital transformation can help firms gain and sustain competitive advantages by improving their organizational flexibility and resilience (briel et al. ) and by enhancing their dynamic capabilities (sambamurthy et al. ; vial ) . in particular, we propose that digitalization is beneficial to firms' dynamic capabilities. first, digitalization helps firms sense environmental changes (vial ; warner and maximilian ; yoo ) . the great advantage of digital resources in volume, velocity, variety, and value makes it possible for firms to collect or retrieve information resources in the external environment at a low cost (gandomi and haider ) . in addition, the application of big data analysis systems and iot technologies helps firms screen valuable information through high-speed computing so that they can sense and predict environmental changes to some extent (george et al. ; sambamurthy et al. ) . second, firms can better seize opportunities in a crisis environment with the help of digital technologies. in the covid- outbreak, digitalization has created plentiful new opportunities (nambisan et al. ) , and areas such as online education, online working, and unmanned delivery have shown great potential. further, the decentralized nature of digital technologies breaks obstacles in time and space, and promotes interactions between focal firms and their value co-creators, thus increasing their opportunities in open networks (zeng and glaister ) . in addition, high-volume big data technology and high-speed cloud analysis technology have greatly improved the accuracy of business analysis, helping firms identify potential opportunities in complex environments (briel et al. ) . furthermore, digital technology has changed the ways in which new opportunities are exploited, in ways that are more novel than predefined (nambisan et al. ) . finally, digitalization enables firms to reconfigure their resources to respond to crises. digitalization improves firms' available resources in scope, scale, and flexibility. for example, it technologies reduce the cost of coordinating activities within firms and promote the flexible allocation of resources (kane et al. ) . in addition, digital technologies have fundamentally reshaped business processes, products, and services, as well as inter-firm relationships, greatly reducing the difficulty and costs of resource shifting (nambisan et al. ) . for instance, the blockchain, cloud computing, and iot technologies have shortened the time required to launch new products and transform businesses, thus enabling firms to quickly adjust their operations with low costs (warner and maximilian ) . during the covid- outbreak, firms with a high degree of digitalization, such as freshhema and meituan, have been able to quickly reshape their businesses to minimize the adverse impacts or even benefit from the crisis. sample and data collection an online questionnaire survey was conducted to collect first-hand data. after reviewing relevant scales in the literature, the survey team discussed scale development and designed the questionnaire. in mid-to late-february , the questionnaire was sent to firm managers through wechat and other online social media channels. the respondents were mainly emba, mba, and ee (executive education) students from key universities in china. by february , valid samples had been collected from smes (with less than employees), accounting for . % of the full sample. as table shows, . % of the sampled smes were private. foreign-invested firms and state-owned firms account for . % and . % of the sampled smes, respectively. in terms of region, more than % of the smes were based in eastern china (including shanghai, jiangsu, zhejiang, fujian, shandong, jiangxi, and anhui) and northern china (including beijing, tianjin, hebei, shanxi, and inner mongolia). the ratio of listed companies is relatively low, accounting for . % of the sample. in addition, about % of the sampled firms' business is conducted offline, leaving much room for digital transformation. the sampled firms are distributed across a wide range of industries, with manufacturing ( . %); information transmission, software, and information technology ( . %); and whole sales and retail trade ( . %) ranking in the top three industries. three alternative measures were adopted to measure the digitalization degree of the sampled firms. first, digitalization can be defined as the overall digitalization degree of a firm, which is reflected through five items. ( ) digital artifacts refers to applications or media content with specific functions and values embedded in digital products or services, such as positioning applications in mobile phones to track travel trajectories during the pandemic (ekbia ; nambisan ) . ( ) a digital platform is a set of shared general services and architectures that plays as an important role as a carrier of digital artifacts (nambisan ; tiwana et al. ) . ( ) the digital infrastructure refers to digital technology tools and systems (nambisan ) . ( ) the digital business model embodies the firm's digital technology-driven value creation logic, such as online retail business during the covid- outbreak. ( ) the digital management model involves the application of digital technologies in the organizational management system, such as the adoption of intelligent office systems. second, we measure a firm's digitalization through its adoption of digital technologies, which is reflected through seven items. these are ( ) social, ( ) mobile, ( ) big data, ( ) cloud computing, ( ) iot, ( ) platform development, and ( ) ai (bharadwaj et al. ; sebastian et al. ; vial ) . third, the digitalization degrees of firms for which the businesses mode is online are thought to be higher. therefore, digitalization can be roughly measured by distinguishing online business from offline business (biswas and burman ). firms' public crisis response strategies can be classified into short-term emergency strategies and long-term strategic ones (müller ; smart and vertinsky ) . while short-term response strategies aim to adapt to the turbulent crisis environment, longterm oriented response strategies endeavor to identify opportunities for future development, suggesting the importance of dynamic capabilities (ginsberg ; müller ; smart and vertinsky ) . in this study, we consider three types of short-term response strategies: production recovery strategies, employee protection strategies, and firm donation strategies (ballesteros et al. ; neise and diez ; wenzel et al. ) . production recovery strategies are reflected through eight items: ( ) reducing production and operating costs, ( ) divesting loss-making/less-profitable business units, ( ) adopting online telecommuting, ( ) optimizing business models to capture new customer needs, ( ) developing marketing channels and removing dependence on offline transactions, ( ) actively investing in technological innovation, ( ) diversifying into new business areas, and ( ) integrating supply chain. firms' employee protection strategies are reflected through six items: ( ) paying wages in accordance with contracts in one pay cycle, ( ) paying basic subsistence allowance in excess of one pay cycle, ( ) retaining employees' jobs, ( ) negotiating with employees or unions to defer payment, ( ) paying wages to employees who are quarantined, and ( ) arranging compensatory leave or overtime paying for employees who cannot take time off. the firms' donation strategies are reflected through their donation amounts. further, two long-term oriented response strategies are included, namely digital transformation and strategic changes (boeker ; kirtley and o'mahony ; romanelli and tushman ; wan and yiu ; wenzel et al. ) . digital transformation is reflected through five items: ( ) strengthening the application of online office tasks, ( ) improving the digitalization of supply chain channels, ( ) adopting digital artifacts, such as digital products or services, ( ) adopting digital platforms, such as digital communication platforms, and ( ) adopting digital infrastructures, such as digital technology systems. strategic changes are reflected through three items: ( ) changing existing product lines, ( ) changing regional market coverage, and ( ) changing external cooperative relations (wan and yiu ; wenzel et al. ) . four alternative measures were adopted to measure firms' performance during the outbreak: cost control status reflected in the extent to which costs are controlled, cash flow status reflected in maintenance of cash flow over time, revenue status in the first quarter of reflected in the extent to which revenue declines are controlled, and overall predicted performance under the crisis reflected in the perception of the firm's overall performance. unless specifically explained, the scales adopted in this study are all five-point likert scales, with indicating "totally disagree," "almost none," or "very poor" and indicating "totally agree," "pretty much," or "very good." we measured the value of each variable by taking the average value of all measuring items. to make the statistical results more intuitive, the average value interval of most variables was converted into an interval from to , except for the minimum and maximum. descriptive and correlational analysis for key variables table shows the descriptive statistics for key variables used in this study. first, in terms of the degree of digitalization, the results show that chinese smes have made initial achievements in digitalization, but there is still plenty of room for improvement. specifically, the mean of digital artifacts (usually embedded in products and services), digital platforms, and digital infrastructure are all above . , indicating that chinese smes have made initial attempts at digitalizing. however, the digital ability in value creation is insufficient, as the business model ( . ) and management model ( . ) are less digitalized. second, our results show that compared to internal r&d, smes often resort to external technologies for digitalization. third, in terms of digital technology adoption, the values for social technology ( . ) and mobile technology ( . ) are higher. this can be credited to the rapid development of china's e-business and internet economy over the past years. however, the adoption of the latest digital technologies such as ai ( . ) and cloud computing ( . ) still requires improvement. finally, considering the modes of business, the online rate is insufficient ( . ). in terms of short-term crisis responses, smes have generally adopted production recovery ( . ) and employee protection strategies ( . ), while the adoption rate of the donation strategy is relatively low ( . ). these results suggest that the most important task for smes is to survive the crisis. for long-term crisis responses, smes prefer to implement digital transformation strategies ( . ) instead of strategic changes in products, markets, and external relations ( . ). these results imply that smes have realized the unique value of digitalization in the covid- outbreak. regarding crisis response performance, the results of the survey show that smes have been adversely affected by the covid- pandemic in various aspects. costs ( . ) have increased less than %, but revenue in the first quarter ( . ) has decreased by %- % on average, and cash flow ( . ) can only be maintained for about half a year, on average. overall, under the covid- outbreak, there is an inevitable decline in predicted performance ( . ) compared with firms' performance in . table shows descriptive statistics with correlations of variables. province (the province in which the firm is located), industry, property, and year (the year in which the firm was established) are set as control variables. the results show that the digitalization of smes is positively associated with the implementation of crisis response strategies and performance, and that crisis response strategies are also positively associated with performance. therefore, it is necessary to continue to explore the functional relationship between variables. we calculated the variance inflation factor (vif) and the result shows that the maximum value is . , far below the cutoff value of . our results show no evidence of multicollinearity among all variables. as this is a survey study, a more intuitive and concise approach should be adopted to clearly present the results in a simple manner. to this end, we divided the value of each variable into three degrees, namely low, medium, and high. for example, the value of the degree of digitalization was sorted from the largest to the smallest, and then divided into three equal groups according to the number of samples, namely the high group, the middle group, and the low group. we then calculated the average value of the variable at each degree. this grouping method has the advantages of simplicity, intuition, and clarity in the presentation of data, which enhances the readability of the paper. the values were reserved to two decimal places, except for special notes. as shown in table , the survey results generally indicate that the digitalization of smes is positively associated with the implementation of crisis response strategies, including both short-term emergency responses (p < . ) and long-term strategic responses (p < . ). in the face of the covid- outbreak, highly digitalized smes can more effectively use short-term responses. for smes with a high degree of digitalization or digital technology adoption, the average scores of short-term crisis responses are . and . , respectively, both of which are higher than those for smes with a medium or low degree of digitalization. specifically, in terms of digitalization, the scores for production recovery strategy, employee protection strategy, and donation strategy are . , . , and . , respectively. in terms of digital technology adoption, the three scores are . , . , and . , respectively. the policy-based shutdown under the pandemic has left many firms facing production stagnation. highly digitalized firms with dynamic capabilities are more likely to integrate their internal and external resources quickly to resume production and operation activities through methods such as adopting online telecommuting or divesting less-profitable units. in addition, digital firms can make donations to the pandemic area through existing digital channels. internet firms, which have attracted plenty of well-deserved attention during this pandemic, have higher philanthropic efficiency than traditional firms and charities. similarly, highly digitalized smes are more determined to make long-term responses in order to deal with the crisis. for smes with a high degree of digitalization, the average scores of digital transformation intention and strategic change intention are . and . , respectively, all higher than those for smes with a medium or low degree of digitalization. similarly, for smes with a high degree of digital technology adoption, the average scores of digital transformation intention and strategic change intention are . and . , respectively, all higher than those for smes with a medium or low degree of digital technology adoption. the agility and openness of digital technologies greatly improves the accuracy of business analysis for the firms that employ such technologies. this explains why highly digitalized firms are more likely to find potential opportunities in disruptive environments and integrate resources for strategic transformation and changes. in addition, smes, with the primary business conducted online instead of offline, will perform better in responding to the covid- outbreak ( . vs. . , p < . ), which is mainly reflected in their long-term crisis responses ( . vs. . , p < . ). specifically, regarding other crisis response strategies, the performance of smes that conduct their business online is no worse than that of those that conduct their business offline, and the difference is not highly significant. responses to the covid- outbreak and response performance table shows the relationship between smes' crisis response strategies and performance outcomes. overall, both short-and long-term strategies in response to the covid- outbreak will lead to improved performance for firms. on the one hand, short-term crisis response strategies can improve the performance of smes, though this does not occur in a uniform manner. production recovery strategies ( . ) and employee protection strategies ( . ) can lead to better predicted performance (p < . ), but do not necessarily help smes improve their current performance. excessive production recovery and employee protection will consume a large amount of a firm's resources, putting even greater pressure on already thinly stretched smes during the outbreak. therefore, a moderate degree of production recovery and employee protection may be a better option. donation strategies have an overall positive impact on sme performance ( . ) and can help smes obtain more revenue ( . ), leading to stronger cash flow ( . ) and better predicted performance ( . ). smes and stakeholders are a community of interests in the public crisis. by donating to relevant causes, smes can obtain support from the government and the community to mitigate the harm of the crisis in an atmosphere of mutual assistance. on the other hand, long-term crisis response strategies have inconsistent effects on smes' performance. digital transformation strategies have the potential to generate better performance for smes, especially related to cash flow maintenance ( . ). however, strategic changes are associated with lower sme performance ( . ), indicating that an sme's intention to change its strategy seems to merely be a reflection of poor performance. strategic changes will consume a large amount of human, material, and financial resources, placing huge pressure on smes that are already struggling in the outbreak. however, this does not mean that strategic changes are not a good choice because the benefits of successful changes may be long-term and sustainable even if the temporal performance may decline. as shown in table , the results of the survey indicate that digitalization is positively associated with smes' crisis response performance. first, a high degree of digitalization will lead to higher sme performance ( . , p < . ). specifically, digitalization could help smes maintain cash flow ( . , p < . ), generate more revenue ( . , p < . ), and lead to perceived best predicted performance ( . , p < . ). as identified above, highly digitalized firms are more likely to resume production, effectively curbing revenue and profit declines. however, the advantage of digitalization for cost control is not obvious, leading to a lack of significant advantages for firms' overall performance. second, similarly, a high degree of digital technology adoption is associated with optimal sme performance ( . , p < . ). specifically, the adoption of digital technologies is associated with a higher degree of cash flow maintenance ( . , p < . ), all higher than the rest. however, this cannot help smes control costs or obtain revenue most effectively. finally, for smes that conduct their primary business online, their performance outcomes are not worse than those whose business is conducted offline, but the difference is not significant. the simple descriptive statistics show that compared to offline businesses, online ones may score consistently higher in cost control ( . ), cash flow maintenance ( . ), revenue ( . ), and predicated performance ( . ). however, these results require further and more rigorous verification. table shows the ways in which the sampled smes have adopted different digital technologies to improve their crisis response performance. the results indicate big data, mobile, and cloud computing technologies are particularly effective in helping smes cope with the crisis, but this does not occur in a uniform manner. first, mobile technologies are particularly helpful for cost control (β = . , p < . ). technologies such like g, instant messaging, and gps together with a variety of mobile applications could support smes in refined management and improve their organizational efficiency. for example, trajectory tracking applications can help smes efficiently conduct human resource management. second, big data technologies are regarded as most useful for maintaining cash flow (β = . , p < . ) and generating revenue (β = . , p < . ). in the digital economy, big data not only helps firms improve the efficiency of their existing businesses, it also enables the creation of new businesses. during the covid- pandemic, big data technology may not only help smes evaluate the spread of the pandemic, guiding them to reactivate work and production, it may also help them identify and take advantage of new business opportunities in the crisis and thus guide the direction of future developments. finally, cloud computing technology seems to be most important for smes' overall predicted performance (β = . , p < . ). this indicates that a new era of cloud computing is being welcomed. descriptive analyses and simple correlation analyses are often conducted in survey research. however, entirely abandoning the regression may cause problems hazarding research rigor such as neglecting the partial correlation variables. therefore, controlling the four variables of province, industry, property, and year, we conducted a simple whole regression test among the main variables as a robustness test. as shown in table , first, the overall degree of digitalization has a significant positive impact on the implementation of crisis response strategies, including both short-term responses (β = . , p < . ) and long-term responses (β = . , p < . ). second, digital technology adoption also has a significant positive impact on the implementation of short-term responses (β = . , p < . ) and long-term crisis responses (β = . , p < . ). third, smes that conduct their primary business online instead of offline will perform better in crisis responses, especially in long-term responses (β = . , p < . ). in addition, overall digitalization degree (β = . , p < . ) and digital technology adoption (β = . , p < . ) both have a significant positive impact on smes' performance. the implementation of crisis response strategies is further positively associated with smes' performance, which is mainly reflected in their short-term strategies (β = . , p < . ). these empirical results support our arguments. we have constructed a theoretical framework based on the above survey results (fig. ) . the survey results show that digitalization is positively associated with smes' public crisis response strategies and performance, which indicates that smes with a higher degree of digitalization are more likely to adopt effective public crisis response strategies and achieve better performance during the covid- outbreak. drawing on the dynamic capabilities perspective, we emphasized the role digitalization activities play in firms' crisis responses. highly digitalized firms can leverage their dynamic capabilities to sense a crisis, seize opportunities during the crisis, and reconfigure resources to cope with the crisis (vial ; warner and maximilian ; yoo ) , which means these firms are more likely to respond to crises quickly and effectively. further, crisis response strategies, including short-term emergency responses and long-term strategic responses (ballesteros et al. ; smith and sipika ) , can lead to improved performance for smes. here, we propose three avenues for future research. digital technologies are at the center of digitalization. sebastian et al. ( ) further builds an smacit framework to classify various digital technologies. the sensitivity and openness of digital technologies provide flexible access to and enable agile responses to the external environment. existing literature shows that digitalization can significantly improve firms' dynamic capabilities (vial ; warner and maximilian ; yoo ) , enabling them to remain flexible in highly uncertain environments (briel et al. ) . compared with traditional firms, digitalized firms are better prepared to sense the environment (george et al. ; nylen and holmstrom ; vial ) , to seize opportunities (briel et al. ) , and to reconfigure resources (vial ) . however, the connotations of dynamic capabilities are changing under the context of digital transformation. on the one hand, the widespread use of digital technologies is reshaping the nature and purpose of dynamic capabilities (warner and maximilian ) , as the powerful aggregation and generation of digital technologies elevate firms' dynamic capabilities. for instance, blockchain, cloud computing, and iot technologies enable firms to exponentially expand the scope and scale of their businesses. therefore, digital dynamic capabilities may become a key source of competitive advantages for firms in the digital economy (warner and maximilian ) . on the other hand, the boundary of dynamic capabilities is also changing. for example, makkonen et al. ( ) reclassify dynamic capabilities into regenerative and renewing capabilities, which are manifested by indicators like reconfiguration, leveraging, learning, sensing and seizing, knowledge creation, and knowledge integration. topic : digitalization and public crisis response strategy firms now operate in "vuca" times, characterized by volatility, uncertainty, complexity, and ambiguity (bennett and lemoine ) . we have witnessed more and more crises in recent years. in fact, at least four public crises have occurred since the start of , including the covid- outbreak, the african locust plague, the collapse of oil prices, and the u.s. stock market meltdown, all reminding us that we should pay attention to responses. firms should build, integrate, and reconfigure resources to cope with complex, turbulent, and highly uncertain environments (bennett and lemoine ; smart and vertinsky ) . the world is undergoing a new industrial revolution-the digital revolution (rindfleisch et al. ) . digitalization creates an abundance of business opportunities, and it is worth questioning whether it may help firms survive or even benefit from crises. however, the existing literature on firms' crisis responses is focused on the firm level, such as bankruptcy, stock price decline, and reputation damage (mayr et al. ; snyder et al. ; wei et al. ) , and public crisis responses have been examined less frequently. further, existing studies on public crises often address issues like governance and community resistance (donaldson ; martinelli et al. ) , overlooking the role of firms. the findings of our survey clearly indicate that digitalization can be useful in public crisis responses. compared with other firms, digitalized firms are more likely to adopt short-term and long-term crisis responses and enjoy better performance outcomes. further, we have bridged digitalization and crisis responses from the perspective of dynamic capabilities. however, research on this theoretical bridge is still in its infancy, with many deficiencies. dynamic capability theory is not the only theory that may help us understand digitalization and crisis responses. other traditional management theories have the potential to be integrated with digitalization, and the theory of digitalization itself deserves more exploration. therefore, it is valuable to examine the relationship between digitalization and firmlevel crisis response strategies. we suggest the following research questions. first, how do different public crises (natural vs. social) reshape the business environment? second, what is the nature of firms' strategic decision-making in crisis environments characterized by sudden and violent environmental changes? third, what are the similarities and differences between short-term emergency response strategies and long-term strategic response strategies? finally, what is the underlying mechanism through which digitalization impacts crisis response strategies? the results of the survey indicate that digitalization has a positive effect on smes' performance. although this paper only focuses on smes' present performance, future research should consider both present and future performance, as digitalization strategies have long-lasting impacts on firms. digitalization has greatly altered the ways in which businesses are run (ofek and wathieu ) , which can be a new source of competitive advantages for firms. on the one hand, digitalization can improve a firm's operational efficiency by automating decision-making, improving the efficiency of business processes, and saving costs (andriole ; pagani ) . for example, cloud computing technology provides elastic resources, which reduces the cost of hiring, managing, and maintaining technology talents (kane et al. ) . big data technology accelerates firms' decision-making processes, increasing the speed at which they can respond to intelligent products and services (bharadwaj et al. ). on the other hand, digitalization involves digital innovation activities (li et al. ) such as creating new products, services, business models, and organizational forms (autio et al. ; yoo ) . as digitalization often occurs at the firm level, firms must make holistic strategic deployments (fitzgerald ) , that is, digital transformation strategies. however, it remains unclear how firms should design and implement their digital transformation strategies. further, although the significance of digital transformations has been recognized, there is still a lack of a theoretical framework that can guide firms to realize this transformation. vial ( ) asserts that digital transformation will contribute to firms' performance through the design of digital business strategies, the adoption of digital technologies, and changes of value creation paths. however, it is unclear how this process works. it is thus a promising research direction to explore digital transformation strategies as well as their impact on firms' competitive advantages. we suggest the following research questions. first, is digitalization a new source of competitive advantages for firms? second, how should firms' digital business strategies be designed? third, how does a digital business strategy guide a firm's adoption of digital technologies? finally, how does digital technology adoption change a firm's value creation paths? the covid- outbreak is a public health crisis that has posed great challenges for the survival and growth of smes. the crisis has also highlighted the important role of digital technologies in the response to the covid- outbreak. data from a questionnaire survey was used in this study to investigate the relationships among digitalization, public crisis responses, and sme performance in the context of the covid- outbreak. the results of our survey show that an sme's efforts towards digitalization, manifested by their degree of digitalization, adoption of digital technologies, and business mode can help them better respond to 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responses to crisis adaptation to climate change: from resilience to transformation case analysis of capability deployment in crisis prevention and response platform heterogeneity, platform governance and complementors' product performance: an empirical study of the mobile application industry. frontiers of business research in china computing in everyday life: a call for research on experiential computing value creation from big data: looking inside the black box. strategic organization publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. guo et al. frontiers of business research in china we appreciate the joint investigation team members for their help in questionnaire design and data collection. notes. in order to show the actual situation in a clear manner, after referring to the opinions of the entrepreneurs, the items and were implemented on a -factor subscale.authors' contributions hg joined the research design, took data collection, undertook the empirical analysis, and drafted the manuscript. zy joined the research design, undertook the empirical analysis, and drafted the manuscript. rh joined the research design, and helped to draft the manuscript. ag participated in the correction of the manuscript and provided the guidance to propose a summary theoretical framework. all authors read and approved the final manuscript. this work is supported by the national natural science foundation of china under grant . the datasets used or analyzed during the current study are available from the corresponding author on reasonable request. the authors declare that they have no competing interests. received: april accepted: july key: cord- -z e z v authors: schilling, megan a.; katani, robab; memari, sahar; cavanaugh, meredith; buza, joram; radzio-basu, jessica; mpenda, fulgence n.; deist, melissa s.; lamont, susan j.; kapur, vivek title: transcriptional innate immune response of the developing chicken embryo to newcastle disease virus infection date: - - journal: front genet doi: . /fgene. . sha: doc_id: cord_uid: z e z v traditional approaches to assess the immune response of chickens to infection are through animal trials, which are expensive, require enhanced biosecurity, compromise welfare, and are frequently influenced by confounding variables. since the chicken embryo becomes immunocompetent prior to hatch, we here characterized the transcriptional response of selected innate immune genes to newcastle disease virus (ndv) infection in chicken embryos at days , , and of embryonic development. the results suggest that the innate immune response h after challenge of -day chicken embryo is both consistent and robust. the expression of ccl , mx , and tlr in lung tissues of ndv challenged chicken embryos from the outbred kuroiler and tanzanian local ecotype lines showed that their expression was several orders of magnitude higher in the kuroiler than in the local ecotypes. next, the expression patterns of three additional innate-immunity related genes, il- , irf- , and stat , were examined in the highly congenic fayoumi (m . and m . ) and leghorn (ghs and ghs ) sublines that differ only at the microchromosome bearing the major histocompatibility locus. the results show that the ghs leghorn subline had a consistently higher expression of all genes except il- and expression seemed to be subline-dependent rather than breed-dependent, suggesting that the innate immune response of chicken embryos to ndv infection may be genetically controlled by the mhc-locus. taken together, the results suggest that the chicken embryo may represent a promising model to studying the patterns and sources of variation of the avian innate immune response to infection with ndv and related pathogens. traditional approaches to assess the immune response of chickens to infection are through animal trials, which are expensive, require enhanced biosecurity, compromise welfare, and are frequently influenced by confounding variables. since the chicken embryo becomes immunocompetent prior to hatch, we here characterized the transcriptional response of selected innate immune genes to newcastle disease virus (ndv) infection in chicken embryos at days , , and of embryonic development. the results suggest that the innate immune response h after challenge of -day chicken embryo is both consistent and robust. the expression of ccl , mx , and tlr in lung tissues of ndv challenged chicken embryos from the outbred kuroiler and tanzanian local ecotype lines showed that their expression was several orders of magnitude higher in the kuroiler than in the local ecotypes. next, the expression patterns of three additional innate-immunity related genes, il- , irf- , and stat , were examined in the highly congenic fayoumi (m . and m . ) and leghorn (ghs and ghs ) sublines that differ only at the microchromosome bearing the major histocompatibility locus. the results show that the ghs leghorn subline had a consistently higher expression of all genes except il- and expression seemed to be subline-dependent rather than breed-dependent, suggesting that the innate immune response of chicken embryos to ndv infection may be genetically controlled by the mhc-locus. taken together, the results suggest that the chicken embryo may represent a promising model to studying the patterns and sources of variation of the avian innate immune response to infection with ndv and related pathogens. keywords: backyard poultry, chicken embryo, newcastle disease virus, innate immune response, transcriptional response introduction newcastle disease virus (ndv) is one of the most important poultry pathogens worldwide, with over eighty countries in north and south america, europe, asia, and africa reporting outbreaks each year (diel et al., ) . ndv infections manifest through a wide range of strain dependent symptoms including those within the respiratory system (coughing, sneezing, and wheezing), the nervous system (such as twisted neck, tremors, and paralysis), and the reproductive system (loss in egg production). mortality rates may reach as high as % in unvaccinated flocks (ashraf and shah, ) . unsurprisingly then, ndv infections are responsible for considerable economic losses to poultry production in both developed and developing countries. for instance, the - ndv outbreak in california resulted in the destruction of million birds and financial losses of over $ million (diel et al., ) . differing levels of susceptibility to ndv have been reported among commercial breeds, as well as between commercial poultry and local ecotypes; however, the mechanisms contributing to these differences remain unknown (zhou and lamont, ; minga et al., ; deist et al., b) . for instance, studies report that the highly inbred fayoumi lines are less susceptible to ndv infection (and other infections) than single comb, highly inbred white leghorn chickens (zhou and lamont, ) . similarly, backyard chickens are generally considered less susceptible to ndv and other infections than commercial chickens that are bred for high productivity. this reduced susceptibility is presumably due to their pre-sensitization with a higher frequency of natural exposure to pathogens in the scavenging environment, as well as through natural selection for hardiness to higher levels of pathogen exposure. however, considerable variation in response to ndv infection has been noted both between and within ecotypes commonly found in backyard settings (minga et al., ) . another breed of chickens hypothesized to be less susceptible to disease is the kuroiler, a dual-purpose chicken, first bred for improving both meat and egg production of backyard poultry in india. the kuroiler, which has multi-colored feathers to help with camouflage in the wild, and thrives in backyard or scavenging environments has recently been introduced in east africa (including tanzania) with reports suggesting that it can coexist with, and out-produce the local chickens in uganda and kenya (dessie and getachew, ) . although differences in susceptibility are observed between these breeds, the underlying immune mechanisms contributing to these differences remain unknown. with the widespread use of ndv vaccines, studies of immunity have focused on antibody production and cell-mediated responses more so than innate immunity (ahmed et al., ; kapczynski et al., ) . one study demonstrated a rapid and robust innate response shortly after virulent ndv infection using a microarray analysis of chicken spleen tissue (rue et al., ) . however, the level of susceptibility to ndv was not examined in the study, hence, whether the innate immune response plays a role in enhancing immunity to ndv in poultry, and particularly in backyard poultry, is presently not understood. current techniques to evaluate the immune response and disease susceptibility in chickens through challenge of live birds are expensive and difficult to interpret due to confounding factors including age, natural exposure to infectious agents, the normal microflora, variability in dosing animals through natural routes of exposure, nutritional status, as well as exposure to other environmental stressors. in contrast, the chicken embryo, enclosed in the protective environment of the shell, is not only considerably cheaper, but also may be influenced by confounding environmental factors that may impact the results of challenge studies in hatched chicks. it has been well described that the developing chicken embryo is able to produce an immune response to a pathogen prior to hatch, a feature that is widely exploited for modern largescale poultry production with the routine administration of in ovo vaccination for multiple pathogens, including marek's disease (md) and infectious bursal disease (ibd) (sharma and burmester, ; stone et al., ; seal et al., ) . embryonic development occurs over days and by the th day, the first signs of the immune system are observed. on days and , t cells and b cells are developed, respectively, with b cell differentiation occurring after the th day of development. by the th day of embryonic development, the chicken embryo is immunocompetent and is capable of producing both an innate and adaptive response to pathogen (davison, ; ribatti, ; mississippi state university extension, ) . importantly, immune responses have been shown to be comparable in birds vaccinated in ovo or at later time points post-hatch (sharma and burmester, ; stone et al., ; gagic et al., ; sharma, ; steel et al., ) . for example, vaccination with an inactivated oil emulsion ndv and avian influenza virus (aiv) vaccine on the th day of embryonic development resulted in high seroconversion rates and antibody titers post hatch (stone et al., ). an md in ovo vaccine was also able to generate a four times greater level of protection than post hatch vaccination (sharma and burmester, ) . even though in ovo vaccination has now been applied for several decades, the mechanisms of induction of protective immunity in the chicken embryo remain poorly understood. to begin to address this knowledge gap, we here transcriptionally profiled the innate immune response of the chicken embryo to ndv infection in both highly inbred and outbred lines. our studies begin to demonstrate the use of the chicken embryo as a tool to examine the immune response to ndv since signatures of a consistent, breed-dependent innate immune response post ndv infection are present. animal use protocols were approved by the pennsylvania state university iacuc committee (protocol numbers and ). specific pathogen free (spf) eggs from white leghorn chickens were sourced through charles river laboratories international, inc. (north franklin, ct, united states). tanzanian local ecotype and kuroiler hatching eggs were sourced from urio cross and pure breeding ltd, a local farm, in tanzania (tengeru, arusha, tanzania). embryonated eggs from two well-defined, inbred leghorn sublines, ghs and ghs , as well as two inbred fayoumi sublines, m . and m . , from iowa state university poultry farm (ames, ia, united states) were also included in this study. eggs were incubated ( . • c, % humidity, rotating hourly) and only temporarily removed from incubation to candle for viability and perform inoculations with virus. the lentogenic lasota strain of ndv was kindly provided by dr. siba samal at the university of maryland, college of veterinary medicine (college park, md, united states). titration of the virus was performed with the final titer of undiluted viral suspension of % egg infectious dose (eid )/ml. the viral suspension was stored at − • c until further use. embryonated eggs were inoculated at days , , or of embryonic development, respectively, with . ml of the viral suspension directly deposited into the allantoic fluid. eggs were sealed with an adhesive glue and placed back in incubators until death or removal for tissue harvest. controls (uninfected eggs) were treated similarly with inoculation of . ml of phosphate buffered saline (pbs). eggs were candled daily once infected and embryo (including dead embryo at that time point) were randomly selected for tissue harvest , , and h post infection (hpi). the embryo selected in the group infected at days and harvested hpi were as close to hatch as possible without allowing the chick to hatch. prior to harvest, the eggs were placed at • c for - h to avoid opening eggs with viable embryos, the harvested embryo was extracted and rinsed three times in x pbs before selected tissues (based on size of the embryo) were harvested (supplementary table s ). tissues harvested at the -day infection included two sections, the body and head, as the chicken embryo was too small and underdeveloped to obtain individual tissue samples. after the -day infection, the spleen, heart, and liver were harvested, and after -day infection, the spleen, heart, liver, and lung tissues were harvested. in the experiment using the spf white leghorn eggs, tissues from three infected and three control embryo were harvested at each time point; for the kuroiler and local ecotype embryos, eight infected and eight control embryos were harvested; and for the leghorn and fayoumi sublines, six infected and six control were harvested in the ghs , ghs , and m . and eight infected and six control embryos were harvested for m . . the tissues were immediately stored at − • c prior to further use. rna from all tissues was extracted using the rneasy plus kit (qiagen inc., germantown, md, united states) following the recommended protocol once tissues were homogenized. tissue homogenization was performed using a mini-beadbeater- (biospec products, bartlesville, ok, united states) for min. each tube placed in the beat beater contained mg of respective tissue, µl of rlt lysis buffer (provided in the rneasy kit), and - . mm silica beads (biospec products, bartlesville, ok, united states). after homogenization, µl of lysate was added to a clean microcentrifuge tube and centrifuged at , rpm for min to remove any debris. the supernatant was transferred to the gdna eliminator column (provided in the rneasy kit) and the recommended protocol was followed from this point forward. cdna synthesis cdna synthesis was performed immediately after rna extraction using the rt first strand kit (qiagen inc., germantown, md, united states) for the samples used with the rt profiler array (one control and one infected embryo at days and harvested h post infection) and the high capacity cdna reverse transcription kit (applied biosystems, carlsbad, ca, united states) for all other samples at all other time points. manufacturer protocols were followed using µg of each respective rna sample. cdna was stored at − • c. the rt profiler array chicken innate and adaptive immune responses (qiagen inc., germantown, md, united states) was used as an initial screen of the immune response of embryo to ndv infection. the array contains a panel of immune-related genes from the innate (pattern recognition receptors, cytokines, other), adaptive (th , th , th , and treg markers, t cell activation, cytokines, other), humoral, and inflammatory responses, as well as the defense response to bacteria and virus (qiagen, ). all recommended kits and procedures were followed (qiagen inc., germantown, md, united states). a master mix was prepared using the rt qpcr master mix and cdna according to manufacturer protocols and added to each well of the well plate, provided by qiagen inc. qpcr was then performed using an applied biosystems stepone plus real-time pcr instrument per recommended protocols. at the pennsylvania state university, the powerup sybr green master mix (applied biosystems, carlsbad, ca, united states) with the stepone plus system (applied biosystems, carlsbad, ca, united states) was used to analyze the spf white leghorn, inbred leghorn, and inbred fayoumi samples. at the nelson mandela african institution of science and technology, the quantinova sybr green rt-pcr kit (qiagen inc., germantown, md, united states) with the quantstudio flex real-time pcr system (applied biosystems, carlsbad, ca, united states) was used to analyze the kuroiler and local ecotype samples. the recommended manufacturer's protocols were followed for both kits. the differences in protocols were due to availability of reagents and machines at nm-aist. the quantinova kit was recommended as most stable for shipment to tanzania. the primers used for analysis can be found in supplementary table s . gene expression was analyzed using the ct method comparing infected ct values (normalized with b-actin) with the average of negative ct values (normalized with b-actin) for each gene studied. the figures were generated in r using log fold change expression data obtained from the ct method for gene expression analysis. statistical analysis performed between the kuroiler and local ecotypes was performed in r using the student's t-test and between the leghorn and fayoumi sublines using the non-parametric pairwise t-test (kruskal-wallis test) in (r core team, ). gene network analysis was performed using the ingenuity pathway analysis (ipa) software (qiagen inc., germantown, md, united states ) (qiagen, ). the data was uploaded to ipa and core analysis was performed to generate the network in figure a . from this the genes with the most gene-gene interactions and highest fold changes were selected ( figure b) . the dataset was filtered for these six genes and the top diseases and biofunctions, in particular related to infectious disease and relevant to this study, were selected and the network in figure c was generated. the same networking was performed for the four sublines (figure ) . studies were performed to define the point during embryonic development when the chicken embryo is capable of producing a robust and consistent immune response to ndv, as well as to identify specific immune genes with the greatest differential expression post infection. developing chicken embryos of various ages were infected with the lasota strain of ndv via the allantoic fluid and tissues were harvested , , and h post infection (hpi) (supplementary table s ). the comparative transcriptional profile of spf white leghorn chicken embryos (one control and one infected at days, harvested hpi) was determined using the chicken innate and adaptive immune responses rt profiler array (qiagen inc., germantown, md, united states) as an initial screen to select for immune genes in the embryo that are differentially expressed during infection since studies have not been performed previously. the upregulated genes range from -fold (irf gene) to -fold (mx gene) increases, and the downregulated genes range from − -fold (il r gene) to − -fold (ccr gene) decreases (supplementary table s ). three of the genes with the highest increase in expression were selected for a more comprehensive analysis of the immune response in the embryo, c-c motif chemokine ligand (ccl ), mx dynamin like gtpase (mx ), and toll-like receptor (tlr ). these three genes are also known to play a role in the innate immune response, in particular the immune response to viral infection (qiagen, ) . this data is publically available at https://doi. org/ . /d h . once the three genes above were selected for further studies, the other collected embryo tissues from the spf white leghorns (three infected embryos and three control embryos per time point) were examined for the expression of these genes throughout the immune development of the chicken embryo (supplementary figure s and table s ). expression of all three immune genes was increased in the infected lung tissues at days of embryonic development hpi with considerably lower variation in expression than other infection time points (supplementary figure s ) . additionally, the mx gene had a greater fold increase in the lung tissues as compared to ccl and tlr (average fold changes of , . , and . , respectively). infection of the chicken embryo at days, in particular with harvest of tissues hpi, was deemed the most suitable time point for examining the immune response of the chicken embryo to ndv infection. after determining a suitable time point to study chicken embryo immune gene expression, the analysis was broadened to include the kuroiler and local ecotypes in tanzania. the same three genes, ccl , mx , and tlr , were examined in the lung tissue of the kuroiler and local ecotype embryos infected at days of embryonic development and harvested hpi (eight infected embryos and eight control embryos per line). striking differences in expression of immune genes were observed between the kuroiler and local ecotypes. the kuroiler consistently expressed ccl , mx , and tlr several orders of magnitude greater ( -, , -, and . -fold increase, respectively) than the local ecotype embryos ( . -, . -, and . -fold increase, respectively) (figure ; data for individual replicates ). pathway analysis using the ingenuity pathway analysis (ipa) software (qiagen inc., germantown, md, united states ) was performed using the rt profiler array data (expression fold change cutoff = ) to determine important pathways and networks in the response to ndv in the chicken embryo (qiagen, ) . the top canonical pathways included the role of pattern recognition receptors in recognition of bacteria and virus, inos signaling, communication between innate and adaptive immune cells, and toll-like receptor signaling (each pathway -p-values < . ). the top regulator effector network was the antiviral response. the identified associated network diseases and functions including infectious disease, the cell-mediated immune response, and organismal injury, were selected based on relevance to the immune response and merged to generate a network map of the gene-gene interactions from the rt profiler array, removing genes that were not included in the array (figure a) . from this, an additional three genes, interleukin- (il- ), interferon regulatory factor (irf ) and signal transducer and activator of transcription (stat ), were selected for future analysis since they were all highly increased in the infected chicken embryo as well as being interconnected in immune response networks ( figure b ). core analysis was performed with filtering for these genes to determine important networks of relevance to this study with ndv ( figure c) . remarkably, however, we note that even though ipa analysis has been previously applied to understand patterns of gene expression in chickens, the pathways have been validated primarily using human/mouse/rat data, and it is also recommended to use at least genes for pathways analyses, and hence, the results should be independently replicated (krämer et al., ) . next, the immune response profiles of -day ndv-infected embryos of the well-defined, highly congenic fayoumi (m . and m . ) and leghorn (ghs and ghs ) sublines were characterized (six infected embryos and six control embryos per subline -ghs , ghs , m . ; eight infected and six control for the m . subline). the congenic fayoumi and leghorn lines are highly inbred commercial poultry with inbreeding coefficients of . , differing within each line only at the microchromosome bearing the mhc (zhou and lamont, ; deist et al., b) . the fayoumi was described as less susceptible to infectious disease than the leghorn (zhou and lamont, ; cheeseman, ) . one study showed the fayoumi line (m . ) to be less susceptible to ndv than the leghorn line (ghs ) based on viral load post-challenge in ocular secretions at (but not ) days post infection (hermann et al., ; deist et al., b) , and have also identified differentially expressed genes in the trachea and lung transcriptomes of these two highly inbred chicken lines (deist et al., a,b) . the results of our current investigation show striking differential expression of select genes in a subline-dependent manner with ghs having consistently higher expression of all genes except il- (figure ; data for individual replicates ). importantly, the results show that the gene expression differences seem to be subline-dependent even more so than breed dependent. similar analysis was performed, as in figure c , with the average fold expression differences between challenged https://doi.org/ . /d h and control lung tissue in the inbred leghorn and inbred fayoumi sublines using ipa (qiagen inc., germantown, md, united states ). interestingly, the high responders, ghs and m . (figures a,b, respectively) , mimic similar patterns as the rt profiler array analysis (figure ) . the low responders, ghs and m . (figures c,d, respectively), differ especially due to the downregulation of one or more of these genes, stat , ccl , and irf , was predicted to increase viral replication and infection in the ghs and m . sublines (qiagen, ). until now, most studies performed to examine the immune response of chickens and other avian species have been through expensive and laborious in vivo experiments or through in vitro cell culture experiments lacking host-pathogen interactions. the animal studies, requiring enhanced biosecurity facilities, are influenced by multiple confounding factors producing results that are sometimes difficult to interpret. therefore, despite current work to examine the chicken immune response to ndv, a knowledge gap remains in the mechanisms of the immune response and the effects they have on susceptibility of chickens to infection. in the present study, we utilized the developing chicken embryo as a controlled and inexpensive approach to evaluate the innate immune mechanisms in response to ndv infection in chickens. through these studies we have found evidence that the chicken embryo is capable of producing a robust signature of the immune response, in particular the innate immune response, to ndv infection, at least after day of embryonic development. in particular, the suitability of the chicken embryo for immune investigation was confirmed through gene expression studies where variation in gene expression was greatly reduced at -day infection as compared to earlier infection time points (supplementary figure s ). since the chicken embryo is immunocompetent and the respiratory system almost fully developed a few days prior to hatch, examining the response of the chicken embryo to respiratory viruses such as ndv may be possible without the introduction of other confounding variables post-hatch. and m . ) sublines. the six immune genes were examined in the lung tissues of each subline (ghs , ghs , m . n = infected and controls per subline; m . n = infected and control). the red line signifies the cutoff between upregulation and downregulation of the genes. statistical analysis was performed according to the materials and methods data analysis and significant p-values noted by the superscripts (p-value < . ) can be found in supplementary table s . using this more controlled approach to examine the chicken immune response, we were able to demonstrate that the innate immune response to ndv in the chicken embryo appeared to be breed- (figure ) and/or subline-dependent (figure ) , with the possibility of having a relation to the mhc type demonstrated by differences in the inbred fayoumi and inbred leghorn sublines, which only differ in the mhc-bearing chromosome. if the gene expression is, in fact, mhc type dependent, this figure | network analysis of the six selected immune genes in the leghorn and fayoumi sublines using ipa software. the data used for this analysis is from the gene expression data from each fayoumi and leghorn subline in figure . the red colored circles represent relative increases in expression of that gene in the infected chicken embryo lung tissues versus control and green circles represent downregulation. dashed lines indicate direct relationships and solid lines indicate indirect relationships. the blue lines represent inhibition, orange represents activation, and yellow represents inconsistent findings with downstream molecules. ghs (a) and m . (b) the highest responders have similar patterns in the network analysis, and ghs (c) and m . (d) show deviations from this network due to the downregulation of some genes. could explain some of the large within breed variation in expression demonstrated by the tanzanian local ecotypes and the kuroiler. these differences in the transcriptional response to ndv infection also demonstrate a possible relation between the innate immune gene expression and level of susceptibility of a particular line, since previous reports demonstrate the ghs leghorn subline and m . fayoumi subline differ in the level of susceptibility to ndv (deist et al., b) . this study also showed il- and mx to be differentially expressed between the two sublines studied (deist et al., b) . a more recent investigation of differential gene expression in the lung transcriptome of these same chicken lines in response to ndv infection also, demonstrated an activation of the il- pathway in the resistant fayoumi line, m . (deist et al., a) . while these previous studies used rna-seq based approaches and longer time-points (days rather than hours) post-infection and performed these studies in hatched chicks and are hence not directly comparable, the similar patterns of expression of at least some of the loci observed in the chick embryo model are encouraging, and need to be confirmed in future investigations. multiple other studies have each discovered at least one of the six genes (mx , ccl , tlr , il- , irf , and stat ) studied here with elevated expression levels in the chicken particularly in response to ndv, aiv, and ibv demonstrating the key role of these innate immune genes in the chicken's response to pathogen (heidari et al., ; rue et al., ; matulova et al., ; cheng et al., ; kang et al., ; ranaware et al., ; deist et al., b) . although the network analysis was only performed with small numbers of genes, relevant pathways to our study were generated. most surprising were the pathways, top diseases, and disorders associated with the genes including ndv infection, paramyxovirus replication, and lung infection since they are closely associated with the study of ndv infection in the chicken embryo lung directly. another significant function of relevance to this study was susceptibility to infection. since determining the feasibility of using the chicken embryo as a new approach to explore the immune response and susceptibility to ndv infection is an ultimate goal of our project, revealing a significant involvement of stat and tlr in this response is promising for future studies. other diseases and disorders include rna virus infection and replication (ndv is an rna virus), measles virus infection and replication (a paramyxovirus, like ndv), and influenza a virus infection (an rna virus of the respiratory tract) (qiagen, ) . in most cases, tlr , stat , and irf are involved in signaling pathways that lead to stimulation of the end targets, ccl , mx , and il- , which then go on to stimulate other immune related cells or pathways (qiagen, ) . multiple studies have recognized the type-i interferon (ifn) inducible response, protecting cells against invading viral pathogens, as important in the innate immune response in chickens, and the six genes examined in this study are involved in that response (xing et al., ; schoggins et al., ; ranaware et al., ) . the six genes studied here have been shown to be important in the chicken immune response to viral pathogens, whether it is ndv, aiv, or ibv (ko et al., ; benfield et al., ; sartika et al., ; cong et al., ; barjesteh et al., ; cheng et al., ; dou et al., ; fulton et al., ; ranaware et al., ; deist et al., a,b) , which demonstrates how the chicken embryo immune response starts to mimic that of hatched chickens, validating the use of the chicken embryo for future studies of the immune response to avian pathogens. the innate immune response is a complex and interconnected network that is dependent on many factors. although the six genes examine in this study are involved in the innate immune response and there are direct interactions between these genes, further validation of the response, including a larger set of innate immune genes, is needed in order to obtain a better understanding of the innate immune mechanisms influencing the chicken embryo and chicken's response to ndv infection. it is interesting to note the differences seen in the pathway analysis from the leghorn and fayoumi sublines and a possible role for these genes in determining the level of susceptibility of a particular line to ndv infection. most interestingly, the ghs subline, previously found to be susceptible to ndv, differs from the other sublines pathway analysis by activating rna virus replication rather than inhibiting it (qiagen, ; deist et al., b) . since only the ghs and m . sublines have been previously characterized for the level of susceptibility to ndv, expanding research in this area to include the other two sublines may provide a better insight into the use of the chicken embryo immune response as a tool to screen for the level of susceptibility to ndv of a particular line, and the role of the mhc complex. it is especially intriguing since the high responders, ghs and m . , differ in the network analysis from the low responder, ghs . a more comprehensive study of the innate immune response of the different chicken lines, including a larger set of genes and larger sample sizes, and correlating these responses to the innate immune response and level of susceptibility of hatched chicks, using phenotypic characteristics such as viral load, mean death time, viral shedding, and antibody titers post ndv infection, will help provide insight into innate immune mechanisms of susceptibility to ndv. other important future studies include assessing whether similar patterns in response using more pathogenic field strains which is especially important in regards to the response in backyard 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infection with salmonella enterica serovar enteritidis biodiversity (variation) in disease resistance in pathogens within rural chicken populations stages in chick embryo development innate and adaptive immune responses pcr aray genome wide host gene expression analysis in chicken lungs infected with avian influenza viruses the chick embryo chorioallantoic membrane in the study of angiogenesis virulent newcastle disease virus elicits a strong innate immune response in chickens selection of mx gene genotype as genetic marker for avian influenza resistance in indonesian native chickens a diverse range of gene products are effectors of the type i interferon antiviral response the avian response to newcastle disease virus introduction to poultry vaccines and immunity disease control in avian species by embryonal vaccination. usa patent application a combination in-ovo vaccine for avian influenza virus and newcastle disease virus in ovo vaccination of chicken embryos with experimental newcastle disease and avian influenza oil-emulsion vaccines modulation of the immune responses in chickens by low-pathogenicity avian influenza virus h n genetic characterization of biodiversity in highly inbred chicken lines by microsatellite markers this research was supported by grant (opp ) from the bill and melinda gates foundation (to vk and ms) and usaid feed the future innovation lab for genomics to improve poultry and hatch project # , usda nifa - - (to sl). the authors thank the kapur lab, especially dr. lingling li, for her guidance and support throughout the study. they thank dr. huaguang lu and dr. suresh kuchipudi and their lab groups for their guidance and technical assistance with the chicken embryo experiments. they also thank dr. beatus lyimo for his support during experimentation at the nelson mandela african institution of science and technology in arusha, tz. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fgene. . /full#supplementary-material key: cord- - gth o authors: puzzitiello, richard n.; pagani, nicholas r.; moverman, michael a.; moon, andrew s.; menendez, mariano e.; ryan, scott p. title: inflammatory and coagulative considerations for the management of orthopaedic trauma patients with covid- : a review of the current evidence and our surgical experience date: - - journal: j orthop trauma doi: . /bot. sha: doc_id: cord_uid: gth o mounting evidence suggests that the pathogenesis of coronavirus disease (covid- ) involves a hyperinflammatory response predisposing patients to thromboembolic disease and acute respiratory distress. in the setting of severe blunt trauma, damaged tissues induce a local and systemic inflammatory response through similar pathways to covid- . as such, patients with covid- sustaining orthopaedic trauma injuries may have an amplified response to the traumatic insult because of their baseline hyperinflammatory and hypercoagulable states. these patients may have compromised physiological reserve to withstand the insult of surgical intervention before reaching clinical instability. in this article, we review the current evidence regarding pathogenesis of covid- and its implications on the management of orthopaedic trauma patients by discussing a case and the most recent literature. the coronavirus disease (covid- ) is a novel viral illness that is precipitated by the severe acute respiratory syndrome coronavirus (sars-cov- ). as of april , , covid- has been diagnosed in nearly . million people worldwide, has led to over , deaths, and has been deemed a pandemic by the world health organization. in its most severe form, covid- is characterized by a cytokine release syndrome (crs) that progresses to multisystem organ failure and death. , recent reports also demonstrate that patients are predisposed to thromboembolic disease through both the direct and indirect effects of covid- . , although some patients require early intensive care, the majority experience a more benign clinical course. a certain subset of patients, however, mount a large inflammatory response despite initially appearing well. early monitoring of inflammatory markers is being used to help predict which patients will eventually necessitate higher levels of care. a similar hyperinflammatory and hypercoagulable response leading to multisystem organ failure is also seen in patients after severe polytrauma. [ ] [ ] [ ] a less severe initial traumatic insult ("first hit") also has the capacity to produce the same systemic response if it is followed by persistent physiologic derangements or subsequent proinflammatory interventions ("second hit"). , , damage control orthopaedics is often used in polytrauma patients to minimize the second hit and prevent subsequent acute respiratory distress syndrome (ards). [ ] [ ] [ ] although a trauma is often thought of as the first hit in the two-hit theory, other hyperinflammatory and hypercoagulable states, such as in covid- , may also act as part of the first hit in patients with orthopaedic injuries. this may be especially relevant in patients who appear well but have developed a large inflammatory response. indeed, recent evidence suggests alarmingly high intensive care unit (icu) admission and mortality rates after elective surgery on asymptomatic patients in the incubation period of covid- . , however, little is known about how orthopaedic trauma and subsequent fracture fixation modulates the inflammatory response in patients with covid- . a better understanding of this relationship can inform the development of evidencebased management strategies in these patients and limit admissions to overcrowded icus. to demonstrate and further define these developing theories on the coagulative and inflammatory risks associated with the surgical treatment of trauma patients with covid- , we will present an unexpected outcome on such a patient at our institution. the purpose of this case presentation is to drive the subsequent discussion and literature review on the management of patients with covid- presenting with orthopaedic trauma. a woman in her s with a history of dementia and myeloproliferative disorder presented with an isolated extraarticular fracture of the left distal femur (fig. ) and acute nonocclusive deep vein thromboses (dvts) of the left popliteal and gastrocnemius veins. on presentation, she was afebrile and denied any respiratory symptoms. her preoperative peripheral capillary oxygen saturation was between % and % on room air and at times requiring l of supplemental oxygen. physical examination revealed that the patient not to be in respiratory distress with clear lung sounds. admission chest radiograph is shown in fig. , demonstrating no major consolidation or infiltrates. laboratory evaluation showed no leukocytosis, anemia, or thrombocytosis. given that she was a nursing home resident with known exposure to multiple covid- -positive residents, she was tested for the disease and found to be positive. a multidisciplinary approach was taken for the care of this patient, including orthopaedics, internal medicine, infectious disease, anesthesia, and vascular surgery. she was deemed an asymptomatic covid+ patient with no concern for her respiratory function. a shared decision was made to proceed with surgical fixation to allow for improved mobility, healing, and pain control. within hours of initial presentation, the patient underwent reamed, locked retrograde intramedullary nailing of her left distal femur fracture. intramedullary nailing was chosen because it is the senior author's preferred treatment for distal third femur fractures and the intramedullary implant would assist in preventing medialization of the distal segment. preoperative templating estimated an isthmus of - mm. plan was for placement of a -mm diameter nail. the patient tolerated the early steps of the procedure well. during passage of the -mm diameter reamer and obtaining cortical chatter, the patient became acutely hypoxic and hypotensive requiring maximal fio (p/f ratio ) and increasing vasopressor requirements. after reaming, the patient improved marginally to the point where she was amenable to intramedullary nail placement. after placement of the intramedullary nail, a heparin drip was immediately initiated for presumed intraoperative pulmonary embolism. she remained intubated at the completion of the procedure and was transferred to the icu. laboratory values obtained immediately postoperatively are provided in table , along with postoperative laboratory trends. the patient's initial leukocytosis and elevated troponin, brain natriuretic peptide, fibrinogen, d dimer, ferritin, and c-reactive protein (crp) are noted. the patient's p/f ratio at that time declined to (pao mm hg, fio %), indicating severe respiratory failure. upon arrival to the icu, a bedside echocardiogram was performed revealing the right ventricular dilation and septal flattening indicative of right heart strain. computed tomography pulmonary angiogram demonstrated a nonocclusive right main pulmonary artery embolus with right heart strain, left upper lobe segmental pulmonary artery embolus, and mosaic, ground glass attenuation of the lung parenchyma concerning for viral pneumonia and fat embolism. subsequent pulmonary angiography redemonstrated the right-sided lobar embolus and elevated mean pulmonary artery pressure. right-sided percutaneous pulmonary suction thrombectomy was performed. blood clot and fat emboli were removed with no significant residual lobar or segmental pulmonary emboli on follow-up angiogram (fig. ). the patient's relatively small embolic burden did not correlate with her clinical presentation of respiratory failure with right heart strain. in addition, the patient's hemodynamic response and lack of improvement after embolectomy was not characteristic of other experiences with similar volumes of thrombus or fat extraction. the patient required inotropic support for the first hours postoperatively. she was successfully extubated on postoperative day and transferred out of intensive care on postoperative day . although the orthopaedic surgeons' role in mitigating the covid- crisis may appear disparate compared with our medical colleagues, the management of patients with covid- undergoing nonelective orthopaedic trauma surgery demands thoughtful consideration. emerging evidence in the medical literature suggests that a cytokine storm, also known as crs, plays an integral role in severe covid- . severe blunt trauma and the resulting surgical intervention similarly initiate a sequence of inflammatory events resulting in clinical instability. in the case we have presented, an asymptomatic covid- patient with a femur fracture urgently treated with intramedullary fixation, as is the standard of care for the treatment of femoral shaft fractures. intraoperatively, she developed pulmonary and fat emboli resulting in a systemic hyperinflammatory response and acute cardiopulmonary collapse. our hypothesis is that the patient's diagnosis of covid- amplified the initial inflammatory response to the low-energy traumatic insult ("first hit") that was not clinically apparent preoperatively. in addition, the hypercoagulable state secondary to covid- and the inflammatory load of intramedullary reaming, fat emboli, and pulmonary embolism resulted in a "second hit" that may have cumulatively pushed our patient past a "tipping point" and into respiratory failure (fig. ) . we would not have expected this type of response during intramedullary fixation of a low-energy fracture in a covid-negative patient without any preoperative respiratory symptoms or illness. the clinical presentation of covid- resembles viral pneumonia, with severe cases rapidly progressing to ards. crs is implicit in the pathogenesis of these severe cases of covid- . although the full immunologic response elicited by covid- is still not fully elucidated, current reports indicate elevations of a distinct set of proinflammatory cytokines, including interleukin (il)- , il- , and tumor necrosis factor (tnf)-a. , treatment protocols for severe covid- are aimed at attenuating this life-threatening inflammation, such as with the il- -inhibiting agent tocilizumab. , these treatments are currently reserved for the population of patients with a declining clinical status paired with worsening inflammatory markers. the overwhelming inflammatory response in these patients is believed to cause diffuse alveolar damage and endothelial dysfunction. the dysfunctional endothelium thus becomes prothrombotic, which predisposes patients to microangiopathy and microthrombi. , the clinical implications of this are profound, as the presence of vasculitis and prothrombotic state can make patients vulnerable to pulmonary embolism, which can exacerbate hypoxemia caused by ards. this is a crucial point for consideration in asymptomatic covid+ patients presenting with orthopaedic trauma, as we hypothesize that subclinical levels of systemic inflammation from covid- may predispose to adverse outcomes. this theory is supported by a recent report out of wuhan that has demonstrated alarmingly high mortality and icu admission rates after elective surgery on asymptomatic patients in the incubation period of covid- . in addition to these microvascular aberrations, significant coagulation abnormalities appear to be associated with the crs and implicated in disease progression. , , , , these hemostatic derangements include increased clot strength, increased fibrinogen and fibrin degradation product levels, elevated d-dimer levels, decreased prothrombin time and international normalized ratio times, as well as patterns of disseminated intravascular coagulation. [ ] [ ] [ ] such changes also predispose these patients to thrombotic events such as venous thromboembolism, much like the previous zoonotic virus outbreaks [sars and middle east respiratory syndrome (mers-cov)]. , this is convincingly demonstrated by an amounting number of reported cases of young patients with large-vessel strokes as a presenting feature of covid- . the most commonly observed hemostatic abnormality in these patients is elevated d-dimer levels (. mg/ml), which have specifically been associated with an increased risk of icu admission, mechanical intubation requirement, and death. , for these reasons, use of empiric anticoagulation at therapeutic doses on patients with highly elevated d-dimer levels is being implemented by some intensivists and is currently supported by some experts in the american college of cardiology. in fact, early reports have indicated decreased mortality in severe covid- patients with coagulopathy who were treated with anticoagulation. it is possible that the coagulation abnormalities associated with this disease may have contributed to the development of the acute dvts in our patient, and that the intraoperative initiation of a heparin drip may have attenuated the effects of pulmonary emboli. these coagulative effects of covid- and proposed benefits of anticoagulative therapies are important to consider when treating orthopaedic trauma patients. in covid+ patients presenting with orthopaedic trauma, the hyperinflammatory and hypercoagulable state caused by the virus may also have significant implications on blunt injury pathophysiology. after severe blunt trauma, damaged tissue induces a local and systemic inflammatory response mediated by the release of the cytokines tnf-a, il- b, and, most importantly, il- . the severity of this inflammatory response and subsequent clinical course is determined by the following factors: ( ) the degree of the initial injury ("first hit"), ( ) the individuals' amounted biological response, and ( ) the type of treatment ("second hit"). these factors contribute toward an amounting inflammatory cascade that increases until a patient's biologic reserve is overwhelmed, and a "tipping point" is reached (fig. ) . the "tipping point" refers to a state of clinical instability associated with microvascular injury, interstitial edema, hemodynamic lability, and end-organ failure. the "first hit" can be reliably quantified in traumatized patients by measuring il- levels, which have been shown to be correlated with increased incidence of multiple organ failure, and patient survival. emerging studies on covid- have similarly observed correlations between il- levels and disease severity, which suggests a potential mutual inflammatory pathway, with that of trauma patients (although stemming from a distinct inciting event). , therefore, covid- may decrease a trauma patient's biologic reserve before reaching a physiologic "tipping point" (fig. ) . in other words, covid- may unfavorably amplify the "first hit" by contributing a significant biologic response before the injury, whether the patient is symptomatic or not. this may manifest clinically as decreased cardiopulmonary capacity in these patients, albeit to variable extents based on the severity of their disease. this hypothesis is supported by a recent case series, demonstrating a % mortality rate in symptomatic covid+ fracture patients. among the aforementioned factors contributing toward the "tipping point," the treatment is the sole modifiable factor. in particular, long bone fractures treated with intramedullary fixation are at risk for fat embolization in addition to the inflammatory response from this "second figure . products removed from percutaneous pulmonary suction embolectomy. the yellow products are presumed intramedullary fat, and the red products are the clot burden. image quality is suboptimal because the camera was required to be in a plastic bag due to covid+ status. hit." , these events have been demonstrated to result in pulmonary insult, changes in markers of coagulation, and, at times, cardiovascular strain. the coagulative effects are due to simultaneous activation of both the fibrinolytic and coagulation pathways, , and the inflammatory effects are mediated by il- . as for the cardiopulmonary effects of intramedullary fixation, intraoperative measurements on humans using transesophageal echocardiogram and cardiopulmonary monitoring have demonstrated consistent pulmonary arterial pressure elevations, and in severe cases-significant hypoxemia and right heart strain, during guide-wire insertion and canal reaming. , these findings are mostly attributable to fat emboli, which occur in approximately % of trauma patients but are only clinically apparent in %- %. in addition, ards is a potential consequence of the inflammatory response from intramedullary fixation of femur fractures. , current outcomes data in the aforementioned study on elective surgery outcomes of asymptomatic covid+ patients, they reported a . % ( of ) icu admission rate postoperatively, which is significantly higher than the reported . % in hospitalized nonsurgical covid- patients. furthermore, the reported . % ( of ) mortality rate was significantly higher than the overall case-fatality rate of . % in nonsurgical covid- patients. patients in that study also developed covid symptoms on average . days postoperatively, and the median time from symptom onset to the development of dyspnea was . days. in comparison, a previous study on nonsurgical covid- patients reported that the median time for symptom onset was . days. the difference is alarming. the most common postsurgical complication in the icuadmitted patients in that study was ards, and over half of these patients received subsequent immunosuppressive medications to attenuate the diseases' inflammatory response. the authors of this study reached a similar conclusion as ours that surgical stress occurring during the incubation period of sars-cov- infection exacerbates disease progression and severity. another recent study out of china by mi et al reported on covid+ fracture patients, of which did not have any signs or symptoms of the disease. three patients underwent surgical fixation of their fractures, and the rest were treated nonoperatively because of their declining clinical status. they reported that of these patients died at and days after admission. abnormal d-dimer levels were present in all patients, elevated crp in % of patients, but normal prothrombin times in % of patients. the authors of this study also concluded that the characteristics and prognosis of covid- patients with fractures tend to be more severe than those reported for covid+ patients without fractures. most recently, catellani et al published their outcomes in italy on the treatment of proximal femoral fragility fractures in patients with covid- . they reported on symptomatic covid+ patients with confirmed viral pneumonia on chest computed tomography. three of these patients died of respiratory failure before surgery, the remaining series underwent surgical stabilization of their fractures with either an intramedullary nail or hemiarthroplasty. nine of these patients were stable postoperatively, whereas patients died of respiratory failure on the first, third, and seventh day postoperatively. the authors of this study concluded that surgical stabilization of proximal femoral fractures resulted in stabilization of respiratory parameters but did mention that elderly patients with comorbidities and symptomatic covid- are not eligible for orthopaedic surgery. the current environment in the health care system is unprecedented. there are little to no data to guide us in our decision making when treating patients with covid- . the disease manifests in ways we would have never been able to predict. the level of cytokine response, hypercoagulability, and pulmonary dysfunction associated with the covid- virus may predispose to a catastrophic "second hit" after even low-energy trauma. this is in line with the previous hypotheses that postsurgical complications are more accurately predicted by assessing objective data covering several physiological systems (coagulation, acid-base changes, softtissue damage, etc.) compared with using data from a single physiologic system (eg, acidemia). regarding our patient, we hypothesize that covid- may have lowered her physiologic reserves to withstand the relatively low intraoperative embolus burden. interestingly, not only did the clot burden fail to correlate with the patient's physiologic status after nailing, she did not show any significant improvement after embolectomy of the thrombus and fat, indicating another disease process such as ards. the following precautions may be appropriate when dealing with unprecedented challenges associated with covid- patients presenting with orthopaedic trauma injuries. . test for covid- in all patients with unknown disease status upon admission. ing il- (if available), d dimer, and crp, which may aid in surgical decision making and prognosis. . consider obtaining lower extremity duplex ultrasound in all patients testing positive for covid- and high-risk fractures. . for patients with confirmed proximal dvt, consider either aggressive intraoperative anticoagulation or placement of an inferior vena cava filter preoperatively. . consider alternative orthopaedic trauma management strategies (eg, damage control orthopaedics and nonoperative treatment) in patients with severe cases of symptomatic covid- , even in low-energy trauma. . consider surgical treatments that avoid canal instrumentation. . consider avoiding excessive reaming if intramedullary fixation is performed. mounting evidence suggests that the pathogenesis of covid- involves a hyperinflammatory response predisposing patients to thromboembolic disease and acute respiratory distress. in the setting of severe blunt trauma, damaged tissues induce a local and systemic inflammatory response through similar pathways to covid- . as such, patients with covid- sustaining orthopaedic trauma injuries may have an amplified response to the traumatic insult because of their baseline hyperinflammatory and hypercoagulable states. careful consideration and risk/benefit analysis, including preoperative evaluation of systemic inflammation and respiratory status, is paramount in patients with covid- presenting with orthopaedic trauma injuries. covid- and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up covid- ): situation report covid- : consider cytokine storm syndromes and immunosuppression covid- infection: the perspectives on immune responses the science underlying covid- : implications for the cardiovascular system clinical characteristics of coronavirus disease in china correlation analysis between disease severity and inflammation-related parameters in patients with 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authors: krieger, elizabeth; vissichelli, nicole; leichtle, stefan; kashioris, markos; sabo, roy; brophy, don; wang, xiang-yang; kimbal, pamela; neale, michael; serrano, myrna g.; buck, gregory a.; roberts, catherine; qayyum, rehan; nixon, daniel; grossman, steven; toor, amir a. title: immunological determinants of clinical outcomes in covid- : a quantitative perspective date: - - journal: nan doi: nan sha: doc_id: cord_uid: zc u g severe acute respiratory syndrome coronavirus (sars-cov- ) has a variable clinical presentation that ranges from asymptomatic, to severe disease with cytokine storm. the mortality rates also differ across the globe, ranging from . - %. this variation is likely due to both pathogen and host factors. host factors may include genetic differences in the immune response genes as well as variation in hla and kir allotypes. to better understand what impact these genetic variants in immune response genes may have in the differences observed in the immune response to sars-cov- , a quantitative analysis of a dynamical systems model that considers both, the magnitude of viral growth, and the subsequent innate and adaptive response required to achieve control of infection is considered. based on this broad quantitative framework it may be posited that the spectrum of symptomatic to severely symptomatic presentations of covid represents the balance between innate and adaptive immune responses. in asymptomatic patients, prompt and adequate adaptive immune response quells infection, whereas in those with severe symptoms a slower inadequate adaptive response leads to a runaway cytokine cascade fueled by ongoing viral replication. polymorphisms in the various components of the innate and adaptive immune response may cause altered immune response kinetics that would result in variable severity of illness. understanding how this genetic variation may alter the response to sars-cov- infection is critical to develop successful treatment strategies. severe acute respiratory syndrome coronavirus (sars-cov- ) has a variable clinical presentation that ranges from asymptomatic, to severe disease with cytokine storm. the mortality rates also differ across the globe, ranging from . - %. this variation is likely due to both pathogen and host factors. host factors may include genetic differences in the immune response genes as well as variation in hla and kir allotypes. to better understand what impact these genetic variants in immune response genes may have in the differences observed in the immune response to sars-cov- , a quantitative analysis of a dynamical systems model that considers both, the magnitude of viral growth, and the subsequent innate and adaptive response required to achieve control of infection is considered. based on this broad quantitative framework it may be posited that the spectrum of symptomatic to severely symptomatic presentations of covid represents the balance between innate and adaptive immune responses. in asymptomatic patients, prompt and adequate adaptive immune response quells infection, whereas in those with severe symptoms a slower inadequate adaptive response leads to a runaway cytokine cascade fueled by ongoing viral replication. polymorphisms in the various components of the innate and adaptive immune response may cause altered immune response kinetics that would result in variable severity of illness. understanding how this genetic variation may alter the response to sars-cov- infection is critical to develop successful treatment strategies. the coronavirus disease (covid- ) pandemic of has created a challenge to humanity like none other in recent times. with its global reach, infections with the severe acute respiratory syndrome coronavirus (sars-cov- ) have exacted a tremendous toll from populations around the world. this single-stranded rna virus gains access to the host intracellular milieu through interaction with the angiotensin-converting enzyme (ace- ) which is expressed on a variety of epithelial and endothelial cells. not only is this novel virus associated with higher mortality than other respiratory viruses, such as influenza, but it also demonstrates a broader variation in its clinical presentation. at disease onset, the symptoms are generally mild and restricted to the respiratory tract. still, later, secondary viremia may involve other organ systems expressing ace- , including the cardiovascular, nervous, and renal systems. several patients experience an exaggerated immune response in the form of cytokine release syndrome. despite this, a vast majority of patients with covid- may be asymptomatic or only mildly symptomatic, with the remainder experiencing a range of clinical manifestations, from moderate-to-severe, life-threatening. mortality rates are high in the elderly and those with comorbidities. still, the young and otherwise healthy may also develop severe disease. there is wide variability in casefatality rates in different populations around the united states ( figure a ) and the world, with rates as high as - % seen in italy, england, and spain, but as low as . - % in iceland, norway and japan (figure b) . this variation in the global death toll from this infection, despite testing and reporting biases, may suggest a genetic component in disease susceptibility. this variation may represent differences in the relative proportions of tested versus non-tested populations, socio-economic, comorbid conditions, and differential public health practices. it may be postulated that the disparity in disease severity could be at least in part due to genetic differences in the innate and adaptive immune responses that individuals may mount to sars-cov- . immune response to sars-cov- , a general overview covid symptomatology may be due to a combination of pathogen and host factors. progression to the more severe forms of disease not only dependent on pathogen factors such as viral inoculum and virulence but, most likely, also on host factors that lead to variable immune response. patients with adequate immune function likely contain the virus without over-reaction. however, in those with severe manifestations of covid , a dysfunctional immune response is frequently observed. the immune system has co-evolved with a multitude of historical pathogens, inherent with redundancies to counteract pathogens which have developed multiple mechanism of immune evasion. this has led to the development of genetic differences and polymorphisms which may explain the variability of the immune response in some patients within the host immune pathway (supplementary table ). it is, therefore, critical to explore the intricacies of the pathways of the immune response to understand the differences in disease severity further. the immune response to viral infections is carefully orchestrated between the innate and adaptive responses, and various steps are required to attain control. failure or suboptimal function of any of the components could lead to an inadequate response. the innate response initially recognizes pathogen-associated molecular patterns (pamp) associated with viruses through pattern recognition receptors (prr) in the tissue-resident antigen-presenting cells (apc), such as macrophages and dendritic cells (figure ) . examples of prrs include toll-like receptor (tlr) - & - , which recognize single-stranded rna, and rna-sensing retinoic inducible gene (rig- ). nod-like receptors (nlr) also help by sensing damage-associated molecular patterns. polymorphisms in the tlr and its downstream signaling molecules such as myd may alter responses triggered pamp recognition. the identification of viral pathogens by tissue macrophages initiates a cascade of cytokine secretion. these cytokines inactivate viral replication through type i interferons (ifn-a & ifn-b), and help activate the next line of innate immune defense, the natural killer (nk) cells, with their repertoire of activating and inhibiting receptors. the nk cell receptors include killer immunoglobulin-like receptors (kir) and the lectin-like, nkg family of receptors, among others. kir receptors recognize the downregulation of human leukocyte antigen (hla) molecules on infected cells and mediate cytotoxicity. nkg receptors recognize non-classical hla molecules, such as hla-e. the innate immune effectors trigger a two-pronged hla dependent adaptive immune response. the hla genes exhibit extreme allelic polymorphisms and present viral peptides on host hla molecules to t cells to trigger an adaptive immune response. the t cell response involves both hla class i and ii molecules, which engage cytotoxic t cell (tc) and helper t (th) cell populations, respectively. the latter promotes both the cytotoxic t cell proliferation, as well as, b cell immunity and generation of sars-cov- specific antibodies. polymorphisms in the hla molecules result in differential antigen binding and presentation, leading to variability in immune responses. antibody production is the task of the b cell arm of the adaptive immune system, which with their unique b cell receptors, engage antigenic epitopes, either solubilized or directly presented by macrophages and dendritic cells. in response, b cells proliferate and differentiate into memory b cells and plasma cells facilitated by interaction with th cells, establishing long term humoral immune response. this humoral immune response is crucial to providing control for any viral infection, including sars-cov- . a subset of patients may not develop long-lasting antibodies and remains unclear if they are at risk for recurrent infection. polymorphisms in the hla and kir haplotypes may be responsible for the significant worldwide variability in the immune response to covid . in other viruses, genetic variation among hla alleles and kir confer differing susceptibility. for example, hla-b* : has been linked to the development and increased severity of sars-cov- . hla-a* : may prevent hiv seroconversion, and hla-b* : -hla-c* : had a protective effect on the progression of hiv in japanese patients. a study using in silico modeling to predict viral peptide-mhc class i binding affinity across all known hla -a, -b, and -c genotypes, has shown that hla-b* : has the fewest predicted binding peptides to sars-cov- . this finding suggests that patients with hla-b* : may be at increased risk for infection with sars-cov- . thus, global haplotypes variation across populations may partly explain the difference in illness severity and case-fatality rates. a similar variety may be observed in the kir haplotype frequencies. as an example, decreased kir dl expression has been linked to increased susceptibility to sars-cov- and kir dl homozygosity in association with its ligand hla-c , is associated with reduced disease progression of hepatitis c virus. the kir dl gene is present in % of europeans and % of ethiopians. % and % of these populations respectively are, at least, heterozygous for its ligand, c . similar hla alleles and hla epitope binding site effects have been observed in dengue fever, transplant outcomes. t cell and b cell repertoire diversity and symptom severity in covid- patients in addition to these innate immune pathways, adaptive immunity with its t and b cell repertoire diversity is critical to mount an adequate immune response to viral infections. the mammalian t and b cell repertoire diversity and ability to recognize pathogen-derived antigens, either directly (b cell receptors) or upon presentation by hla (t cell receptors), is derived from a unique process of t and b cell receptor gene rearrangement. each of these genetic loci (for the immunoglobulin heavy and light chain and t cell receptor a & b loci respectively) are comprised of variable joining and diverse gene segments. these segments are recombined and further modified through the addition of non-templated nucleotides to yield an enormous repertoire of t and b cell receptor-bearing clones capable of recognizing the pathogens associated antigens with high precision and fidelity. a decline in repertoire diversity with age may create dominant oligoclonal t and b cell populations and predispose the elderly to develop symptoms with potentially higher severity of illness. further, inadequate t and b cell clonal responses in immunocompromised patients may put patients at risk for insufficient viral clearance, and a prolonged, severe, clinical course. as noted above, patients with covid- develop significant lymphopenia, which may be due to a pan-t cell-suppressive effect. a reduction in circulating t cells may represent migration to the site of inflammation, hyperfunction, or direct virally mediated lethality, and it is known that t cells also express markers of exhaustion in the face of a severe viral infection. inflammatory cytokines during cytokines are key signaling molecules that orchestrate cellular and humoral immune responses to viral infections. the cascade of pro-inflammatory cytokines is triggered by the engagement of tlr on macrophages and by nk cells not finding their cognate autologous inhibitory ligands on infected cells. this first wave of proinflammatory cytokines includes tnf-ail- , il- , il- , il- , and ifn-g. these stimulate th cells to produce downstream cytokines, such as the pro-inflammatory il- , and , and the anti-inflammatory il- and . these cytokines provide the critical third signal to trigger both th and tc responses to clear the infected cells. however, a generalized systemic inflammatory response may also be observed in viral infections. while optimal cytokine secretion will trigger an appropriate immune response, excessive cytokine release, i.e., "cytokine storm," may develop in some patients with covid- , resulting in fulminant disease with high mortality. sars-cov- may also infect monocytes and dendritic cells, altering the cytokine expression patterns and contributing to lymphopenia observed. importantly, this results in overproduction of inflammatory cytokines il- and downstream release of mcp- , vegf, and il- , eventually culminating in a cytokine storm. secondary hemophagocytic lymphohistiocytosis may ensue in these patients. patients with severe disease have higher levels of il- , il- , il- , il- , and tnf-a compared with patients with milder disease. these high cytokine levels are associated with lymphopenia, with a deficit of both th, tc subsets, including naïve t cells and regulatory t cells. elevated cytokine levels are also associated with a decrease in hla-dr expression. in parallel, the nk cell population is also depleted, which may be the result of viral replication. while those with severe disease have significantly higher sars-cov- rna load and lymphopenia has directly correlated with viral load, these cytokine differences between moderately and severely ill patients may be due to polymorphisms in the cytokines involved. a quantitative approach relating differences in cytokine levels and polymorphisms in the immune response pathways may help identify patients at risk of severe disease. the immune response to infection may be broadly classified into a signaling component and an effector component, with the balance between the two determining the eventual outcome (figure ) . the signaling function is determined by the cytokine and chemokine secretion by cells of the target tissues and the innate immune system in response to the infection. the effector component, on the other hand, is characterized by a pathogen-specific t and b cell response. the ability to maintain control of the virus and successfully recover from infection, relies on this sequential feed-forward loop nature of the signaling and effector components, with the virus attempting to inhibit these processes simultaneously. mathematical models have been proposed to understand the quantitative nature of these processes. these models generally study the growth of viruses as an exponential function of time. viruses enter their host cells, grow exponentially in these cells, and are released into the surrounding milieu and infect an equal number of cells, where this exponential growth and release are repeated. thus, viral replication exhibits exponential growth occurring in an exponentially rising number of target host cells until a limit is reached by target cell exhaustion. in sars-cov- , because of the widespread expression of ace- , a large tissue reservoir is at risk of infection, amplifying the viral burden manifold over time. the corresponding immune response to the viral proliferation in the host has multiple components, and each of these components involves the growth of a population of immune effectors such as dendritic cells, nk cells, t cells, and b cells. the growth of each of these components will have to reach a threshold in an optimal period (e.g., by time, t , t , t or t, t', t'', t''' and so on for each cell type) to ensure timely and complete clearance of the virus (figure ) . as the first line of defense, interferons slow down viral replication. virus, driven by their receptor affinities. for nk cells, this represents the balance of inhibitory and activating signals from kir and nkg family of receptors. in some individuals with a kir b haplotype and a larger component of activating receptors, this may be a more effective process as opposed to those with a dominant component of inhibitory receptors. for the t cell responses, the ability of the hla haplotype of the individuals to present sars-cov- derived peptides will be critical. this encompasses both the antigen-binding affinity of the hla molecules in the host as well as antigen abundance. the requisite t cell response will be proportional to this antigen affinity and the affinity of the t cell receptor to the viral antigen-hla complex. while the nk and t cell subset proliferation may catch up with an exponentially rising number of infected cells, cellular immunity may eventually fail under the pressure of rapidly replicating sars-cov- , with widespread tissue involvement due to extensive ace- expression. thus, in addition to an efficient cellular immune response, a robust humoral immune response is essential to control and eliminate the infection eventually. the humoral response is characterized by a proliferating b cell and plasma cell population. each plasma cell makes large amounts of pathogen-specific antibodies, which finally allows the host to match the growth rate of the virus and neutralize the viral particles being generated. failure or suboptimal rate of any one of these pathways will lead to inadequate immune response and delay in viral clearance. the suboptimal response will potentially lead to earlier components in the pathway over-compensating for the failure of downstream mechanisms, caught in a feedback loop leading to phenomenon such as cytokine storm. the immune pathways are all susceptible to genetic polymorphisms that have functional consequences, such as variability in cytokine expression, antigenbinding affinities, the strength of receptor ligation and downstream signaling. thus, functionally consequential polymorphisms in these interconnected immune pathways may impede the development of an optimal immune response to covid- . the immune response to viral infections is a multi-step, precisely coordinated process, which results in viral clearance through initial innate and later adaptive immune mechanisms. this has been modeled mathematically to a high level of precision using ordinary differential equations. the immune system behaves like a dynamical system when both t cells and nk cells are considered. small changes in parameter values, for instance, in sars-cov- antigen -hla binding affinity or the initial il- or ifn-levels produced in response to infection, may have a profound impact on the eventual clinical outcome. thus, polymorphisms in critical immune response genes may alter the clinical outcome in a significant manner, mainly when they are considered together in a comprehensive mathematical description of the total immune response. quantifying the number of polymorphisms across variables can, therefore, be used to assess differences in disease risk. as such, it may be assumed that there are a set of variables (innate and adaptive) that confer an advantage in terms of viral clearance without cytokine storm and reduce the risk of severe disease, as compared to the set of variables with no common elements ( figure ) . in patients who have common elements across the entire set of immune response determinants (overlapping sets), the risk of severe disease may be equivalent when adjusted for age and comorbid conditions such as obesity, lung disease, and diabetes. among patients who either have very few or no overlapping elements, the risk of disease may be significantly different. these considerations become evident in a thought experiment to model the growth of any virus. if rv is the viral growth constant (number of virions replicated, for each cell infected, for each iteration of the growth process), then the total viral burden v at time t can be given by the equation where h is the target tissue reservoir, and ie is the infection efficiency for that virus (proportion of virions that end up infecting a new cell). with an initial inoculum of viral particles, cells (h) get infected (ie= . or %); in the second iteration virions will be produced with , cells infected, and it this keeps growing over time t until the limit of h is reached. interferon (ifn) would directly suppress v, by a fraction, sc (cytokine-induced suppression), starting at a later time t' (t' < t), and depending on how ifn much is produced, modifies vt to vt(ir) (diminished viral burden following the immune response) along with ifn production, a cascade of cytokine signaling will also be initiated, by the tissue-resident dendritic cells (dc) as well as the innate immune cells migrating into the tissues, where c is the unit production of cytokine per unit increase in v. are the tissue-resident macrophages, and are the macrophages/inflammatory cells that migrate into the tissues. dc then is the dendritic cell ( ) production of cytokines in response to v. as v gets larger, dc gets higher with t and constitutes the feed-forward loop depicted in figure and figure . unless the overall feedback loop depicted in figure slows down the growth of v over time, this can lead to cytokine storm. it is also important to note that dc is a vector matrix made up of many different cytokines, which all work on their respective receptors in the different target cell populations. the cytokines will trigger an nk cell response, which will slow down v. of note, this variable, and others, represent growth or change over time and is more accurately referred to by dv/dt. still, for simplicity, we will simply consider absolute value vt. nk cell growth and response may be considered as a function of the sum of activating and inhibitory receptors, the kir and the nkg family of receptors. these are modeled as vectoroperator equations, where the nk cell with its kir molecules constitutes a 'vector,' and the target with its kirligand molecules, an operator. the 'operator' modifies the 'vector' upon interacting with it; for example, it may either inhibit or activate it. each individual kir-kirl interaction may be described as follows; if an inhibitory kir (ikir) on the nk cell encounters a ligand on its target, this results in an interaction which may be scored, (− ) × ( ) = − , this will give the nk cell an inhibitory signal, assuming constitutively active basal state for nk cells; if there is no ligand for an inhibitory kir, i.e., missing kirl (mkirl), the interaction score will be (− ) × (− ) = + because of the abrogation of the inhibitory signal, and finally, activating kir (akir) interacting with its ligands will be scored, ( ) × ( ) = + , when the ligand is present, and ( ) × ( ) = , when the ligand is absent since no signal is given. each of these different scores constitute a distinct component of the total kir effect on individual nk cells expressing them, and while akir and ikir function independently the cumulative effect calculated by taking their sum, determining the eventual outcome of nk cell-target interaction. activating kir are not required for a robust nk cell immune response in most instances, evident as individuals with haplotypes containing no functional akir are common and even people with haplotypes that include akir have nk cells in their nk cell repertoire without any akir present. depending on the degree to which the virus down regulates hla class i molecules to evade tc mediated killing, it may lead to ikir-missing kir ligand mediated killing or may not through viral immune escape mechanisms such as, no hla class i down regulation or hla decoy expression. though viral evasion through decoy hla expression can lead to nk cell medicated viral killing as in m protein expression by mice with mouse cmv infection. m has been shown to strongly associate with an activating mouse ly receptor (analogous to kir) and confer host protection against mcmv. so, the nk cell effect may be summarized by here time, t'' is a later period in time at which the nk cell effect becomes manifest, it is later than the time, t when viral proliferation begins. this means that the impact of an immune checkpoint triggered later than t (i.e., t' and t'') will require for those processes to outpace the viral proliferation, which already had a lead on these in time. the another mechanism for cytokine storm in the former situation (with optimal, timely viral control) will be autologous tissue destruction perpetuating the cytokine cascade in a different feedback loop. it is important to recognize that dc is a matrix, where many different cytokines with both suppressive as well as growth promoting effects are represented. thus the overall effect of dc on t cell or b cell growth is not always in the positive direction, rather depending on the cytokines dominating in the milieu at any given time one may observe growth suppression, a phenomenon commonly seen in severely ill covid patients. the whole immune cascade may thus be mathematically described as a system of matrix vector operator equations. this schematic depicted in figure . this exercise demonstrates the multiplicity of responses involved in controlling infections in general, as it is in this particular case. further, they indicate the many redundant pathways at work in the immune response to the same. it is also imperative to note that pathogens have evolved mechanisms to evade the immune response at multiple checkpoints. because of genetic variation, not all individuals are equally well endowed with the ability to control infection. a multi-targeted approach may be needed to overcome the redundancies encountered. the notion of performing randomized trials with a single intervention tested at a time is at the heart of medicine, and agents are failing such trials are often discarded for not having met efficacy endpoints. the thought experiment above suggests that for a pathogen that has the capability of outpacing or disabling several different immune mechanisms, a combination of many agents and modalities may be required to control the disease. attention to this aspect of infectious disease management needs to be given in future trial design. a quantitative approach, such as outlined here, may be used to identify variation in immune response and genetic components that may be responsible for the range of illness severity that is observed in covid- . obtaining a better understanding of the differences in the host response is vital to identify those at risk for developing severe disease and target treatment strategies. with differential susceptibility to covid- . polymorphism associated syndrome interferon alpha hepatitis b [ ] [ ] [ ] [ ] [ ] hepatitis 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polymorphisms on the progression of hcv infection in egyptian patients lack of tnf-alpha gene polymorphism (rs ) association with sustained virological response in iranian patients with chronic hcv infection il and tnfa gene polymorphisms and the risk of graft-versus-host disease after allogeneic haematopoietic stem cell transplantation the effects of tumor necrosis factor-&#x b ; (tnf-&#x b ;) rs and rs polymorphisms on sepsis risk structural basis for the recognition of sars-cov- by full-length human ace cryo-em structure of the -ncov spike in the prefusion conformation covid- -navigating the uncharted sars-cov- viral load in upper respiratory specimens of infected patients full spectrum of covid- severity still being depicted substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) covid- : towards understanding of pathogenesis the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab 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roadmap for influenza research a qualitatively validated mathematical-computational model of the immune response to the yellow fever vaccine mathematical modeling provides kinetic details of the human immune response to vaccination group as part of the h africa consortium. classical hla alleles are associated with prevalent and persistent cervical high-risk hpv infection in african women an increased frequency in hla class i alleles and haplotypes suggests genetic susceptibility to influenza a (h n ) pandemic: a case-control study cytomegalovirus infection in ireland: seroprevalence, hla class i alleles, and implications mathematical modeling provides kinetic details of the human immune response to vaccination a qualitatively validated mathematical-computational model of the immune response to the yellow fever vaccine modeling influenza virus infection: a roadmap for influenza research dynamical system modeling of immune reconstitution after allogeneic stem cell transplantation identifies patients at risk for adverse outcomes dynamical system modeling to simulate donor t cell response to whole exome sequencing-derived recipient peptides: understanding randomness in alloreactivity incidence following stem cell transplantation dynamical system modeling to simulate donor t cell response to whole exome sequencing-derived recipient peptides demonstrates different alloreactivity potential in hla-matched and -mismatched donor-recipient pairs killer immunoglobulin-like receptor-ligand interactions predict clinical outcomes following unrelated donor transplantations stem cell transplantation as a dynamical system: are clinical outcomes deterministic? understanding how combinations of hla and kir genes influence disease direct recognition of cytomegalovirus by activating and inhibitory nk cell receptors determining the quantitative principles of t cell response to antigenic disparity in stem cell transplantation key: cord- -o ffxgnn authors: lorts, angela; cornell, timothy t.; shanley, thomas p. title: sepsis date: - - journal: pediatric critical care study guide doi: . / - - - - _ sha: doc_id: cord_uid: o ffxgnn the health care provider faced with the management of a child with septic shock relies on a comprehensive understanding of the numerous disciplines embodied in the practice of pediatric critical care medicine. the child with septic shock may have simultaneous derangements in the function of virtually every system of the body including: cardiovascular, respiratory, immune, renal, coagulation, hepatic, metabolic and neurologic. the degree to which physiologic alterations are manifest in a given patient is variable and influenced by multiple host and non-host factors including: the developmental stage, the presence of co-morbidities, pathogen-related factors, and genetic influences on both the host inflammatory response as well as the response to pharmacologic agents, all combining to have a profound influence on outcome. the clinician must possess a systematic and multifaceted approach to these critically ill patients. the goal of this chapter is to provide a comprehensive description of the epidemiology, biology and pathophysiology (at both the cellular and organ level) of sepsis, as well as outlining the current principles of managing septic shock. it will be apparent that optimal management requires a strong working knowledge of cardiovascular physiology, infectious diseases, multiple organ interactions, immunity, coagulation, pharmacology, and the molecular biology of inflammation. the health care provider faced with the management of a child with septic shock relies on a comprehensive understanding of the numerous disciplines embodied in the practice of pediatric critical care medicine. the child with septic shock may have simultaneous derangements in the function of virtually every system of the body including: cardiovascular, respiratory, immune, renal, coagulation, hepatic, metabolic and neurologic. the degree to which physiologic alterations are manifest in a given patient is variable and infl uenced by multiple host and non-host factors including: the developmental stage, the presence of comorbidities, pathogen-related factors, and genetic infl uences on both the host infl ammatory angela lorts , timothy t. cornell, and thomas p. shanley response as well as the response to pharmacologic agents, all combining to have a profound infl uence on outcome. the clinician must possess a systematic and multifaceted approach to these critically ill patients. the goal of this chapter is to provide a comprehensive description of the epidemiology, biology and pathophysiology (at both the cellular and organ level) of sepsis, as well as outlining the current principles of managing septic shock. it will be apparent that optimal management requires a strong working knowledge of cardiovascular physiology, infectious diseases, multiple organ interactions, immunity, coagulation, pharmacology, and the molecular biology of infl ammation. before reviewing the epidemiology of pediatric sepsis, it must be appreciated that the conclusions of prevalence studies have been obscured in the past by several factors including a lack of a reliable case defi nition. it has only been in the s that consensus defi nitions for sepsis and septic shock were achieved. it was hoped that the development of standard defi nitions would not only enable accurate characterization of the epidemiology of sepsis, but also serve to stratify patients early in the course of sepsis for the purpose of clinical studies aimed at testing novel therapies. the most widely used defi nition of pediatric sepsis/septic shock is based on the american college of chest physicians/society of critical care medicine (accp/sccm) consensus conference, with adaptations for the pediatric population. the following four defi nitions resulted from these discussions: sirs , sepsis , septic shock , and severe sepsis . although there is overlap between some of these terms (particularly between septic shock and severe sepsis), each is intended to defi ne a particular patient population. longstanding clinical observations have identifi ed the presence of tachycardia, tachypnea, hyperthermia and leukocytosis as signs of infection, though these responses may also be present in the absence of any apparent infectious source. as a result, this physiologic response was defi ned as the systemic infl ammatory response syndrome (sirs). sirs defi nes a state of infl ammation/immune activation in a child and is based on the presence of at least two of the four criteria listed in table - . thus, patients with diverse clinical conditions such as sepsis, pancreatitis, burns, or severe trauma can meet criteria for sirs. it has been argued that the sirs defi nition is non-specifi c and that too broad a range of patients are ultimately classifi ed as having sirs. nevertheless, the criteria have been widely used in both prescriptive and interventional studies to enhance the "capture" of all patients at risk for the subsequent development of severe sepsis or septic shock. sirs i s a state of infl ammatory/ immune activation and is based on the presence of at least two of the four following clinical criteria: temperature > °c or < °c, heart rate > th percentile for age, respiratory rate > th percentile for age, or hyperventilation to paco < mm hg. the defi nition attempts to "capture" all patients at risk for the subsequent development of severe sepsis or septic shock. sepsis is defi ned as a sirs response which is secondary to an infection, either documented by microbiology cultures or other clinical evidence of infection. severe sepsis is defi ned by sepsis criteria plus evidence of insuffi cient end organ perfusion (table - ) . finally, septic shock is defi ned by sepsis criteria plus hypotension (two distinct measurements < rd percentile for age) after the administration of at least ml/kg of crystalloid or colloid, in addition to the criteria listed for severe sepsis (table - ) . these criteria have been used extensively for conducting clinical investigations and have proven to be of value despite criticism for lack of both sensitivity and specifi city. the latest consensus conference was convened in to further refi ne the diagnostic criteria and therapeutic recommendations, with specifi c considerations for the pediatric population. published in , the surviving sepsis campaign aims to improve the outcome in sepsis worldwide. the refi nement of pediatric-specifi c criteria for septic shock is also intended to aid future clinical trials and epidemiologic investigations in pediatric sepsis. the few published pediatric-specifi c studies illustrate the importance of sepsis in this age range. proulx analyzed the incidence and outcome of sirs, sepsis, severe sepsis, and septic shock in a single institution. over , admissions were analyzed over a -year period. sirs was present in % of patients, while % had sepsis, % had severe sepsis, and % had septic shock. the overall mortality for this population was % with a majority of deaths occurring in patients with multiple organ dysfunction syndrome (mods). an epidemiologic study using discharge international classifi cation of disease, th revision (icd- ) codes reviewed hospital records from seven large states representing nearly one-quarter of the united states population. while the criteria used for inpatient coding at discharge are not identical to accp/sccm consensus conference criteria, the study estimated an incidence of , cases of severe sepsis in individuals less than years of age ( . cases/ , population). the highest incidence was in neonates ( . cases/ , population), compared to children ages - who had an incidence of . cases/ , population. the overall mortality rate was . % ( , deaths nationally) consistent with the frequent observation that the mortality rate remains lower than comparable adult data. the study estimated an annual national health care cost of $ . billion associated with severe sepsis in children. a follow-up study with the same methodology appeared to show a % increase in the absolute number of cases of severe sepsis from and with the majority of this increase accounted for by severe sepsis in children less than year of age. the mortality rate had decreased to . % during this time period. collectively, these data illustrate that sepsis is a major health problem on the basis of incidence, mortality, and health care costs. there remains a need for further, well-designed epidemiologic studies of pediatric sepsis. future studies will enhance our understanding of not only epidemiology, but also the impact of new diagnostic and therapeutic approaches resulting from improved design of interventional trials specifi c to the pediatric population. sepsis is a systemic disease and can impact the functioning of all organ systems. the most common clinical manifestations of sepsis include: fever or hypothermia, tachypnea, tachycardia, leukocytosis or leukopenia, thrombocytopenia, and change in mental status. one of the earliest signs of infection is fever which results from the pyrogenic effect of cytokines, particularly interleukin (il)- b and tumor necrosis factor (tnf)-a . presentation with hypothermia can also occur, but is more common in infants. one traditional classifi cation of shock states divides this clinical state into three broad categories: hypovolemic, cardiogenic and distributive shock. the shock associated with c hapter • s eps is sepsis is unique in that all three forms are likely to be present. hypovolemic shock results from capillary leak, increased insensible losses, and decreased effective blood volume secondary to venodilation. cardiogenic shock is related to direct myocardial depression, the cause(s) of which remains the focus of investigation. finally, distributive shock is often apparent as brisk capillary refi ll, widened pulse pressure and bounding peripheral pulses and is caused by abnormally decreased systemic vascular resistance from pathologic vasodilation. the particular pattern of these hemodynamic physiologic perturbations manifested by any individual patient can be variable. some children have increased cardiac output with diminished systemic vascular resistance characteristic of distributive shock or the so-called "warm" shock state. in stark contrast to adults, in which this hemodynamic profi le (increased cardiac output/decreased systemic vascular resistance) is most common, children more frequently present with depressed cardiac output and elevated systemic vascular resistance. these patients appear cool with diminished pulses and poor capillary refi ll that is characteristic of the "cold" shock state. while important to recognize that patients may transition from one state to another, the presence of hypotension is often a late and particularly ominous sign that requires prompt intervention as its presence is associated with increased mortality. patients with sepsis often present with alterations in their respiratory system, notably tachypnea that refl ects a compensatory respiratory alkalosis aimed at neutralizing a metabolic acidosis related to hypoperfusion and anaerobic metabolism. chest x-ray fi ndings can reveal a small heart in the presence of hypovolemia with few vascular markings. alternatively, the combination of capillary leak, decreased myocardial function and the result of fl uid resuscitation in some children with sepsis can result in pulmonary edema. rapid progression to acute respiratory failure from ards is not uncommon. all organ systems and ultimately cellular functions are affected by poor perfusion and decreased oxygen delivery related to depressed cardiac and respiratory function. in addition, there may be direct injurious effects of bacterial toxins and circulating cytokines such as triggering of programmed cell death or apoptosis. the neurologic state of a child with sepsis is frequently altered and can range from agitation or irritability to frank obtundation. this depressed mental status can be present even in the absence of meningitis as a manifestation of cerebral hypoperfusion. skin manifestations are not uncommon and can include petechiae and purpura that are ominous signs of disseminated intravascular coagulation (dic) and purpura fulminans secondary to meningococcemia. diffuse erythema secondary to toxic shock syndromes can be present. there is also an increasing appreciation of sepsis-induced microvascular angiopathy contributing to distal skin and organ ischemia. an initial thorough and detailed physical exam provides both important clues to the diagnostic possibilities of pediatric septic shock and the underlying hemodynamic profi le. however, serial exams are imperative to follow pathophysiologic changes and to gauge the impact of therapeutic interventions in reversing the manifestations of shock. data from both clinical and basic science studies have supported the hypothesis that pathogens and/or their products initiate a host immune response that triggers widespread infl ammation causing tissue injury and organ dysfunction. potential initiating pathogens include gram-negative and gram-positive bacteria, viruses, fungi and protozoa. in some cases, overwhelming spread of pathogens (e.g. bacteremia) with release of toxins (e.g. endo-or exotoxins) may directly injure the host resulting in organ dysfunction. higher order organisms have evolved an immune system to eradicate pathogens which has evolved to include two systems: the innate or natural immune system and the acquired or adaptive immune system. the innate immune system is responsible for the highly conserved function of recognizing pathogens and mounting an effector response. it includes a series of molecules located on the cell surface termed pattern-recognition receptors (prr) which are capable of recognizing a broad array of conserved structures on a variety of children with sepsis may have hemodynamic characteristics that transcend traditional classifi cation. they often have elements of hypovolemia, cardiac dysfunction and abnormal vascular tone. in the septic child, the combination of capillary leak, decreased myocardial function and the result of fl uid resuscitation may result in rapid progression to acute respiratory failure. pathogens (so-called pathogen-associated molecular patterns, or pamp's). examples of pamp's include: lipopolysaccharide (lps) on gram-negative bacteria, lipoteichoic acid on gram-positive bacteria, mannans on yeast, double-stranded rna of rna viruses and unmethylated, cpg dna from bacteria. the effector responses that are regulated by the innate immune system (e.g. phagocytes, complement) are activated immediately upon infection and are designed to rapidly inhibit the replication of microorganisms. these cell surface pattern-recognition receptors (prr) are expressed on most antigen presenting cells of the innate immune system and represent diverse families of proteins. one group of prrs, the toll-like receptors (tlr), has been identifi ed as perhaps the most critical pathogen recognition receptor family in the context of sepsis biology. other families of prr include the c-type coll agenous lectins (collectins) that bind to a variety of carbohydrate moieties on cells, bacteria and viruses. most members of this family share structural homology to the complement protein c q and can functionally substitute for c q in activating the complement cascade. another family of prr possesses leucine-rich regions critical for protein-protein interactions that are necessary for immune recognition. examples of these leucine rich receptors include cd , a receptor on the cell surface of macrophages that binds to lps and the macrophage scavenger receptor that binds to bacterial cell walls. unbound circulating prrs exist and include pentraxins, such as c-reactive protein, an acute phase reactant synthesized by the liver and lipopolysaccharide-binding protein (lbp) which binds to lps to optimize its binding to the cd /toll-like receptor cellular complex. another key component of innate immunity is the complement system. the complement system is a complex cascade of proteins that possesses a broad array of anti-pathogen activities including: opsonization (c ), neutrophil chemotaxis (c a), perforating cytotoxicity (c - , mac complex) and the ability to bind to and directly lyse viruses (c ). an in depth discussion of the role of complement in the response to infection is beyond the scope of this chapter, but has been recently summarized. in summary, the host possesses a ubiquitous and diverse set of pathogen recognition receptors which function to protect the host from infectious challenges, but at the expense of triggering powerful effector responses. paramount to effector responses of the innate immune system is a proinfl ammatory action of numerous cytokines and chemokines. these biologically active proteins are critical to the activation and recruitment of cellular components of the adaptive immune system. while necessary for pathogen clearance, this acute, proinfl ammatory immune response must also ultimately subside in order to reestablish homeostasis and avoid cellular and tissue damage. a key pathophysiologic feature of sepsis is that this immune response often appears to become unregulated resulting in an overwhelming proinfl ammatory response and host autodestruction. this characteristic systemic infl ammatory response seen frequently in response to infection can also be observed in association with non-infectious triggers (e.g. trauma, burns, pancreatitis, cardiopulmonary bypass). lps recognition: recent epidemiologic surveys of the causative agents of sepsis have indicated an increase in the incidence of gram-positive organisms such that there is a roughly equivalent prevalence between these and gram-negative organisms. historically, sepsis research has focused on the role of gram-negative bacteria in evoking a pathologic response. the structure of endotoxin shows three domains: an outer polysaccharide hydrophilic chain which determines the o-antigenicity, an acidic core region, and a lipid-rich region. gramnegative organisms possess endotoxins with variable repeats of mono-and heteropolysaccharides with complex side chain structure to provide a basis for distinct antigenicity. the o-region is linked via an acidic core to the lipid a region that is highly conserved and responsible for much of the toxicity attributed to intact lps. a series of seminal observations have determined the molecular mechanisms by which the classic pamp, lps, initiates a proinfl ammatory response. first, a strain of lps-resistant mice, the c h/hej strain, was identifi ed and its resistance was found to be attributed to a single genetic mutation. second, it was shown that the lethal effects of endotoxin could be conferred by transfer of hematopoietic cells. endotoxin tolerant mice could be rendered the cells of the innate immune system contain cell surface molecules termed patternrecognition receptors (prr). these receptors are capable of recognizing a broad array of conserved structures on a variety of pathogens (so-called pathogenassociated molecular patterns, or pamp's). examples of pamp's include: lipopolysaccharide, lipoteichoic acid, viral rna and bacterial dna. toll-like receptors (tlr) are pathogen recognition receptors that have a critical role in sepsis. tlr is active in recognition of lps on gram-negative bacteria whereas tlr is active in the recognition of lipotechoic acid on gram-positive bacteria. a hallmark of sepsis is an immune response that appears to become unregulated resulting in an overwhelming proinfl ammatory response and host autodestruction. this characteristic systemic infl ammatory response is seen frequently in response to infection, but can also be observed in association with non-infectious triggers (e.g. trauma, burns, pancreatitis, cardiopulmonary bypass). c hapter • s eps is lps-sensitive after reconstitution with hematopoietic cells derived from the monocyte/macrophage lineage from an lps-sensitive strain. third, stimulation of monocyte-derived cells with endotoxin resulted in production of several cytokines and chemokines critical to the systemic infl ammatory response. among these, tnf and il- were shown to be critical initiators of the septic response and could in fact mimic the endotoxin response. finally, the elucidation of the lps receptor assisted the identifi cation of those signal transduction pathways by which endotoxin triggers infl ammatory gene expression. lps receptor: membrane bound cd- was shown to be required for lps signaling. however, it lacked a transmembrane extension required for cytoplasmic signaling indicating the presence of additional components of the receptor complex. investigators working with drosophila had identifi ed a gene, toll, which was responsible for dorsoventral polarization in embryonic development. when toll was functionally mutated, it was demonstrated to play a key role in host defense against aspergillus fumigatus . homology between the toll-like receptors and the mammalian il- family of receptors was discovered and provided additional evidence that this family was crucial to the human innate immune response. finally, it was determined that the c h/hej mouse strain which is hyporesponsive to lps possessed a mutation in toll-like receptor (tlr ), providing further evidence that this receptor was necessary for lps signaling. tlr is one of ten mammalian toll-like receptors that have been cloned to date, each being activated by a specifi c set of ligands. since these discoveries, other members of the lps-receptor complex have been elucidated and include both md- and myd . it is also known that circulating lpsbinding protein (lbp) facilitates lps binding to the cell surface receptor complex. together these components are able to "sense" lps at the cell surface and transmit this signal via a series of complex pathways. similarly, the products of gram-positive organisms, notably the cell wall component lipotechoic acid, activate cell activation through the related toll-like receptor (tlr ). after engagement of cell surface receptors (e.g. tlr and tlr ), several important signal transduction pathways are activated that elicit a number of transcriptional factors responsible for infl ammatory gene expression. among these, the nuclear factor-k b (nf-k b ) and the mitogen activated protein kinase (mapk) pathways play a prominent role in regulating the expression of a number of infl ammatory gene products key to propagating the sepsis response. in the case of nf-k b, stimulation of the lps receptor causes phosphorylation of the inhibitor of k b kinases (i k k) which in turn phosphorylates the intracellular inhibitor of nf-k b, i kappa b (i k b). upon phosphorylation, i k b undergoes poly-ubiquination followed by proteosomal degradation. the removal of i k b effectively unmasks a nuclear translocation sequence on nf-k b enabling it to proceed into the nucleus to bind to nf-k b consensus sequences present on the promoter regions of many infl ammatory genes: cytokines including tnf, chemokines including il- , adhesion molecules including e-selectin and others such as inos (see fig. - ). the role of nf-k b in sepsis is supported by studies demonstrating that survivors and non-survivors of sepsis are distinguishable on the basis of nf-k b binding activity in peripheral blood mononuclear cells. in addition, in sepsis-induced ards, increased activation of nf-k b in macrophages obtained by bal is found in ards patients when compared to icu controls. to a similar degree, the mapk signaling pathways are important in mediating the septic response. three mapk pathways exist: p protein kinase, extracellular-regulated protein kinase (erk), and c-jun-terminal kinase (jnk). evidence exists for the role of each of these signaling pathways in sepsis. tnf production by neutrophils and macrophages is dependent on p activation. lps stimulation of monocytes activates jnk with downstream activation of activating protein- (ap- ) and subsequent il- b production. lps induction of tnf is in part dependent on erk pathway activation. together, these two pathways, nf-k b and mapk's, appear to be critical to the propagation of signals from the cell surface to the nucleus where expression of infl ammatory gene products occurs. as such, these pathways remain valid targets for future strategies in modulating the septic response. nuclear factork b (nfk b ) and the mitogen activated protein kinase (mapk) pathways play a prominent role in regulating the expression of a number of infl ammatory gene products key to propagating the sepsis response. while numerous proteins have been shown to play a role in the septic response, a full review of each protein's function is beyond the scope of this study guide. instead, we aim to highlight some known principle mediators in this cascade. evidence that tnf mediates the septic response stems from numerous observations: it is produced by hematopoietic cells, its expression is temporally related to the development of septic shock, recombinant tnf induces experimental septic shock in animals, and passive immunization against tnf attenuates endotoxin-mediated responses. tnf possesses numerous functions in infl ammation such as driving adhesion molecule and chemokine expression to facilitate leukocyte-endothelial cell adhesion; upregulating tissue factor and inhibition of protein c to create a pathologic procoagulant state in the vasculature; and inducing nitric oxide synthase (inos) which mediates pathologic vasodilation. in human studies, levels of tnf have been shown to correlate with mortality, with the development of shock and purpura fulminans and with the development of sepsis-induced ards and shock. the name, il- , is now used to describe the family of proteins including two agonists (il- a and il- b ) and one antagonist, the il- receptor antagonist protein (il- ra). il- b which is secreted, mediates much of the systemic effects attributed to il- release in sepsis. synthesized as a propeptide, il- b requires proteolytic cleavage by the il- converting enzyme (ice) to become bioactive. il- b utilizes the -kda type i receptor which is tnf possesses numerous functions in infl ammation such as inducing adhesion molecules and chemokines that facilitate leukocyte-endothelial cell adhesion and inducing nitric oxide synthase (inos) which mediates pathologic vasodilation. tnf also upregulates tissue factor and inhibits protein c to create a pathologic procoagulant state in the vasculature. clinically, levels of tnf correlate with mortality, the development of shock and purpura fulminans and with the development of sepsis-induced ards and shock. p p iκ κ κ κk associated with a number of adapter proteins (e.g. myd ,tnf receptor-associated factor (traf ) and interleukin- receptor-associated kinase (irak)) to propagate signals through both the nf-k b and ap- pathways. il- b infusion elicits fever, hypotension and leukocytic infi ltration to the lungs. in a manner similar to tnf, il- stimulates monocyte activation and phagocytosis, increases adhesion molecule expression, and increases tissue factor expression while inhibiting thrombomodulin secretion, thus creating a procoagulant state. when detected in the circulation of septic patients, il- levels also correlate with mortality. of note, the il- ra is a circulating inhibitor of il- b that binds to the il- receptor without initiating a signal. the expression of il- ra has been shown to follow peak expression of il- . it is speculated that il- ra is an endogenous regulator of il- effects. however, in clinical trials, il- ra infusion failed to improve mortality in sepsis. furthering our molecular understanding of sepsis-induced organ dysfunction was the identifi cation of the "leukocyte-endothelial cell adhesion cascade". this cascade is characterized by cytokine activation of the selectin family of adhesion molecules (e.g. e-selectin) on the endothelium which initiate a process of neutrophil "rolling" via interaction with sialyated moieties constitutively present on circulating neutrophils. activation of the "rolling" neutrophil results in both increased expression and activation of the integrins which in turn bind to intercellular adhesion molecule (icam)- that is upregulated on the endothelial cell surface by tnf and il- b . this integrin-icam- interaction mediates fi rm adhesion of the neutrophil to the endothelial cell surface. finally, in response to various chemotactic cytokines or chemokines, neutrophils migrate to the site of infl ammation. release of both oxygen-and nitrogen-based radical species and proteases by the neutrophils may ultimately contribute to cellular injury and organ dysfunction. nitric oxide (no) is responsible for endothelium-derived relaxation of blood vessels. three isoforms of nitric oxide synthase are responsible for production of no: type i, a neuronal isoform (nnos); type ii, an inducible isoform (inos) and type iii, a constitutive, endothelial isoform (enos). tnf and il- b are capable of inducing inos and increased levels of circulating stable byproducts of no are found in both septic adults and children who simultaneously display low systemic vascular tone. this supports the hypothesis that no plays a principal role in septic shock via pathologic vasodilation. it has also been suggested that tnf and il- b may be the so-called "myocardial depressant factors" by increasing circulating no through induction of inos ; however, it is not clear that no is the exclusive mediator of these effects. in light of the evidence supporting the role of no in septic shock, clinical trials employing no synthesis inhibitors in septic shock were initiated. though early clinical reports and small studies reported that nos inhibitors could signifi cantly improve blood pressure, this was at the expense of decreasing cardiac output secondary to increased afterload. as a not uncommon hemodynamic profi le in pediatric septic shock is decreased cardiac output and elevated systemic vascular resistance, it is not cleat that nos inhibitors will have a therapeutic role in pediatric (or adult) sepsis in the future. studies employing agents directed against the early mediators of the septic response have been mostly ineffectual. this has led to the hypothesis that additional molecules with delayed kinetics of expression may infl uence the outcome in sepsis. as an example, it was observed that lps-challenged mice often die long after peak expressions of tnf and il- b suggesting that late-acting proteins may contribute to endotoxin-induced mortality. investigators searching for late expressed proteins identifi ed a member of the high mobility group (hmg)- non-histone chromosomal protein family in conditioned media h after lps-stimulation of macrophages. this protein renamed hmgb is a known ligand for the receptor for advanced glycation end products (rage). the rage receptor is expressed on monocytes and vascular smooth muscle. binding by hmgb activates both the nf-k b and mapk pathways. increased expression of hmgb was found in endotoxemic mice and in critically ill patients with surgical sepsis where increased levels correlated with non-survival. in animal models, the blockade of hmgb can inhibit the infl ammation associated with endotoxemia, cecal ligation and puncture and lps-triggered acute lung injury. identifi cation of hmgb and similar "late" mediators may provide a broader therapeutic window for successful immune modulating therapy in sepsis. regulatory processes and mediators exist for the purpose of modulation and eventual resolution of infl ammation and the septic response. an absence in the decline of proinfl ammatory mediators such as tnf and il- over the course of sepsis is an associated risk factor for mortality. monocyte activation results not only in production of proinfl ammatory cytokines, but also expression of a number of endogenous cytokine antagonists including soluble tnf receptors, the il- ra and additional anti-infl ammatory cytokines, such as il- and transforming growth factor-β (tgf-b ). il- has multiple anti-infl ammatory properties including inhibition of cytokine production from activated monocytes. il- inhibits expression of those cytokines known to contribute to sepsis, as well as important chemokines, including il- . in addition, il- increases expression of other anti-infl ammatory molecules such as il- ra and soluble tnf receptors. exogenous administration of il- in various experimental models has been used in an attempt to decrease infl ammatory cytokines and diminish organ injury. human studies showed that patients who did not survive ards had lower levels of il- in their bal fl uid compared to survivors. furthermore, the inability to increase il- in response to meningococcal infection was associated with increased mortality. thus, il- and additional regulatory cytokines (e.g. tgf-b , il- ) possess a number of anti-infl ammatory properties and are important contributors to the endogenous regulation of the acute septic response. dysregulation of the coagulation cascade occurs in sepsis as refl ected by activation of procoagulant pathways, consumption of clotting factors, alterations in fi brinolysis, and reduced anticoagulant activity. a common hematologic alteration in sepsis is the development of disseminated intravascular coagulation (dic) which is an acquired state of activation of coagulation and intravascular fi brin formation resulting in vascular thrombosis. in addition to proinfl ammatory cytokines, tissue factor (tf) activation also plays a prominent role in activating the coagulation cascade, initiating fi brin formation and contributing to the development of dic. concurrent with enhanced production of fi brin, there is decreased fi brinolysis related to increased plasminogen activator inhibitor type (pai- ), as well as dysfunction and/or depletion of antithrombin iii, protein c, protein s and tissue factor pathway inhibitor (tfpi). at iii which inhibits thrombin by forming thrombin-antithrombin (tat) complexes is decreased in sepsis related to degradation by elastases from activated neutrophils, dilution secondary to volume resuscitation, and impaired hepatic synthesis. despite a correlation between low at iii levels and mortality in patients with sepsis, replacement trials of at iii have failed to show a signifi cant effect on improving mortality. protein c is also noted to be depleted among patients with sepsis and septic shock. regulation of activation of protein c to activated protein c (apc) in the coagulation cascade is mediated in a complex manner and will not be discussed here. apc, upon dissociation from its receptor, binds to its co-factor, protein s, to subsequently inactivate factors va or viiia, thus playing a key role in inhibiting coagulation. it is both antithrombotic and profibrinolytic. apc also possesses anti-infl ammatory activity. in models of endotoxemia, apc infusion decreases cytokine production and attenuates neutrophil activation. these antiinfl ammatory effects appear to be independent of apc's anticoagulant effect. following an absence in the decline of proinfl ammatory mediators such as tnf and il- over the course of sepsis is an associated risk factor for mortality. monocyte activation results not only in production of proinfl ammatory cytokines, but also expression of a number of endogenous anti-infl ammatory cytokines including soluble tnf receptors, the il- ra and additional anti-infl ammatory cytokines, such as il- and tgfb . important anti-infl ammatory molecules include il- , il- ra and soluble tnf receptors. il- inhibits expression of proinfl ammatory cytokines known to contribute to sepsis, as well as important chemokines, including il- . in addition, il- increases expression of other anti-infl ammatory molecules such as il- ra and soluble tnf receptors. at iii inhibits thrombin by forming thrombin-antithrombin (tat) complexes. at iii is decreased in sepsis due to degradation by elastases from activated neutrophils, dilution secondary to volume resuscitation, and impaired hepatic synthesis. despite a correlation between low at iii levels and mortality in patients with sepsis, replacement trials of at iii have failed to show a signifi cant effect on improving mortality. these encouraging pre-clinical studies, clinical trials examining the effect of apc on mortality from sepsis were commenced culminating in the protein c worldwide evaluation in severe sepsis (prowess) trial. in this study, apc was associated with a statistically signifi cant reduction in -day mortality in septic adults. however, a recent pediatric study was stopped after an interim analysis showed that apc administration was highly unlikely to show improvement in outcome ( fig. - ). it is not uncommon to observe that patients exposed to seemingly identical pathogen insults display strikingly different pathophysiology and outcomes. it is believed that genetic differences among hosts are at least in part responsible for this variability in sepsis responses. as mentioned previously, the insensitivity to lps in the c h/hej mouse line was mediated by a mutation in the coding sequence for tlr . similar fi ndings of an attenuated response to pathogen stimulation have now been reported in patients with mutations in both the tlr and tlr gene. the polymorphism in tlr appears to confer an increased predisposition to severe gram-positive bacterial infections. more recently, a polymorphism within the cd promoter gene (c to t transition at base pair - ) was identifi ed with a particular genotype over-represented among septic shock patients compared to healthy controls. among the septic patients, the presence of this genotype also was associated with a signifi cantly higher mortality ( % versus %). these studies support the concept that genetic alterations in those genes known to participate in the septic response affect the host immune response and likelihood of survival. for a more complete review of the numerous examples of genetic alterations in key infl ammatory genes, the reader is directed to the suggested readings. the administration of activated protein c was associated with a statistically signifi cant reduction in -day mortality in septic adults. however, a recent pediatric study was stopped after an interim analysis showed that apc administration was highly unlikely to show improvement in outcome. the coagulation cascade in sepsis (reprinted with permission from bernard ) as the cellular response to sepsis has become better understood, the approach to treatment of sepsis has become broader. the treatment of sepsis involves four important components: initial resuscitation, elimination of pathogen, maintenance of oxygen delivery, and carefully directed regulation of the infl ammatory response. as reviewed above, sepsis is an immunologically complex response to an invasive pathogen necessitating tight physiologic regulation in order to eradicate the organism while maintaining cellular and organ homeostasis. in cases where the immunologic and infl ammatory responses continue to escalate, numerous pathways are altered and may ultimately prove amenable to immune modulating therapy, but this approach has been unsuccessful to date. the initial priority in the treatment of the septic child is respiratory and cardiovascular stabilization. the primary goals of therapy in those initial hours following clinical presentation are to maintain oxygenation and ventilation, achieve normal perfusion and blood pressure, and re-establish appropriate urine output for age. children with sepsis may have altered mental status which, if profound, raises concern about the ability to protect the airway. tachypnea associated with a primary or compensatory respiratory alkalosis is commonly present. the combination of increased lung vascular permeability and aggressive fl uid resuscitation to restore intravascular volume and maintain blood pressure may contribute to the subsequent development of pulmonary edema. in children with lung edema, the related changes in lung compliance and loss of functional residual capacity can dramatically increase the work of breathing ultimately necessitating tracheal intubation and mechanical ventilatory support. arterial blood gas analysis may show hypoxemia and metabolic acidosis; however, the decision to provide mechanical ventilatory support should not be based solely on laboratory fi ndings. the presence of increased work of breathing, hypoventilation or obtundation are all indications for instituting mechanical ventilatory support which holds additional benefi t in decreasing the overall oxygen consumption, especially when combined with sedation and paralysis. it should be stated, however, that children with warm shock can commonly be managed without endotracheal intubation so long as they are not obtunded or fl uid overloaded. disorientation or lethargy with intact responsiveness does not require placement of an artifi cial airway as many institutions manage these patients without intubation. the work of breathing associated with hyperventilation in the absence of pulmonary edema is not clinically signifi cant. furthermore, there is no evidence that decreasing work of breathing in the presence of distributive shock will result in redistribution of nutrient fl ow to vital organs, the very nature of distributive shock. however, it is more common for infants to present with cardiac dysfunction and pulmonary edema or seriously altered mental status requiring endotracheal intubation and mechanical ventilation. correction of intravascular volume depletion should be made prior to the institution of positive pressure ventilation. the decrease in venous return after the initiation of positive pressure ventilation may lead to further hemodynamic compromise in the child with intravascular volume depletion. caution should also be taken in choosing sedative agents for intubation, using agents that have the least impact on tenuous hemodynamics (e.g. ketamine). controversy exists over the adrenal suppressive effect of a single dose of etomidate when used for intubation in septic children and adults. the pediatric critical care clinician should be aware of the concern for adrenal suppression following a single dose of etomidate used for the intubation of children with septic shock and the published guidelines which do not recommend its use in this setting. following intubation, attention must be paid to matching the mechanically provided minute ventilation to that which was present during spontaneous respiratory effort so that respiratory compensation of acidemia is preserved. if it is deemed that positive pressure ventilation is not needed, supplemental oxygen should be provided to maintain normal oxygen saturations. treatment of sepsis involves four important components: initial resuscitation, elimination of pathogen, maintenance of oxygen delivery, and carefully directed regulation of the infl ammatory response. with regard to fl uid status, septic children have decreased effective intravascular volume related to a number of causes. poor oral intake of fl uid for a period of time prior to clinical presentation is common. increased vascular permeability leads to intravascular volume loss due to extravasation of fl uid from the vascular space, so-called "third spacing". finally, the no-mediated vasodilation reviewed above increases vascular capacitance thereby decreasing the effective circulating volume. thus, when sepsis is suspected, it is imperative to expeditiously achieve vascular access and initiate fl uid resuscitation with ml/kg of isotonic fl uid as quickly as possible. while debate continues as to the most effective fl uid for resuscitation, no pediatric literature exists to support colloid over crystalloid, the latter of which was recently demonstrated to be equally effective in a large adult icu trial. there is some support for using colloid fl uid in patients with a narrow pulse pressure; however, this practice is not supported by any large, well-designed clinical studies. while following the clinical exam for signs of intravascular volume overload (new onset of rales, increased work of breathing, development of a gallop, or hepatomegaly), fl uid should be administered quickly with the goal of monitoring heart rate response, urine output, capillary refi ll time and level of consciousness. initial fl uid resuscitation of the child with septic shock commonly requires a volume of up to or greater than ml/kg in the fi rst hour and one retrospective study demonstrated an increased survival in children given fl uid volumes of ml/kg or more within the fi rst hour. the accm clinical practice parameters for hemodynamic support of pediatric and neonatal septic shock recommend that the child with septic shock be repeatedly examined for the development of "rales, gallop rhythm, hepatomegaly, and increased work of breathing" during volume loading, and that in the absence of such fi ndings, volumes up to ml/kg can be administered in the fi rst hour. these guidelines further state that "the rate of fl uid administration should be reduced substantially when there are clinical signs of adequate cardiac fi lling without hemodynamic improvement." despite on-going fl uid resuscitation, hypotension and inadequate organ perfusion may persist requiring the initiation of inotropes and/or vasopressors. in children, vasoactive medicines should only be given in addition to fl uid resuscitation. however, consensus guidelines recommend that vasoactive infusions may be necessary in some cases to sustain perfusion pressure even when hypovolemia is not yet resolved. dopamine is the most common fi rst choice agent selected for hemodynamic support in those patients with fl uid-refractory shock. dopamine provides inotropic support at lower concentrations; however, it is often necessary to increase it to higher doses that provide vasopressor activity (up to m g/kg/min) to maintain adequate tissue perfusion. the decision of which agent to add in the setting of dopamine-refractory shock should be based on the underlying cause of cardiovascular compromise. for example, if hemodynamic instability is related to low cardiac output from direct cardio-depressant effects then increased inotropy from dobutamine or low-dose epinephrine may be indicated. if hypotension persists secondary to decreased vascular tone, then agents such as epinephrine and norepinephrine dosed in the alpha agonist range should be considered. finally, in children who demonstrate low cardiac output and/or increased afterload from vasoconstriction (i.e. increased systemic vascular resistance), agents with primarily inotropic or vasodilator function including milrinone, dobutamine or short-acting nitrovasodilators can been considered in the fl uid-resuscitated, normotensive child. as in evaluating the adequacy of fl uid resuscitation, similar clinical parameters should be followed in titrating vasoactive medications. appropriate endpoints include: capillary refi ll time less than s, normal peripheral pulses, warm extremities, urine output greater than . ml/kg/h, improved mentation, resolving acidemia, decreasing serum lactate and when available, superior vena cava (svc) oxygen saturation greater than %. invasive monitoring can further assist the clinician with goal directed endpoints. although frequent beside examination remains integral to the care of the child in septic shock, an additional task in the initial resuscitation phase is placement of appropriate and necessary vascular access and monitors. central venous access is a necessity for the child with fl uid refractory shock to provide for delivery of vasoactive medicines and large volumes of fl uid. these catheters can be useful for following the central venous pressure (cvp) during fl uid adequate volume resuscitation of the child with septic shock commonly requires a volume of up to or greater than ml/kg in the fi rst hour. appropriate endpoints of sepsis resuscitation include: capillary refi ll time less than s, normal peripheral pulses, warm extremities, urine output greater than . ml/kg/h, improved mentation, resolving acidemia, decreasing serum lactate and when available, superior vena cava oxygen saturation greater than %. administration. finally, when the tip is located in the superior vena cava, blood sampling can provide an approximate measure of the mixed venous oxygen saturation which has been validated as a critical target in adult shock resuscitation. the decision regarding the access site for a central venous catheter is dictated by a number of mitigating factors such as the experience level of the operator and the presence of coagulopathy. femoral catheters, in the absence of abdominal pathology, can be used to estimate cvp with good correlation. the cvp measured in the abdominal inferior vena cava must be assessed carefully as a low cvp can be a reliable indicator of hypovolemia, however, a normal or high cvp in the presence of abdominal distention does not automatically exclude the presence of hypovolemia. multiple adult studies have demonstrated that even an accurate intra-thoracic cvp may be a poor approximation of left ventricular end diastolic pressure and volume. the cvp can be elevated despite the presence of hypovolemia if pulmonary hypertension, right ventricular dysfunction with poor diastolic compliance, tricuspid regurgitation, cardiac tamponade or an intracardiac left-to-right shunt exists. even though precise determination of the true mixed venous saturation requires the presence of a pulmonary artery catheter, the approximations derived from the svc saturation have proven a useful target in septic adults. in contrast, because of differences in oxygen extraction between the upper extremities, abdomen, and lower extremities, venous oxygen saturations from a low lying femoral line do not accurately correlate with those measured in the pulmonary artery. consensus guidelines recommend therapeutic endpoints of superior vena cava oxygen saturation > % or mixed venous (pulmonary artery) oxygen saturation > %. placement of an intra-arterial catheter provides continuous monitoring of systemic blood pressure, pulse pressure and hemodynamic variation with respiration, as well as a means for drawing arterial blood gases, lactate levels and additional laboratory studies. the arterial blood also provides the most accurate measure of arterial oxygen content and can be used to both assess the function of the lungs and to maximize oxygen delivery. in the ventilated patient, variation in the amplitude of the arterial waveform has been found to correlate closely with intravascular volume status (see also chapter ). systolic pressure variation (spv), also referred to as "reverse pulsus paradoxus," is the variation in beat-to-beat amplitude of the arterial pulse during positive pressure ventilation. a single positive pressure breath normally affects the arterial pressure in a biphasic manner. the initial response to a positive pressure breath is to "squeeze" pulmonary vascular blood into the left atrium (the opposite, "pooling" of blood, occurs with negative pressure inspiration) leading to a rise in systolic pressure. in addition, positive intrathoracic pressure reduces the afterload on the left ventricle by virtue of the pressure gradient from the thorax outward further augmenting this early rise in arterial pressure. these two effects produce an upward movement of the systolic blood pressure coincident with the positive pressure breath, referred to as the d up component of spv. following this d up, a fall in systolic pressure occurs a few beats later as the decreased venous return (preload) to the right ventricle that occurred during positive pressure inspiration is now evident as decreased preload to the left ventricle after a few cardiac cycles. the transient reduction in right ventricular volume and output leads to a smaller left ventricular stroke volume and a brief reduction in arterial pressure that occurs later in the ventilator cycle ( d down). an exaggerated spv (> mm hg) has been seen early in the setting of hypovolemia. this is due to a greater d down component. several studies have shown that an increase in the spv occurs prior to a fall in arterial pressure and may a better predictor of hypovolemia than a low pulmonary capillary wedge pressure (pcwp) (< mm hg). an increase in the spv due to a greater d down component can also occur due to high airway pressures causing decreased venous return. recently, pulse pressure variation (ppv) has also been found to be a sensitive indicator of preload, and more importantly, fl uid responsiveness. ppv is defi ned as the maximal pulse pressure (systolic minus diastolic blood pressure) less the minimum pulse pressure divided by the average of these two pressures. the use of systolic pressure variation and pulse pressure variation is limited to those patients on mechanical ventilation. these measurements should occur when there is no spontaneous breathing. in the presence of a consistent delivered tidal volume, systolic pressure variation can be used to track the adequacy of intravascular volume over time (fig. - ) . the decision to use a pulmonary catheter (pac) with the goals of optimizing left ventricular preload, monitoring cardiac index and measuring oxygen delivery remains controversial. caveats to interpretation of pac data include the presence of an intracardiac shunt and an abnormally functioning mitral valve or other obstructed left heart lesion as either shunting, regurgitation, or inaccurate pressure determinations will alter cardiac index and/or the pulmonary capillary wedge pressure measurements. as previous data in adults have shown no benefi t of pac use, a recent consensus statement regarding their use stated that the role of pac remains unclear. studies of children with septic shock have shown that the information obtained from a pac aided in identifying hemodynamic profi les different from those presumed by care givers and has directly infl uenced care decisions. it was concluded by a recent consensus panel to be of potential benefi t in improving the management of pediatric patients. pulmonary artery catheter placement should be considered for pediatric patients who remain in shock after resuscitation and initiation of the usual vasoactive agents in whom the fl uid status and cardiac function remains unclear. in this setting, therapeutic endpoints are a cardiac index of > . and < . l/min/m and systemic and pulmonary vascular resistances within the normal range. early identifi cation of a possible offending pathogen and aggressive source control represent a crucial component of septic shock therapy. prompt initiation of appropriate antimicrobial therapy against the causative pathogen has been shown to be one of the most important predictors of outcome. in a 's study of over , adults, providing appropriate systolic pressure variation (spv) and pulse pressure variation (ppv) can accurately predict which patients will be responsive to further fl uid resuscitation (gunn and pinsky ) . antimicrobial coverage at least day prior to identifi cation of the organism was associated with improved survival. the pathogen itself has prognostic signifi cance. fungal infections, while accounting for only a minority of sepsis cases, carry the lowest survival rate, followed by gram-positive and gram-negative bacteria. survival rate has been reported to be the highest in patients in whom no pathogen was identifi ed. because of the importance of appropriate antimicrobial therapy, the decision of which agents to empirically start must balance potential side effects versus maximizing coverage. in this respect, it is important to be familiar not only with the most common causative pathogens, but also the local icu nosocomial risks and pathogen resistance patterns. initially, broad antibiotic coverage is initiated. neonates are most frequently placed on ampicillin and an aminoglycoside (e.g. gentamicin) or a third generation cephalosporin such as cefotaxime. in infants and children over the age of - weeks, the decision to start vancomycin empirically should be considered in light of the increasing antibiotic resistance of streptococcus pneumoniae and rising incidence of community acquired methicillin resistant staphylococcus aureus (mrsa). in addition, a rd or th generation cephalosporin (e.g. ceftriaxone) should be used. suspicion of a gram-negative infection or nosocomial infection requires additional coverage, usually in the form of an aminoglycoside, for the possibility of pseudmonas species and other resistant gram-negative organisms. because of its broad coverage, including many anaerobic species, and low renal toxicity, piperacillin/tazobactam is empirically administered with increasing frequency. the antiviral agent, acyclovir, should be administered if there is suspicion of a herpes virus infection. in immunocompetent children, the decision to start empiric antifungal therapy remains controversial. in the child who is not improving over the initial days of empiric coverage or in whom there is a higher risk for fungal infection (e.g. presence of indwelling devices, immunosuppression or other signifi cant co-morbidities), antifungal coverage may be indicated. the development of agents equally as effective as amphotericin, but with substantially reduced nephrotoxicity such as fl uconazole and caspofungin, may ultimately sway the risk/benefi t analysis towards more aggressive, earlier initiation of empiric antifungal coverage in select, high risk populations. the ability to narrow the spectrum of treatment once the causative organism has been identifi ed will reduce the number of potential side effects and curtail the development of pathogen resistance related to imprudent use of broad spectrum antibiotics. the current mainstay of supportive care in sepsis remains the maintenance of adequate oxygen delivery in the face of myocardial depression, capillary leak, acidosis, and massive cytokine release. while some early adult studies have suggested improved outcomes when achieving supra-normal levels of oxygen delivery, this approach in pediatric sepsis remains unproven. this is likely due to the fact that septic patients may have a perturbed ability to extract oxygen in addition to suboptimal oxygen delivery. clinically, this impairment in cellular oxygen uptake may be refl ected by an inappropriately high central venous oxygen saturation (s cv o ) in the face of a progressive and therapy refractory acidosis. optimizing appropriate oxygen delivery remains a clinical goal and incorporates the need for maximizing oxygen carrying capacity. while there is no recommended hemoglobin level for children, the most recent nih consensus conference suggested a hemoglobin concentration of g/dl for adults with cardiopulmonary compromise as part of a protocol toward achieving the therapeutic goal of s cv o > %, with improved outcomes demonstrated when this goal was achieved during initial resuscitation. in the context of fl uid loading with blood transfusion, empiric administration of diuretics to eliminate extra fl uid should be avoided until hemodynamic stability has been achieved or if the child exhibits signs of intravascular volume overload defi ned earlier in this chapter. similar clinical parameters can be assessed in response to blood transfusion. perhaps the best assessments are determinations that provide indirect evidence of the balance between oxygen delivery and consumption such as lactate levels and mixed venous oxygen saturation. in the context of fl uid loading with blood transfusion, empiric administration of diuretics to eliminate extra fl uid should be avoided until hemodynamic stability has been achieved or if the child exhibits signs of intravascular volume overload. finally, the nutritional status of the septic child must be addressed. patients with sepsis often have poor nutrition prior to admission to the picu and often may not be fed in the fi rst few days of illness. this state combined with the increased metabolic rate associated with sepsis place the septic patient at risk for protein calorie malnutrition. intestinal hypoperfusion in combination with absence of local enterocyte nutrition can cause mucosal barrier dysfunction and may contribute to translocation of bacteria and endotoxin from the intestine into the blood stream. while the use of enteral feeding in critical illness has been shown to improve survival and decrease hospital stay, its use must be balanced with the risk of stressing intestinal function in the face of poor splanchnic perfusion, especially in the child requiring the use of vasopressors such as epinephrine and norepinephrine. regardless of which mode of nutrition is chosen, the goal of achieving nitrogen balance is important for allowing recovery and return to physiologic homeostasis. in the absence of enteral feedings, protection from stress-related gastrointestinal ulcer formation is advised. because poor outcome in sepsis has been attributed to a dysregulated proinfl ammatory state, anti-infl ammatory agents, such as corticosteroids, have long been proposed as a potential therapeutic strategy. anecdotally, it has been observed that some patients treated with antibiotics appear to acutely worsen in a time frame consistent with the onset of antibiotic activity. this observation has been attributed to massive release of bacterial products following the lysis of high numbers of bacteria. to this end, investigators had shown that animals treated with anti-infl ammatory drugs prior to receiving antibiotics demonstrated a less severe response to bacterial lysis. despite encouraging preclinical studies, two subsequent large adult trials using high dose steroids early in sepsis showed no improvement in mortality. more recently, studies using lower doses of steroids over a longer period of time have suggested a possible benefi t including a reduced time to cessation of vasopressor therapy. these more recent observations have stimulated a resurgence in the use of corticosteroids in sepsis. adrenal insuffi ciency is frequently unrecognized in children with septic shock and a low basal circulating cortisol level, especially in association with an abnormal corticotropin stimulation test, has been associated with higher mortality rates. therefore, identifying "at risk" patients with a corticotropin stimulation test and treating this group may improve outcome in sepsis. a study in which adult septic shock patients who were classifi ed as "nonresponders" based on corticotropin stimulation results were treated with hydrocortisone and fl udrocortisone for days and had a signifi cantly reduced risk of death. however, this initial observation was not observed in larger follow-up studies. thus, the use of hydrocortisone and the application of a corticotropin stimulation test in septic patients remains highly controversial. in pediatrics, it is currently recommended that any child with fl uid-and catecholamine-refractory shock (inadequate response to two or more vasoactive agents), has a known history of adrenal insuffi ciency, or has previously received exogenous steroids should be considered for steroid replacement with hydrocortisone (usual dose between and mg/m /day divided every h). because the host response to sepsis is mediated by circulating infl ammatory molecules, it has been hypothesized that extracorporeal removal of these mediators via hemofi ltration or exchange transfusion may affect outcome. case reports suggest that arterial oxygenation and hemodynamics can be improved with use of hemofi ltration during sepsis and multiple organ failure. however, there exist many mitigating factors in evaluating the pediatric experience and the effi cacy of hemofi ltration remains unproven. challenges with instituting extracorporeal hemofi ltration include diffi culty with vascular access in smaller children, potential fl uid and electrolyte imbalance, hypothermia, anticoagulation requirements and acutely compromised hemodynamics during initiation. in addition, it is not known whether benefi cial proteins such as albumin, immunoglobulins, clotting factors and counter-regulatory cytokines are removed during this process. while experience shows that hemofi ltration can be safely performed in children with sepsis, it remains unclear if it will improve outcome. part of the infl ammatory response involves cytokines that cause widespread activation of the coagulation cascade with suppression of fi brinolysis as reviewed above. it is encouraging adrenal insuffi ciency is frequently unrecognized in children with septic shock. a low basal circulating cortisol level, especially in association with an abnormal corticotropin stimulation test, has been associated with higher mortality rates. that administration of activated protein c in adults with septic shock was associated with a signifi cant decrease in -day mortality. though activated protein c should not be routinely used in pediatric sepsis, indications for its use may be determined from further trials in pediatric sepsis. in the meantime, many other potential immune modulating therapeutic agents have been identifi ed and are currently under investigation. unfortunately, many of the antiinfl ammatory agents tried to date (anti-il- , anti-bradykinin, anti-endotoxin, anti-tnf-a , soluble tnf receptor and anti-platelet activating factor) have not shown any benefi t in large, randomized clinical trials. it is hoped that improvements in study design which include thoughtful stratifi cation of patients, timely identifi cation of the presence or absence of a pathogen and consideration of genetic factors that infl uence outcome will eventually assist in discovering and targeting pharmacologic agents that ultimately improve the outcome of the pediatric patient with septic shock. vascular bed-specifi c hemostasis: role of endothelium in sepsis pathogenesis corticosteroids for severe sepsis and septic shock: a systematic review and meta-analysis immunological therapy of sepsis: experimental therapies effi cacy and safety of recombinant human activated protein c for severe sepsis signal transduction during innate and adaptive immunity role of nfkappab in the mortality of sepsis new insights into its pathogenesis and treatment the accp-sccm consensus conference on sepsis and organ failure stress doses of hydrocortisone reverse hyperdynamic septic shock: a prospective, randomized, double-blind, single-center study clinical practice parameters for hemodynamic support of pediatric and neonatal septic shock: update from the american college of critical care medicine toll-like receptors: molecular mechanisms of the mammalian immune response prognostic values of tumor necrosis factor/cachectin, interleukin- , interferon-alpha, and interferon-gamma in the serum of patients with septic shock. swiss-dutch j immunoglobulin study group hemodynamic support in fl uid-refractory pediatric septic shock surviving sepsis campaign guidelines for management of severe sepsis and septic shock surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock organization and regulation of mitogenactivated protein kinase signaling pathways lps induction of gene expression in human monocytes implications of arterial pressure variation in patients in the intensive care unit signal transduction by the c-jun n-terminal kinase (jnk) -from infl ammation to development the i kappa b kinase (ikk) and nf-kappa b: key elements of proinfl ammatory signalling use of corticosteroid therapy in patients with sepsis and septic shock: an evidence-based review myocardial dysfunction in septic shock: part ii. role of cytokines and nitric oxide rationale for restoration of physiological anticoagulant pathways in patients with sepsis and disseminated intravascular coagulation genomic polymorphisms in sepsis the contrasting effects of dopamine and norepinephrine on systemic and splanchnic oxygen utilization in hyperdynamic sepsis early enteral nutrition in acutely ill patients: a systematic review source control in the management of severe sepsis and septic shock: an evidence-based review role of the coagulation system in the local and systemic infl ammatory response new rationale for glucocorticoid treatment in septic shock anti-infl ammatory cytokines pediatric considerations epidemiology of sepsis and multiple organ dysfunction syndrome in children pulmonary artery catheter consensus conference: consensus statement drotrecogin alfa continuous plasmafi ltration in sepsis syndrome. plasmafi ltration in sepsis study group early goal-directed therapy in the treatment of severe sepsis and septic shock innate immune mechanisms triggering lung injury effects of nitric oxide in septic shock signal transduction pathways in acute lung injury: nf-k b and ap- sepsis remains one of the most pressing clinical challenges for the pediatric intensivist. it is apparent that while a great deal is now understood about the biological and molecular mechanisms involved in sepsis, this knowledge has not yet had a dramatic impact on improving outcome. at present, therapeutic modalities for sepsis remain largely supportive and founded on the fundamental physiologic principle of providing adequate oxygen delivery. with this approach, mortality in pediatric sepsis improved modestly over the past decades. however, the fact that over , children per year continue to die in association with severe sepsis argues that further advances be made. realization of the goal of improving survival requires investigators committed to achieving further mechanistic insights into the physiologic, molecular, and genetic biology of sepsis, in concert with large pediatric-specifi c interventional trials. a year old female is admitted to the pediatric intensive care unit with septic shock. she is well oxygenated on a % oxygen face mask. she has already received ml/kg of . % normal saline and has been started on a dopamine infusion at a rate of mcg/kg/min. in monitoring her response to these interventions, which of the following should not be used as a therapeutic endpoint to monitor her progress? a. capillary refi ll time b. echocardiographically measured ejection fraction c. mental status d. serum bicarbonate level e. urine output ³ ml/kg/h . a year old male with acute lymphocytic leukemia is admitted to the pediatric intensive care unit with vancomycin resistant enterococcus bacteremia. his vital signs reveal a temperature of . °c, a heart rate of bpm, a respiratory rate of breaths/min, and a blood pressure of / mm hg. he is lethargic, but arousable. his pulses are bounding and his capillary refi ll is brisk. an arterial blood gas reveals a ph . , a paco mm hg, a pao mm hg, an oxygen saturation of %, and a base defi cit of (− ). the oxygen saturation of venous blood sampled from the superior vena cava is %. a. the young man is bacteremic, but not in shock as evidenced by his bounding pulses, brisk refi ll, and normal systolic blood pressure. b. the young man is in shock with inadequate oxygen extraction at the tissue level evidenced by the elevated superior vena cava saturation. c. the young man has a primary metabolic acidosis, but has a normal oxygen extraction as oxygen saturation in the superior vena cava is normally higher than elsewhere in the body. d. the young man has a primary metabolic acidosis, but is not in shock, evidenced by his high superior vena cava saturation. e. the young man has a primary respiratory alkalosis that would benefi t from supplemental oxygen therapy. . there is suffi cient data to justify the use of which of the following adjuvant therapies in pediatric sepsis? a. the administration of activated protein c to a child in septic shock without thrombocytopenia or coagulopathy b. the administration of anti-tnf-a monoclonal antibodies to a septic patient to decrease the proinfl ammatory response c. the administration of stress dose hydrocortisone to a septic patient whose serum cortisol level fails to increase sufficiently in response to a corticotropin stimulation test d. the early initiation of high volume, continuous veno-venous hemofi ltration to remove proinfl ammatory cytokines e. the transfusion of packed red blood cells to maintain a hemoglobin ³ g/dl in order to provide supranormal oxygen delivery . it has become clear that dysregulation of the coagulation cascade occurs in sepsis as refl ected by activation of procoagulant pathways, consumption of clotting factors, alterations in fi brinolysis, and reduced anticoagulant activity. which of the following components of coagulation is increased during sepsis? a. antithrombin iii (at iii) b. plasminogen activator inhibitor type (pai- ) c. protein c d. protein s e. tissue factor pathway inhibitor (tfpi) key: cord- -lle mha authors: martinon, fabio title: the endoplasmic reticulum: a sensor of cellular stress that modulates immune responses date: - - journal: microbes infect doi: . /j.micinf. . . sha: doc_id: cord_uid: lle mha many inflammatory and infectious diseases are characterized by the activation of signaling pathways steaming from the endoplasmic reticulum (er). these pathways, primarily associated with loss of er homeostasis, are emerging as key regulators of inflammation and infection. recent advances shed light on the mechanisms linking er-stress and immune responses. loss of cellular integrity is a hallmark of severe pathogenic insults and leads to the activation of immune defenses aimed at repairing tissue damage and fighting infections. it has been postulated that the release of danger signals by damaged cells could lead to the mounting of immune defenses [ e ] . these danger signals also known as damage associated molecular pattern (damps) are molecules that trigger specific receptors including receptors that initiate inflammatory and immune responses. many damps are nuclear proteins such as hmgb or cytosolic molecules including, for example, uric acid and atp. when released outside of the cell following tissue injury, damps are sensed by immune cells and trigger innate immune receptors such as the toll-like receptors (tlrs) or the nlrp inflammasome [ , ] . the release of damps by cells is often described as a passive process, occurring as a consequence of sustained cell damage. this process is irreversible and correlates with the death and elimination of the damaged cell. however, before losing membrane integrity, cells can experience various states of malfunction that are reversible. we can define these cellular states as stress. typically, stressed cells are malfunctioning, but are also characterized by the activation of adaptation responses to the stress aimed at repairing cell damage. stress can be a consequence of perturbations in basic cellular functions including the availability of nutrients and oxygen as well as the capacity to communicate with neighboring cells and to respond to changes in the microenvironment. if the cell is able to sense these perturbations it mounts a response (hereby referred to as a stress response) that, by definition, is an active cellular adaptation to the abnormal conditions. if this repair/adaptation fail or if the stress is too severe, the cell will eventually die possibly releasing damps. tissue stress and malfunction promotes low-grade inflammation which helps the tissue to adapt to the damage and restore tissue function [ , ] . this low-grade inflammation also termed para-inflammation illustrates how stress and possibly stress response pathways can contribute to immune responses (fig. ). however the mechanisms and the role of stress responses in controlling innate immunity and inflammatory pathways are poorly understood. the endoplasmic reticulum (er) is emerging as a key organelle that maintains cellular homeostasis and contributes to the regulation of innate immune response [ , , ] . perturbations that affect the er trigger a specific response known as the er-stress response. in this review we discuss how infectious and inflammatory signals affect the er and focus on how perturbations and signaling pathways emerging from this organelle regulate innate and inflammatory pathways. the er is an essential organelle that controls the biogenesis of cellular components including proteins, lipids and carbohydrates. it is connected with the nuclear membrane and via mitochondria associated membranes (mams) to the power source of the cell: the mitochondria. in eukaryotes, resident proteins of the endocytic and exocytic organelles as well as cell surface and secreted proteins are synthetized on polysomes anchored to er membrane and then, translocated across the membrane into the er lumen. the er lumen is rich in calcium and its oxidizing environment catalyzes posttranslational modifications such as the addition of n-linked glycans and the formation of disulfide bonds [ , ] . the nascent protein must fold via a complex process that is monitored by resident ereprotein chaperones and enzymes that prevent aggregation and regulate proper folding of newly synthetized proteins. this process being essential for the production of virtually every secreted protein and membrane receptor, the er is therefore an essential hub controlling various aspect of cellular biology including virtually all aspects of cellecell communication and signal transduction pathways steaming from membranes. in immunology the er is best known for its role in antigen presentation, it is the main site for the assembly of mhc class i and mhc class ii molecules and is the location of peptide loading onto mhc class i molecules. conditions of er stress occur when the amount of proteins entering the er exceeds its folding capacity. this imbalance induces a protective signaling cascade collectively termed the unfolded protein response (upr) or the er-stress response [ , ] . experimentally, many conditions can trigger the erstress response. for example, conditions that inhibit glycosylation including low glucose as well as pharmacological compounds such as tunicamycin, affect the maturation and transport of most secreted proteins causing er-stress. other drugs and conditions that perturb er ca þ levels have profound effects on er homeostasis and protein folding. drugs such as thapsigargin can trigger depletion of er calcium stores leading to acute er-stress responses. the er presents an oxidizing environment that can favor protein folding and the formation of disulfide bonds. reducing agents such as dithiothreitol (dtt) affect the oxidizing potential of the er leading to the accumulation of misfolded proteins and the subsequent engagement of the er-stress response. in addition, various physiological and pathological insults that perturb the cell protein homeostasis network, a group of interconnected pathways collectively termed the proteostasis network may trigger the er-stress response [ , ] . these insults include alterations in cellular ph, inhibition of the proteasome pathways, infections with various pathogens as well as metabolic changes associated with cancer. ersignaling pathways and the er-stress response are among the major and best-characterized components of the proteastasis network pathways. the er-stress response is a highly controlled process that adapt to specific insults to optimize and orchestrate an appropriate response. while this response may differ from one condition to another, a general picture of signaling events emerge. the attenuation of translation is among the first things that are observed upon induction of er-stress. this occurs within minutes to hours of er-stress activation and prevent further translational overload of the er. perturbation of erhomeostasis also results in the up-regulation of genes involved in er biogenesis, in chaperoning and folding of proteins as well as in the quality control mechanisms that target and ensure elimination of malfunctioning proteins including the endoplasmic-reticulum-associated protein degradation (erad) pathway. the aim of these responses is to help the cell cope with the stress, remove the accumulated protein load, increase er capacity and restore normal function of the er. in conditions of acute and prolonged stress, the response changes from promoting survival to inducing cell death. the point at which the 'apoptotic switch' is activated has not yet been determined, and may vary form cell to cell. the mechanisms of er-stress mediated apoptosis are yet poorly understood but may involve the intrinsic apoptotic pathway that lead to release of cytochrome c from the mitochondria and activation of the caspase- apoptosome [ e ]. in mammalian cells, the er-stress response is mediated by at least three signaling pathways that operate in parallel (fig. ) . each pathway steams from a class of transmembrane proteins anchored at the er: ire (inositol-requiring enzyme ) perk (pkr-like er kinase), and atf (activating the state of a tissue can range from healthy, to stressed or malfunctioning, to irreversibly damaged. damaged cells release damp, that directly engage innate immune receptors such as tlrs, nlrs or rage receptors. in contrast, tissue stress or malfunction is a reversible state that triggers an adaptation response aimed at restoring tissue homeostasis. stressed tissue may also trigger danger signals or stress signals that could contribute to the upreglation of inflammatory and innate immune responses. transcription factor ). activation of these three signaling branches control the expression of a few proximal er-stress related transcription factors and signaling events that orchestrate the transcriptional upregulation of the universe of genes involved in restoring er functions [ , , ] . ire is the most conserved branch, the only one present in lower eukaryotes and probably the best-studied branch of the er-stress response. in yeast, ire triggers the full er-stress response program, whereas in higher eukaryotes the program requires cooperation between the three branches of the er-stress response. ire transmembrane region is flanked by a sensor unit at its n-terminus that localize in the er lumen and a c-terminal portion that reside in the cytosol. the cytosolic portion of ire contains two functional units: a ser/thr protein kinase and an endoribonuclease. oligomerization of ire at the er, triggers its ribonuclease activity and cleaves, at two closely located and distinct sites, the mrna of the x-box binding protein- (xbp ) gene. this cleavage results in the excision of a mini intron of nucleotides in mammals. upon ligation, mature xbp mrna encodes a functional transcription factor. ire mediated removal of the nucleotides mini intron open a new translation reading frame at the c-terminus of the protein coding for a potent transactivation domain. therefore, by a unique mechanism in mammals, unrelated to regular splicing, ire converts an inactive xbp protein sequence into an active transcription factor. active xbp referred hereby as xbp s, contribute to the regulation of genes involved in different processes including protein folding, mechanisms controlling protein quality as well as a broad array of genes involved in almost every aspect of er function, physiology and biogenesis [ , ] . early studies on xbp highlighted the role of the er-stress response pathways in the development and regulation of secretory cells including plasma b cells. xbp -deficient b cells are unable to differentiate into antibody-secreting plasma cells [ , ] and antibody production in vivo in response to antigenic challenge is impaired by xbp deficiency. importantly it was shown that activated b cells undergo xbp mrna maturation to produce xbp s in order to promote er expansion and develop the secretory capacity of plasma cells [ , ] . however, what drives ire activation in b cells is still unclear. in b cells, xbp activation is not impaired by igm deficiency [ , ] suggesting that massive immunoglobulin production per se is not the cause of xbp s activation and that ire /xbp activation may by part of a preemptive differentiation program rather than as a consequence of er overload. in addition to xbp activation, ire ribonuclease activity has other functions. upon sustained and acute er-stress, ire may contribute to the degradation of membrane-associated mrnas through a process known as regulated ire dependent decay or ridd [ , , ] . the exact role of ridd is unknown. it is unclear if it solely contribute to decreasing protein load in the er or is part of the induction of the apoptosis phase as suggested by experiments showing increased er-stress induced apoptosis in cells in which ire activity was manipulated to promote ridd activation [ , ] . perk is an er anchored kinase that upon activation oligomerizes and autophosphorylate. it kinase activity phosphorylates the translation initiation factor eif a, transiently decreasing overall protein translation. this helps reducing folding requirements in the er. by phosphorylating eif a at serine perk inhibits the guanine nucleotide exchange factor eif b, a factor involved in the recycling of eif a to its active gtp-bound form [ e ]. eif a phosphorylation can be targeted by phosphatases, such as growth arrest and dnadamage inducible protein- (gadd ) [ , ] that contribute to the resolution of the stress responses. some mrna containing short open reading frames in their untranslated region are induced when eif a is inhibited. eif a phosphorylation can therefore promote the synthesis of a specific subset of genes by increasing the translation of selective mrnas whose translation is inhibited in unstressed cells. for example this mechanisms promote the translation of activating transcription factor (atf ) a major regulator of er-stress. atf is involved in various cellular responses including, glutathione biosynthesis, amino acid import, and resistance to oxidative stress [ e ]. perk also phosphorylates nuclear factor (erythroid-derived )-like (nrf ), a transcription factor that regulates oxidative stress responses and a critical regulator of innate immune responses [ , , ] . the third branch of the er-stress pathway is mediated by atf , the best-known member of a transmembrane protein fig. . er-stress signaling pathways. accumulation of unfolded proteins or loss of homeostasis in the er triggers a stress response that activates ersignaling pathways including ire , atf , and perk. active ire lead to an unconventional splicing of the transcription factor xbp and the translation of an active transcription factor. ire can also activate additional pathways such as the degradation of er-related mrnas through a process termed ridd. perk activation induces the translation of the transcription factor atf and dampen the translation initiator factor elf a activity, leading to decreased translation and protein synthesis. perk also promotes the activation of the transcriptional regulator nrf . the transcriptionally active form of atf is produced by proteolysis. when these three main pathways are activated simultaneously they orchestrate the er-stress response characterized by the transcriptional induction of genes aimed at restoring er homeostasis. the outcome of the response is repair or, if repair fails, cell death and apoptosis. family that encodes membrane anchored bzip transcription factors. upon er-stress, atf translocates to the golgi where it is processed by site and site proteases that sequentially remove the luminal domain and the transmembrane anchor. these cleavages release the transcriptionally active n-terminal fragment of atf (atf n) which relocates to nucleus where it upregulates er-stress induced genes related to protein folding and lipid synthesis [ , e ] . beyond the adaptation phase that restore er homeostasis, er-stress as well as the activation of er-signaling pathways can impact on host immune functions and promote inflammation [ , ] . er-stress observed in inflammatory pathologies determine the size, nature and duration of immune response [ , , ] . several studies have showed that er-stress might augment inflammation. mutations in mice that produce defects in protein folding or in the er-stress response pathways lead to spontaneous inflammation. mice with a mutation impairing the folding of muc , a mucin expressed in the paneth and goblet cells, show signs of er-stress in the intestine and develop an inflammatory phenotype similar to the inflammation observed in inflammatory bowel disease (ibd) patients [ , ] . patients that carry misfolding mutations in the surfactant protein-c (sftpc) gene are characterized by er-stress activation in epithelial cells and develop hyper-inflammatory responses upon viral infections [ , ] . viruses are believed to contribute to the onset of idiopathic pulmonary fibrosis (ipf) in these patients. however the exact contribution of the infection in the induction of er-stress or the contribution of er-stress to the upregulation of inflammation-mediated lung fibrosis in these patients are still open questions. similarly mutations in the cystic fibrosis transmembrane conductance regulator (cftr), an ion transporter, may cause er-stress in bronchial epithelia cells that become hyper-responsive and more susceptible to respiratory infection. hyper-responses to inflammatory stimuli have also been shown in macrophages. studies showed that hla-b misfolding can induce er-stress responses in macrophages resulting in enhanced cytokine production and increased release of type i interferon, upon stimulation with the tlr agonist lps. hla-b misfolding is associated with susceptibility to spondyloarthritides, a group of heterogeneous autoinflammatory diseases [ , ] . in an other autoinflammatory disease, er-retention of the tnf receptor (tnfr ) was shown to contribute to increased cytokines production observed in tnfr -associated periodic syndrome (traps) patients [ ] , suggesting that perturbations in the er could be directly linked to the development of autoinflammatory syndromes. most autoinflammatory diseases have been associated with aberrant inflammasome activation [ ] . the inflammasome is a cytosolic multiprotein complex that senses pathogens and cellular damage and leads to the activation of a central proteolytic enzyme, caspase- . the main function of the inflammasome is the cleavage and maturation of a few key cytokines, including il- b. these cytokines drive the initiation of inflammatory cascades in patients. because the inflammasome has been shown to become activated upon er-stress [ ] , it is possible that loss of er-homeostasis could directly drive il- b dependent inflammation and contribute to the pathogenesis of diseases including autoinflammatory syndromes such as traps, by directly driving inflammasome activation. er-stress is also involved in regulating another important branch of the innate immune system: the toll-like receptors (tlrs) response. tlrs are type i transmembrane proteins characterized by an extracellular leucine-rich domain and a cytoplasmic domain harboring a tir domain similar to the cytoplasmic domain of the mammalian interleukin receptor (il- r). tlrs activation upon recognition of "pathogen-associated molecular patterns" or pamps, results in initiation of cellular signaling events including activation of transcription factors, cytokine modulation, and upregulation of interferonstimulated genes, leading to inflammatory responses and the release of antimicrobial molecules. regulatory loops connecting tlrs and er-signaling have been described. for example, tlr and tlr promote the mrna maturation of xbp s by selectively triggering the ire branch of the er-stress response while inhibiting the other branches of the er-stress pathway [ , ] . therefore the activation of ire and xbp s by tlrs does not trigger a full er-stress response but an xbp s dependent program that synergizes with tlr signaling pathways. the induction of xbp s amplifies tlr responses leading to increased cytokine production [ , ] . this demonstrates that specific engagement of er-signaling pathways in absence of erstress can contribute to immunity. xbp s activation in the context of er-stress can also enhance tlrs responses. macrophages treated with pharmacological agents disrupting er homeostasis display a dramatically enhanced response to tlr and tlr activation [ , ] . the mechanisms by which xbp s enhances cytokine production is still unclear but may likely involve xbp binding at cytokine promoters [ ] . in addition to xbp , the er stress-induced transcription factor c/ebp homologous protein (chop), is involved in the upregulation of cytokines including il- in the context of dendritic cells undergoing both er stress and tlr stimulation [ ] . knockdown of chop in a monocytic cell line significantly reduced the synergistic effects of er-stress and tlrs on il- expression. therefore diverse er stress-related transcriptional pathways can affect the nature of immune responses (fig. ) . the er-stress response and inflammation are interconnected through various additional mechanisms. the production of reactive oxygen species (ros), the activation of the transcription factor nuclear factor-kb (nf-kb) and the induction of acute-phase proteins have being linked to both er-stress and inflammatory responses [ ] . immune cells can also respond to stress signals released by other tissues. a recent report suggests that stressed cells can transmit (via a yet to be defined stress signal) er-stress conditions to macrophages, increasing inflammatory responses [ ] . incubation of macrophages with cultured conditioned medium from erstressed cells promotes ire activation and protein chaperones upregulation in the macrophages that deploy a more sustained proinflammatory response. similarly injection into mice of media supernatant harvested from erstressed cells elicit the upregulation of genes typically associated with er stress responses in the liver [ ] . these observations may indicate that loss of er homeostasis in tissues can be transmitted to the microenvironment and immune cells to augment immune responses [ ] . whether this phenomenon is relevant to the pathology of inflammatory diseases characterized by the loss of er-homeostasis in tissues is an exciting question that remains to be addressed. er-stress pathways are also implicated in regulating immunogenicity. it has been shown that in tumor cells dying upon treatment with anticancer therapy, er-stress promotes cell-surface localization of factors involved in immunogenic cell death [ , ] . calreticulin is the best-characterized immunogenic signal emerging from the er. this erresident chaperone delocalize on the cell surface where it can act as an "eat me" or "danger" signal that primes immune responses directed against the stressed cell [ ] . the exposure of calreticulin on the cell surface is associated with the induction of er-stress [ , ] and may directly involve the activation of er-stress signaling pathways such as the perk pathway [ ] . therefore er-stress specific signal may increase immunogenicity by tagging the cell as stressed [ , , ] . pathogens activate er-signaling pathways such as ire by directly engaging innate immune receptors as it was shown for tlrs, however live pathogens affect also directly er functions as part of their infectious and replicative cycle [ ] . in plants, the ntbzip gene (a signaling branch of the plant er-stress response related to atf ) is activated upon infection of tobacco leaves with pseudomonas cichorri. moreover, silencing of ntbzip allows higher multiplication of p. cichorri compared to control plants [ ] , suggesting that er-stress pathways may represent an ancestral mechanism of immune regulation. in caenorhabditis elegans infected with bacteria expressing pore-forming toxins, xbp s and atf are induced [ ] . genetic studies have shown that these er-stress signaling pathways augment survival of infected worms probably by promoting a protective restoration of er homeostasis or by increasing immune responses. similarly in c. elegans infected with pseudomonas aeruginosa, activation of ire and xbp was noted [ ] . in this model, xbp deficiency decreases viability, possibly due to a reduced ability to cope with loss of er-homeostasis in these worms [ ] . several types of virus, parasite and bacterium that infect humans have been shown to perturb the er and to manipulate er-signaling pathways as a mechanism aimed at interfering with host immune responses and supporting replication. viruses are obligate intracellular pathogens that depend on the host machinery to produce large amounts of infectious viral particles. many viruses depend on er-membranes for translation and budding of viral particles, a process that can result in the alteration of er-homeostasis and therefore affect immune responses [ ] . in humans, er-stress was detected in tissues infected with different viruses. for example, xbp splicing and chop upregulation was detected in duodenal biopsies from individuals infected with hiv compared to uninfected persons [ ] . er-stress was also observed in the liver of patients with chronic hepatitis c (hcv) [ ] . parasites such as toxoplasma gondii activate the er-stress response [ ] . loss of er-homeostasis was also observed upon infection with different bacteria. chop induction, eif a phosphorylated and ire activation was observed in macrophagerich areas of granulomas in lungs of mice infected with virulent mycobacterium tuberculosis (mtb) [ ] . er-stress activation by live bacteria can also occur prior to cell host invasion. the facultative intracellular pathogen listeria monocytogenes (lm) was found to induce er expansion and erstress signaling pathways prior to host cell entry [ ] . the monitoring of genes typically associated with erstress responses is the main feature used to identify the activation of er-signaling pathways. however recent studies demonstrate that pathogens can trigger a specific branch of the er-stress program independently of the other branches without engaging a full er-stress response and upregulating genes characteristic of er-stress responses. influenza a virus activates the ire pathway with little or no parallel activation of the perk and the atf pathways. ire activation is apparently important for viral replication. inhibition of ire blocked influenza a virus replication [ ] , demonstrating that the virus may manipulate the ire and benefit from it. acute infection with lymphocytic choriomeningitis virus (lcmv) was shown to trigger a selective induction of the atf regulated branch of the er-stress response, whereas perk and ire pathways are neither activated nor blocked [ ] . fig. . tlr responses and er-signaling are connected and synergize to promote cytokine production. tlr activate the transcription factor xbp by selectively activating the ire kinase while decreasing the activation of atf and perk pathways as well as elf a activation. the activation of ire and xbp does not contribute to an er-stress response. however, activated xbp amplifies tlr signaling by enhancing cytokine production. similarly, if chop and xbp are induced upon treatment with pharmacological agents, they both may contribute to cytokine production. how viruses trigger specific er-signaling pathways is unclear. in the case of lcmv, activation of atf may involve the viral glycoprotein precursor (gpc) [ ] , further suggesting that some viruses may directly regulate these pathways independently of the induction of er-stress. while many pathogens trigger er-stress and/or features associated with the er-stress response, the exact function of these pathways are still poorly understood. activation of erstress and er-signaling pathways may increase immunity and reduce the cellular stress associated with the infection. this is a recent hypothesis and few experimental evidences demonstrate the physiological importance of these pathways in regulating pathogen replication and immunity in vivo. importantly, the key role of these pathways is also highlighted by the fact that pathogen have evolved mechanisms to manipulate er-signaling pathways optimizing survival and replication. the role of the er-stress pathways in shaping immunity is underscored by the fact that pathogens as well as innate immunity pathways regulate er-stress responses. it has been shown for example that the atf -chop branch of the perk pathway is specifically inhibited by tlr signaling [ ] . activation of trif by tlrs activates a phosphatase, pp a, that dephosphorylate a subunit of eif b (guanine nucleotide exchange factor for eif a) leading to the inhibition of phosphorylated eif a activity and downregulation of perk mediated atf and chop upregulation [ ] . in macrophages tlr signaling has been found to inhibit atf and perk activation by a mechanism yet to be identified [ ] . this regulation of er-signaling responses by innate immunity may explain why tlrs in macrophages only activate ire and in absence of concomitant activation of perk and atf , therefore, favoring an inflammatory response rather than a full er-stress response. viruses are also able to manipulate the er-stress response by altering specific pathways or changing cellular homeostatic setpoints. dengue fever virus (denv) for example, manipulates the er-signaling pathways to specifically activate and suppress the three different branches of the er-stress response. importantly each branch is regulated differently depending on timing and the infectious stage [ ] , suggesting that the virus can specifically manipulate the system to increase survival and prolong the viral life cycle. hcv provides us with another example of time-dependent regulation of the er-stress response. this virus triggers the erstress response in a wave-like fashion, which peaks a few days after infection. then the hepatocytes become tolerant to the stress induced by the virus or by chemical agents. suppression of viral replication restore the ability to engage an er-stress response suggesting that, the virus itself is can regulate the response [ ] . in line with these observations, livers from patients with untreated chronic hepatitis c exhibit activation of the three er-stress branches without apparent induction of downstream er-related responsive genes [ ] . these findings could be explained by specific dampening of the downstream response by viral effectors. it has been shown that hcv reduces xbp transcriptional activity [ ] and that the hcv envelope protein e may regulate perk activity [ ] . similar observations were reported for coronavirus mouse hepatitis virus (mhv). infection by mhv induces ire -mediated splicing of xbp mrna and the maturation of atf , without upregulating significant downstream target genes [ ] . likewise, the influenza virus and the enterovirus (ev ) modulate the stress response in the setting of a preexisting stress by dampening the activation and processing of the atf as indicated by its inhibitory effect on atf -dependent genes [ , ] . parasites can also modulate er-signaling responses. the host atf pathway is targeted by the t. gondii virulence factor rop [ ] . rop is a kinase that triggers proteasome-dependent degradation of atf a process that apparently is required for parasite virulence. these examples demonstrate that pathogens have evolved distinct mechanisms to regulate and benefit from er-signaling pathways. cells that undergo acute pressure on the er may trigger an apoptosis program as part of a cellular response to acute dysfunction. it is therefore likely that the dampening of this response by pathogens may increase it life cycle by promoting survival of host cells. alternatively, the pathogen may also manipulate the system to specifically control transcriptional programs triggered by the er-stress signaling branches independently of the er-stress response. the exact function of these programs in the context of an infection in vivo is unclear. the regulation of inflammatory and immune functions as well as the induction of pathogen specific transcriptional programs may represent key mechanisms orchestrated by pathogens takeover of er-signaling pathways. the overall message of this review is that er-signaling pathways steaming from the er are often tightly linked to inflammation and immunity. the er is therefore a central stress sensor that detects cellular insults and trigger specific responses involved in restoring homeostasis as well as promoting immunity. however, there are many knowledge gaps that need to be investigated in order to have a comprehensive picture of how innate immune signaling pathways intersect with signaling branches emerging from the er. the challenge for the future is to characterize at the molecular level the proximal pathways that emerge from the er-stress signaling pathways and to understand how these branches are activated and regulated by pathogens, pathogen virulence factors as well as innate immune signaling component such as tlrs. er-stress is often defined by the induction of the erstress response, however it is now clear that pathogen as well as inflammatory condition can operate er-signaling pathways in absence of er-stress. the development of new techniques and reagents to monitor the stress in the er as well as the activation of er-signaling branches will foster a better characterization of these pathways in immunity. deficiencies in the main components of the er-signaling pathways are generally lethal in mice. the development of new mouse models with conditional deletion of specific er-signaling pathways may therefore help assess the physiological importance of these responses during the course of infections as well as in autoimmune and autoinflammatory diseases. it will be also important to understand how er-signaling pathways affect immune responses in cooperation with other stress responses related to the proteostasis network such as autophagy and the oxidative stress response. finally, it is tempting to speculate that the development of drugs to alleviate erstress or regulate specific branches of the er-stress response may provide new therapeutic approaches in the regulation of infectious and immune-related diseases. regulation of innate immunity by signaling pathways emerging from the endoplasmic 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secretion in immunogenic cancer cell death activating transcription factor mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death nrf is a critical regulator of the innate immune response and survival during experimental sepsis coordination of er and oxidative stress signaling: the perk/nrf signaling pathway selective activation of the transcription factor atf mediates endoplasmic reticulum proliferation triggered by a membrane protein atf alpha optimizes long-term endoplasmic reticulum function to protect cells from chronic stress transcriptional induction of mammalian er quality control proteins is mediated by single or combined action of atf alpha and xbp the interplay between endoplasmic reticulum stress and inflammation ntbzip , an endoplasmic reticulum-localized transcription factor, plays a role in the defense response against bacterial pathogens in nicotiana tabacum activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo an essential role for xbp- in host protection against immune activation in c. elegans physiological ire- -xbp- and pek- signaling in caenorhabditis elegans larval development and immunity viruses, endoplasmic reticulum stress, and interferon responses inflammation and epithelial cell injury in aids enteropathy: involvement of endoplasmic reticulum stress in vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis c atf beta is a host cellular target of the toxoplasma gondii virulence factor rop induction of er stress in macrophages of tuberculosis granulomas activation of the unfolded protein response by listeria monocytogenes influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme (ire ) stress pathway role of the host cell's unfolded protein response in arenavirus infection adaptive suppression of the atf -chop branch of the unfolded protein response by toll-like receptor signaling toll-like receptor activation suppresses er stress factor chop and translation inhibition through activation of eif b dengue virus modulates the unfolded protein response in a time-dependent manner hcv causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response hepatitis c virus suppresses the ire -xbp pathway of the unfolded protein response protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e through the eukaryotic initiation factor alpha kinase perk coronavirus infection modulates the unfolded protein response and mediates sustained translational repression endoplasmic reticulum stress is induced and modulated by enterovirus key: cord- -rz unqaz authors: gupte, jaideep; mitlin, diana title: covid- : what is not being addressed date: - - journal: environ urban doi: . / sha: doc_id: cord_uid: rz unqaz as the number of confirmed covid- cases nears million, there is a rush to answer (what next) and a rush to act (to solve the immediate problems of covid- ). this paper discusses, with a specific focus on urban areas in the global south, what is missing to date from this response. that includes an identification of things that there are too much of, things that are not being done at all, and things that are unbalanced. there has been an enormous upsurge of academic research papers and opinions on covid- . “technological” and “scientific” solutions tend to overshadow other approaches, even if people know that “social is important”. based on our analysis to date, our primary concern is that there is too little understanding about the importance of building dialogue, exploring collaboration and co-producing solutions. there is too little understanding as to why social and cultural responses are important, and how the recognition that they are important can be actioned. over per cent of all covid- cases globally occur in urban areas. ( ) the very nature of pandemics is that they are dependent on the interactions of humans with their environment and with each other. these interactions can become intensified in the built environment, making epidemic control a key consideration in city making. the indirect and long-term impacts are likely to be more pronounced and protracted, requiring more complex responses, in lower-and middle-income countries (lmics) due to their more limited ability to respond to the risks. globally, almost one billion people live and work in conditions of urban informality and precarity. hundreds of millions more living in lmic cities can just about afford homes with formal services such as piped water, electricity and access to healthcare, but these services are patchy and reliant on deteriorating infrastructure. while these challenges are location-or place-specific, it is misleading to limit the assessment of the ecology of risk, the direct and indirect impacts of the disease, or the implications for long-term responses, to city limits alone. we recognize that urban systems share a deeply symbiotic relationship with peri-urban and rural contexts, not least through their infrastructural links and dependencies, but also more directly as a result of food systems and patterns of human mobility, and it remains important to take account of these wider contexts. take the example of jaideep gupte is a fellow of the institute of development studies, where he co-leads the cities cluster. he is the principal investigator of the smart data for inclusive cities project (european union/european commission), and coinvestigator of the global challenges research fund (gcrf) accountability in urban health (arise) hub. he is currently seconded to lead the cities and sustainable infrastructure portfolio of gcrf, uk research and innovation (ukri). . keil et al. ( ) the first human-to-human transmission of the coronavirus in germany, which reportedly occurred in january in a car parts company in bavaria, when a worker asked a colleague to pass the salt. ( ) however, as roger keil and colleagues point out, the coronavirus had travelled to this distant bavarian suburb from the periphery of wuhan, where . million cars were produced last year. ( ) hence the spatial analysis of risk has to include the very local as well as the global. as two urban scholar-activists, we are motivated to write this paper because we are concerned that covid- will exacerbate existing inequalities and absolute deprivation and disadvantage, while fuelling a misinterpretation of the nature of societal relationships that produce and sustain agglomeration. we have observed this happening both through the movement of the epidemic on the ground in the context that we know (i.e. low-income neighbourhoods), and through the ways in which local and national governments, as well as external agencies, are responding to this situation. we take note of a misconception that covid- is caused by agglomeration and therefore requires "dedensification" ( ) or heightened surveillance ( ) in cities -it is not. it is a global health crisis, whose impacts on urban populations are exacerbated by debilitated -or outright nonexistent -health infrastructures and, as we expand upon in this paper, the unequal nature of socio-political and economic relationships that categorize the circumstances faced by the lowest-income urban residents. in this context, there are implications for the local actions global compacts aspire to in order to achieve sustainable development, as well as for our understanding of urban living itself. the impacts will be most dramatic where health systems are inadequate, unreliable, under-resourced and mismanaged. while most reported covid- deaths have occurred in europe and north america, there are numerous concerns about the accuracy of global estimates of both mortality and infection. the data that we do have suggest that latin america, where the number of deaths surpassed , in june and the number of infections doubled to . million in less than a month, ( ) and increasingly south asia, ( ) are particularly badly hit. guayaquil and rio de janeiro illustrate why we are concerned. low-income residents are unable to protect themselves. overcrowding and lack of access to basic services prevent the implementation of two key measures that populations are being urged to adopt: social distancing and increased hygiene, particularly handwashing. in terms of state interventions, curfews and lockdown make income generation difficult to impossible. informal traders have been stopped from operating, while formal shops selling food and sometimes other goods are allowed to be open. formal manufacturing businesses have closed, laying off workers, and subcontracting is not taking place. low-income households face acute difficulties with limited savings; the need to pay for food, rent and services; and limited access to social protection. given what feels to be a rapidly moving set of activities and reversals, we wish to contribute our reflections on current debates. the required speed of our response means that this contribution is not based on a methodologically robust research design carried out in multiple locations in a comparative analysis. rather, we draw on our research network and our research experience to survey the information available to us, test out emerging observations and lessons, recraft our conclusions and test them again. this means that we are reading, analysing and synthesizing reports from a network of colleagues that has been built up over - years. many of these reports are from people we are working with, or have worked with for many years as research collaborators, whose capability and integrity we trust. in other cases, we are using sources of information that we have also worked with for some time, although we may not have worked directly on the same research projects. as we have come to know them, we have found these sources to be accurate as providers of information, and their analysis and conclusions are verified by other information and by our colleagues who know that context well. a typical day over the months since early march brings at least five notifications of new reports, blogs and webinars. we have each been checking these sources regularly, scanning them to understand what is going on in towns and cities across the global south, and the responses there to the health and economic crises. these reports have adopted a variety of research methods including ethnographic reporting, analysis of medical data, community monitoring and a host of other techniques. all of these sources were published between february and august . tables and summarize the set of blogs, grey literature and media sources we have consulted; a complete list is available in the online supplement. we have also checked these findings against peer-reviewed academic papers about pandemics, recent and longstanding, and through discussions with other scholars, particularly those in the medical profession. moreover, we have not been passive listeners. we have ourselves lectured or presented on these topics, written blogs and articles, and provided policy advice over the past months. indeed, our ongoing research activities have also directly or indirectly engaged with covid- . we are deeply appreciative of the multitude of activities and accounts that have been sent to us, the emerging understandings that have been shared with us and the invitations that we have received. the conclusions here draw on these sources, as understood over the last three months, captured through conversations, blogs, reports, webinars (both presentations and discussions) and news stories. the next section outlines our key concerns with covid- responses in lmics and associated discussions. the subsequent section explores the failure to identify and understand what is taking place in towns and cities in the global south, and the potential consequences of this lack of understanding. as we write this in august , the number of confirmed covid- cases globally stands at over million, with , deaths thus far. ( ) "by one estimate, the covid- literature published since january has reached more than , papers and is doubling every days". ( ) this constitutes one of the largest upsurges in research on any topic. technology-based responses to covid- seem to play a particularly dominant role. one commentator groups the digital responses to the pandemic into five categories: ( ) solutions for effective and efficient contact tracing, responding to the need to track the pathogen exchange faster than traditional systems of disease reporting; testing and disaster responder capacity to improve, adapt or invest in medical devices, tests and protective gear; early warning and surveillance systems to better understand the pathogen and monitor the outbreak; quarantine and social control as important elements of the human side of a pandemic response; and technical advancements in vaccine, mitigation and treatment research. we find there is a critical gap between the technological solutions being suggested and whether they contribute to inclusive, resilient and sustainable responses from the perspective of economically and socially disadvantaged urban residents. we see that tech-based responses are often based on uncritical and unnuanced techno-utopian understandings of what are deeply unequal relationships. ( ) at the same time, technoutopian narratives are an "easy sell", particularly to those who do have access to digital infrastructures and therefore stand to benefit from technological interventions, and they serve as an illusory alternative for meaningful local action. app-based track and trace "solutions" proposed in various countries are an illustration of this. compare for example, the aarogya setu mhealth app ( ) promoted by the government of india, ( ) and the increasing unease with its use and impact on digital freedoms. ( ) these interventions highlight the need for an engagement with issues of inequality and social relations, themes of concern to the social sciences. they mistakenly assume equality of access to digital infrastructures, which in fact are ta b l e . this finding comes from discussion between medical academics at the university gendered and unequal: across low-and middle-income countries, women are per cent less likely than men to own a smartphone or use mobile internet. ( ) the gender gap is exacerbated in low-income areas; a study of informal settlements in nairobi, kampala, lagos, yaoundé, maputo, cairo, bogotá, new delhi, jakarta and manila revealed women are about per cent less likely to be connected to the internet and a third less likely to use a smartphone to connect, compared to men in the same age group with similar levels of education and household income. ( ) track and trace approaches assume that the disease is spread through contact resulting from voluntary movement alone and that people do not live in overcrowded dwellings, and can therefore be isolated once identified. this completely disregards the compulsory nature of the economic and sociocultural relationships that force people to continue living and working in precarious locations despite the risk of contracting or spreading the disease. track and trace approaches also severely misunderstand the magnitude of the involuntary movement of people often forced by authorities or landlords who, at least in the initial stages of lockdown, continued evictions unabated. ( ) the reflections of alice wanini, a community health volunteer working in nairobi's mukuru village (home to , households), are captured in a blog series by the muungano alliance, a civil society group in kenya that is affiliated to the transnational network sdi. ( ) this is a neighbourhood in which. . . ". . .space is a luxury-and social distancing is nearly impossible, both within the households and on the narrow streets around them. [alice] tells of an incident that recently transpired where a water boozer [bowser or water tanker] supplying free water in the community, brought hundreds of residents scrambling for access to free water. in an area notably known for its erratic water supply, the free supply of water to support regular hand washing and other household usage foresaw residents crowd at the water supply area each aiming to get their water cans full with little care to prioritize social distancing." ( ) earlier research details the high densities and lack of services that residents face in this neighbourhood. ( ) these challenges run deeper than in relation to specific technologies. covid- demands and has secured a wealth of research projects. ( ) it appears that many of those with solely medical interests have little understanding of the realities of people living in informal settlements, ( ) where low wages and relatively high costs of housing and essential services mean that cash-at-hand and savings are inadequate. ( ) measures to control population movements and their consequences for income generation have therefore triggered an economic emergency. here, we recognize the need to look beyond "the immediate health consequences of the pandemic", particularly as these "begin to be superseded by the impact of public health containment measures" ( ) -notably through the inability of those subject to these measures to buy essential goods and services. social and economic interdependencies skew health and wellbeing outcomes, but in our opinion, and as we elaborate upon in later sections of this paper, are likely to be underestimated by those without an engagement with the social sciences. of manchester and one of the authors with respect to a research project: specifically on how the sample might need to be adjusted to take account of different social categories, and how the questions need to be framed given local realities. these realities lie behind the formation of the online social science in humanitarian action platform (https://www. socialscienceinaction.org/ about), accessed august . see also wilkinson ( ) . furthermore, the lack of literacy and/or the deep suspicion of formal processes on the part of many low-income residents mean that preferred "modern" approaches to ethics as developed in the global north are not effective in securing the desired ethical response. northern ethics research requires informed consent and documentation to show that this has been secured. typically, this is a form signed by the interviewee. people who are illiterate are asked to listen to a reading of the form and indicate their agreement in some way. however, it is rare that allowance is made for their fear related to officialdom. if their vulnerabilities are recognized, then it may be suggested that this group is left out of the sample. however, excluding those who are illiterate and/or who are unable to take part for these reasons is likely to bias the results, as the same people may have significantly lower incomes. researchers must engage with those who are familiar with local contexts, and those who have been trained in appropriate research methodologies. we believe that societal relations are key to addressing covid- -and that these relations have implications for technology and science. solutions have to be locally owned for them to have any chance of success, as shown by matt nohn ( ) in his analysis of responses to ebola in monrovia. this level of behavioural change cannot be imposed from above; the required modification of everyday practices has profound implications for all relations including the most intimate. in addition, strong local engagement will enable generalized solutions to be modified so that they are effective within local realities. such modifications to designs are essential to -and as important as -local ownership. the coproduction of responses is essential to secure the required modifications to designs and their effective adoption. too little attention has been given to this. this is the context in which we understand al jazeera's report from june that there have been killings in kenya to enforce the covid- related curfew. ( ) in sum, there is an oversimplistic approach to the urban context by "non-urban" professionals and academics. too little attention is given to the multiplicity of ways in which the "urban" nature of settlements and livelihoods influences what is possible in terms of responses to the health and economic emergencies. there needs to be a substantively greater consideration of what it means to seek to intervene in an urban location. we put forward several points in this regard: agglomeration and the nature of societal relationships: it is critical to recognize the nature of social relations, the intensity of -and interaction between -government agencies, and the interconnections between multiple and cascading risks and structural disadvantage. this includes a recognition of the nature of relations between urban residents, the intensity and diversity of state agencies, the creativity of people through interactions and opportunities for capability development, and the dynamic nature of (some) urban contexts. ( ) we take note that grassroots organizations are doing much to address local needs. isabel duque franco et al. ( ) document and analyse over community initiatives to address local needs related to the covid- pandemic across latin america. thirty-seven per cent are related to food security and per cent to public health. a further example is provided by a community leader from mumbai who discusses the ways in which members of mahila milan (a collective of women's savings groups) are helping to distribute food donated by local politicians, focusing on those who do not have ration cards and therefore cannot benefit from state support. ( ) another survey of "slum leaders" across informal settlements in two north indian cities (jaipur and bhopal) finds that community leaders in these settlements have persisted in their problem-solving roles during the pandemic and relied upon their informal authority and political networks to request urgently needed government relief. ( ) the study also finds however, that community leaders "are not equally positioned to secure information, make claims, and command government responsiveness in the context of the pandemic. . . leaders that are more deeply entrenched in partisan networks also appear to be more likely to contact, and be contacted by political elites, as well as report having received assistance to pandemic-time claims". ( ) this raises concerns regarding the inclusiveness and fairness of these efforts. in the philippines, the homeless people's federation did not wait for the government but immediately bought supplies for the neediest households in the neighbourhoods in which they had active organizations. while also working with state agencies providing welfare and relief. ( ) these local efforts notwithstanding, in our opinion the nature of urban organizations -and the potential for resistance and collaboration they display -remains understated in present pandemic responses, requiring a recalibration of interventions towards co-producing solutions to covid- . agglomeration and the commodification of labour: low incomes make it impossible for many living in towns and cities of the global south to secure adequate accommodation. poorly informed government strategies to reduce poverty lay too much emphasis on cash incomes and not enough on the social wage (i.e. access to affordable basic services including water, sanitation, energy, health and education). without access to these public services, households cannot afford safe and secure housing. there is insufficient information on the costs of adequate access to basic services. while community groups have sought to improve access to the services required for health, ( ) without state investment this cannot be done effectively and at the scale required. as noted in the following section, there have been multiple reports that low-income households -embedded in economic and spatial informality -have not received social protection. cash transfers are seen as the key source of support (regardless of whether they are actually received). as analysed by wiego, a transnational network working with women employed in the informal economy, government measures have been partial. ( ) insufficient attention has been given to the heightened vulnerability profiles due to multiple and cascading risks drawing residents further into destitution -and this may be true even for those who were just about surviving before the pandemic. in peru, for example, informal workers have been denied access to government emergency covid- grants because the government does not have lists of those in extreme poverty in urban areas, as it does not consider there to be extreme poverty in these areas. ( ) insufficient consideration is given to urban social assistance going beyond cash transfers, "to focus on generating jobs (especially for young people and women) and to link low-income urban residents to basic services such as health care, through subsidies, vouchers, or case management". ( working in precarious conditions. these trade-offs not only underpin their risks and vulnerability to disease in the present situation, but also speak to a much deeper history of marginalization and exclusion. there is a need to draw on the longstanding experiences of these trade-offs to better understand the ongoing health and economic pressures, and to prepare for recovery. wiego also highlights the need for state support for economic recovery for informal sector workers. ( ) what is telling is that, despite their comprehensive engagement with government measures, they are only able to identify three countries (burkina faso, jordan and uruguay) that exemplify the measures that might be taken. there is a real fear that lockdown measures will exacerbate inequality. for example, in peru, supermarkets have been allowed to remain open and sell a range of essential and non-essential goods, but the only informal workers allowed to remain trading are those selling essential goods. ( ) a key constraint to effective response is identifying and utilizing the appropriate and adequate space for pre-emptive measures (e.g. construction of appropriate wash sites with drainage, strategies to isolate patients who have tested positive), responses (e.g. routes to and from hospitals and parking spots for ambulances; transfer of unwell or indisposed patients from residences), distribution of aid (food, water or other essential distribution), and recovery interventions (e.g. disinfection or sanitization of spaces; opening-up of markets; staggered lifting of other lockdown policies). the lack of adequate consideration of informal vendors who are dependent on street spaces is just one example; wiego reports on the devastating consequences of this lack of understanding for the livelihoods of these low-paid street workers. ( ) early experiences from news websites show that the demand for space can change rapidly: the sudden crowds of migrants gathering at the anand vihar bus station in delhi (on march), ( ) long queues developing suddenly as food aid was distributed in dharavi, mumbai (on april), ( ) and similarly in alexandra township, johannesburg (on april). ( ) moreover, space considerations vary by location as well as by gender, age and other socioeconomic markers that influence risks and vulnerability profiles. the visualization of informal settlements in many covid- discussions, however, is of homogenous highdensity inner-city shacks, with insufficient attention given to lowerdensity settlements (more likely to have urban agriculture) that may also face health and economic emergencies. furthermore, decisions over the use of space are being carried out in real time and usually from a distance in compliance with lockdown. given the short supply of open space in dense urban areas, intervention sites are being used for multiple actions simultaneously. however, not enough resources are being devoted to rationalize clearance times, to formulate patterns for safe distancing, and to manage perimeters. the onset of rains will change drainage and access patterns, while soil conditions, especially in informal settlements, will increase the risk of other water-related infectious disease, such as malaria, dengue and cholera. ( ) proximity to other vulnerable areas or hotspots is also an important consideration, though it has been understated in determining the efficacy of response spaces and strategies, including those involving the eventual easing of lockdown policies. . muungano wa wanavijiji ( a). ( ). . iied ( ). ( ). . visagie and turok ( ). the potential within urban contexts: the rapid response to the need for masks -in multiple countries -is one example of the potential processes of production, consumption and creativity that exist. the guardian's report on mask making in multiple locations by diverse groups illustrates this, ( ) although it may not give sufficient attention to individual entrepreneurship. but this requires an understanding of this context, a sensitivity to the way in which governments intervene, and attention to the unintended and unanticipated consequences of such interventions. in kenya, the muungano alliance, in recognition of the impossibility of informal settlement residents isolating at home, has produced maps of informal settlements indicating places that are appropriate for isolation centres. ( ) in lima, street vendors faced a strict curfew introduced on march, as well as state actions against informal workers and a lack of access to government emergency grants. they responded by negotiating with local government for the return of confiscated goods, launching a media campaign to demonstrate their willingness to comply with hygiene measures, and articulating their potential contribution to a safe and secure food supply within a more equitable city. ( ) the wealth of grassroots responses to covid- is elaborated by the international institute for environment and development (iied), which has drawn on experiences from across its partners in the global south, who provide evidence of local groups stepping in to reduce health risks and provide emergency access to food and hygiene. ( ) as a consequence of the failure to adequately contextualize, responses are deficient in three ways. first, there is a tendency to focus concerns on issues of density without adequate attention to lack of services and overcrowding. both the press and the experts have recognized that urban areas are particularly at risk as the disease is spread by close contact, to which social distancing is the solution. the urbanization focus of covid- is exemplified by the financial times (in its free-to-read coronavirus updates ( ) ), which has been reporting "excess deaths" in urban centres, including five from the global south. the crude aggregation of population density and impact pays insufficient attention to the circumstances under which risk and vulnerability occur. as elaborated by harvard university professor mary bassett in the new york times ( ) for the us, the context is everything. this is a context in which poverty leads to residential overcrowding and lack of access to adequate levels of the basic services required for hygiene. density itself is not the problem; it is the overcrowding that is a result of poverty and the lack of infrastructure and services from state neglect. the climate emergency adds to the need for clarity on this point. there has been a recognition that a successful transition to a low-carbon economy is facilitated by more compact cities, and that densification is a solution rather than a problem. however, as noted by justin visagie and ivan turok, ( ) densification requires careful planning, especially given the implications of the irregular plots that are frequently associated with informally developed urban neighbourhoods. infrastructure networks and housing need to be planned both to be affordable and to be consistent with overall development of the town or city. ( ) in the current . ntseku ( ). ( ). . moser and peek ( . connolly ( ) . also, see for example klaus ( ). context, de-densification is viewed with trepidation by those who fear that covid- will be associated with evictions legitimated through this rationale. see, for example, the response of south african civil society when their government highlighted the potential of "de-densification" as a response to the financial times reports that guayas province in ecuador has had , excess deaths, which is per cent of what would have been anticipated. ( ) the vulnerabilities of the population in the provincial capital, guayaquil, are elaborated by university of manchester emeritus professor caroline moser and architect olga peek in an iied blog post, and relate both to globalization (travel to and from spain, italy and the us in particular) and poverty that puts healthcare out of reach. ( ) in response to overcrowding at home, young people have been on the streets, potentially adding to risks. in nairobi, the scale of deprivation in informal settlements is evident. in mukuru, for example, only per cent of residents have access to a private water tap and private sanitation. the lack of provision means that a public tap serves an average of households, and there is one public latrine for every households. ( ) this study also documented just how expensive it is to be "poor" and the premium paid to access housing and informal services -not a new story in any respect. more recent research has pointed to the reality that this premium may be related not just to the higher cost of informal services but also to the adverse circumstances under which low-income households access formal services. ( ) rigorous community monitoring has shown that despite these adverse conditions, there have been relatively few cases of covid- in nairobi, kisumu and nakuru. ( ) the impacts of the responses to covid- , as with historical responses to public health crises such as ebola (in ), ( ) h n (in ) or sars (in ) ( ) -or even further back historically, the cholera pandemics in cities (for example in london and new york in the mid- th century) ( ) -are filtering through to built environment design, planning and building processes. in the initial weeks of the covid- pandemic, much accusatory attention was directed towards highdensity urban areas and mobilities within such areas. ( ) subsequently, however, "the contemporary processes of extended urbanisation, which include suburbanisation, post-suburbanisation and peri-urbanisation", have also been linked with "increased vulnerability to infectious disease spread". ( ) given this, creighton connolly writes, "governance is a more important factor than density in determining the severity of outbreaks". ( ) but there is no consensus as yet on the practical steps required to rethink the material production of the city, nor on how managing density (and overcrowding) and installing new ways of transportation, relate to the vulnerabilities of the lowestincome urban residents living and working in precarious sites. how do we prevent a response driven by environmental health imperatives that is fundamentally hostile to informal dwellers, condemning them because of their poverty? the required response has to prioritize essential investment in basic services and the provision of affordable housing so as to assist lowincome households to secure access to the services and housing that they need for good health and wellbeing. "affordable" in this context cannot be synonymous with "low-cost" unless low-cost is indeed affordable to the lowest-income households. second, the actions and messaging are too frequently top-down, with little attention being given to questions of local ownership. in kenya, the muungano alliance have established a monitoring system ( ) in settlements where their membership is strong, to ensure that the government and communities have accurate information. on june , they collected data from community leaders (youth, community, health workers and village elders) from villages in three cities (nairobi, nakuru and kisumu). their data highlight that the areas with the most restrictions are not those with the highest reported rates of infection. specifically, per cent of informants reported cases of covid- in mathare, but informants for this area were less likely than those in other areas to report the presence of isolation centres, social distancing and restrictions on movement. pressure from the communities has meant that the alliance is sending the information back to the key informants with summaries of the situation along with updates on both the situation and what is being provided nationwide. going forward, the ability of grassroots organizations to engage with digital information is an opportunity to build new relations between responsible state agencies and organized community groups. the significance of co-productive responses, and the need to ensure strong local ownership, means that this should be explored. however, attention also has to be given to the digital divides, women's relative lack of access to mobile phones, and the risk that some of the older vulnerable groups in the population will not be equally able to represent their needs and interests. third, while recognition is being given to the economic emergency, actions do not acknowledge the depth of the crisis that is facing the urban poor. it is evident that considerable numbers of informal workers and enterprise owners are either unable to sell anything at all because they are forcibly closed, or are selling considerably less than usual due to reduced hours and/or consumers having less to spend. ( ) in nairobi, for example, a curfew from : to : has considerably reduced the numbers of clients and customers, and a complete lockdown in areas such as eastleigh has meant that domestic workers have lost their incomes -with obvious knock-on effects within, in this case, the adjacent informal area of mathare. ( ) the impacts are similar in middleincome countries such as south africa. ( ) however, while governments promise income support and emergency cash transfers, many households living in informal settlements have not benefitted from this provision. in nigeria, justice and empowerment initiatives have worked with the nigeria slum/informal settlement federation and the physically challenged empowerment initiative to track the neglect of those living in informal settlements and their inability to either earn a living during lockdown or access government funds. a survey of leaders in communities found that " per cent reported people are unable to meet basic needs. meanwhile, the vast majority of urban poor communities ( %) reported government-provided "palliatives" intended for the vulnerable had not reached them." ( ) similar findings are evident in brazil where, for example, low-income favela residents in são paulo are not receiving the monthly emergency basic income payment (worth us$ ), despite the shutdown by the city authorities of informal trading on april . ( ) longer-term impacts for low-income residents are likely to be severe, particularly for those engaged in informal livelihoods. the economic . rutherford ( . goñi ( ) . . see https://www. justempower.org (accessed september ) and @ justempower on twitter for consequences of lockdown are already evident in testimonies from those living and working in the informal sector. an early analysis of dhaka, bangladesh is provided by stuart rutherford, a longstanding expert in financial services for low-income households. ( ) the lockdown here began on march and lasted until may, although it was progressively less actively respected and enforced. data from individuals who keep financial diaries (a process ongoing since august ) showed a significant collapse in total flows of money. in april , the first full month of the covid- lockdown, total money exchanged (inflows and outflows) aggregated across all diarists fell to . million bangladeshi taka (us$ , ) . for comparison, the average equivalent amount of money (from august , when the diaries began) was . million taka (about us$ , ). average household expenditure of taka a month was financed by income ( taka), loans ( taka) and savings withdrawals ( taka), with the balance being met by gifts and a small payment of taka for the completion of the diary. however, these average figures are deceptive as one-third of all expenditure was made by one family that included two government employees. the remaining families spent four times more than they received in income during april, with per cent of non-construction-related expenditure ( ) being spent on food. dramatic falls in income for low-income families in brazil are also reported by rioonwatch, which regularly updates readers on the challenges faced by favela residents. in this case, rioonwatch draws on a national survey to report, "four out of five favela families ( %) across the country are living on less than half the income they received prior to the pandemic, the study found. of those %, more than a third reported losing all of their family income, while only % of the total experienced no change in income or only a minor drop." ( ) one result of this loss of income, combined with a lack of social protection, is that low-income residents are forced to find work despite the risks to themselves and their communities. john taylor, chief technical advisor for the food and agriculture organization (fao) in bangladesh, reports that some low-income workers in dhaka have been working as "rogue rickshaw drivers". ( ) he notes that households and communities have turned to urban agriculture as a way to secure access to food. furthermore, national strategies have required a homogenized (and often brutal) enforcement of order under lockdown. this has led to a heightened incidence of violence, fear and anxiety, experienced particularly by those who were already targets of violent evictions or enforcement of cityplanning and state practices, and who suffered sociocultural marginalization and stigmatization as a result prior to the pandemic. as noted by the un high commissioner for human rights, there has been a troubling increase in cases of police brutality and rights violations under the cover of exceptional or emergency measures. ( ) an inability to rely on informal arrangements with non-state actors, including illegal and extra-legal groups, has meant the police are hindered in their ability to provide safety and security during the covid- pandemic. on the one hand, the police are failing to protect communities from criminal gangs, where they pose an increased risk. ( ) at the same time, they have hampered aid efforts when these have come from non-state or criminal groups. ( ) city police are tasked with placing physical constraints to stop people from reaching their places of work, ( ) while the closure of markets, demolition of informal shops ( ) and prohibition of street vending have prevented people from earning a living. ( ) there is also a notable increase in domestic violence, violence against children, gender-based updates on this in lagos, such as the demolition of shops in ijora badia on may . . see, for example, this report from uganda: hayden ( violence (gbv) and antisocial behaviour. ( ) we note that in these examples, police and other security services were described as overburdened. trust was noted to be low between communities and enforcement officials, who have been exposed to high levels of violence, service delivery protests and criminal activities that have existed since before covid- , while at the same time being exposed to covid- -related risks themselves. ( ) maintaining law and order has proven doubly complex given that legal and procedural practices have also needed to adapt extensively and repeatedly to situations of social distancing and lockdown, as exemplified in south africa. ( ) in most lmic contexts, police personnel, particularly those holding lower ranks, have limited opportunities to access mental health and other wellbeing support. it is under these difficult circumstances that city police forces have become central to national covid- response strategies. they are required to enforce regulations that are often unpopular in public, private and commercial spaces, in lived environments as diverse as informal settlements and gated communities, including their own poorly serviced living quarters, as well as online in response to new and increased digital activities. as authors of this paper, we remain deeply sceptical that the pandemic, and the policy or programmatic interventions in response to it, will trigger the sort of system-wide rethink required to uplift urban futures, particularly for people who live and work in conditions of urban informality and precarity. on the contrary, the most likely outcome is an ossification of pre-existing inequalities, persecutions and structural failures. yet again, we see "the local" placed as the frontline of crisis response. and yet again, the frameworks that guide our collective thinking seem incapable of adequately devolving political authority with adequate resources to this level. without a meaningful and sustained political shift towards the local, crisis response remains debilitated. we despair as we note parallels with the plague epidemic in bombay all the way back in , when colonial policies aimed at supressing the disease showed explicit class bias against the urban poor. ( ) those experiences could have triggered a new way of city planning, involving a rethinking of urban service infrastructures, and a re-strategizing of crisis responses, to engage the politics and negotiations of agglomeration. more than a century later, however, mumbai finds itself in the same position. what then is the potential to see the shock of covid- as an opportunity? there is -it seems -a new attention to towns and cities in the context of the covid- pandemic. for example, we see this in nairobi, where the national government has committed to informal settlement upgrading. in august , the government declared a special planning area with two years to prepare and deliver an integrated plan. that period has now been extended for a further two years. what is exciting in mukuru is that infrastructure improvements in water and sanitation, roads, drainage and electrification have now been finalized, and implementation by nairobi metropolitan services began in may . ( ) and the kenyan president announced upgrading plans for both korogocho and mathare on march . meanwhile nairobi metropolitan services announced the suspension of all existing development plans in four further informal settlements in kibera on may , in order to enable a more participatory integrated approach. ( ) on february, the city hall (nairobi county) and the ministry of transport and infrastructure announced their intent to scale up the upgrading of nairobi's informal settlements with a special planning area for the neighbourhoods around nairobi central station. covid- , then, has been associated with an acceleration of plan implementation in mukuru and significant new upgrading initiatives across nairobi. once new external agencies and their interventions are better informed about the urban context and more successfully embedded within urban institutions (both formal and informal), the situation might be recognized as an opportunity. this requires addressing the unequal socioeconomic and political relationships that have come to characterize our cities with a set of reforms that have broad ramifications and that can lead to a realization of the local objectives of key global compacts such as the sustainable development goals. we think that closer attention to such reforms will be helpful as long as it recognizes the insights of the social sciences and the knowledge created and owned by local communities. there is an urgent need to invest in the infrastructure required to make low-income housing safer, especially dwellings in informal settlements. and there is a need to support better-quality housing, through access to more affordable improvements. at the same time, for an effective response to the health emergency, there is a need for residents, community health workers and/or volunteers, and the health authorities to work together. it is critical that those who are knowledgeable about effective urban development approaches are asked to share their expertise. successful interventions are those that recognize the need for targeting financial support, and devolving authority to respond to the municipal (even sub-municipal) level, while at the same time proceeding based on knowledge about economic and spatial informality and building on the efforts, knowledge and 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recovering from the impacts of the covid- outbreak. in this opinion, the basics of biodefense as well as specific opportunities for the data science community to contribute are discussed. the coronavirus outbreak is sweeping the globe with outbreaks reported on every continent except antarctica as of march . data scientists are uniquely and diversely skilled in ways that can be highly effective in minimizing, combatting, and recovering from the impacts of the covid- outbreak. in this opinion, the basics of biodefense as well as specific opportunities for the data science community to contribute are discussed. there is no doubt that covid- is an unprecedented challenge for humanity, and one where data scientists can play an active and useful role. in an earlier piece, i provided a brief overview of the field of biodefense with a focus on the importance of risk assessment and resilience, the ability to maintain operations during disasters or other unanticipated crises. this piece will dig deeper into biodefense policy as well as suggest specific actions that the data science community can take to contribute to covid- resilience, response, and recovery efforts. starting at the top and looking more deeply into risk and resilience in the united states, much of the policy stems from the homeland security presidential directive , which outlines the policy and strategy for public health and medical preparedness. based upon hspd , the biodefense for the st century directive, signed into law by george w. bush in , the critical components of the plan are biosurveillance, countermeasure distribution, mass casualty care, and community resilience. an excellent source for understanding these initiatives is the biodefense policy landscape analysis tool, which provides significant detail on these policies, including the complex web of relationships of roles and responsibilities between and across federal agencies. one criticism of these policies that is highly relevant to the current outbreak is that they are far too broad and that the original focus, protection from biological weapons, is not aligned with threats such as the current coronavirus outbreak and that an information-driven biosafety strategy that focuses on all threats during peacetime would serve the public better. to me, these criticisms are highly valid, especially when considering the fractured appearance of the us government responses to covid- to date. what is less clear is whether the government response is fractured due to poor execution or because the plan is indeed a poor fit for peacetime public health threats. much of the current peer-reviewed literature on minimizing disruption and ensuring business operations during a crisis and most comprehensive toolkits focus upon ensuring biosafety, which refers to the safe handling of biological agents, or on traditional biodefense, tactics against manmade biothreats. traditional approaches to disaster preparedness often focus upon institutional business continuity plans (bcps). as covid- continues to spread through undocumented community transmission, many have turned to social distancing as their primary prevention and resilience strategy. globally, organizations are limiting travel, having employees work from home, distributing personal protective equipment, and taking other measures that focus upon containment. these measures are helpful and important, but as we are seeing significant community spread, public health officials are blending their focus from containment only to containment and mitigation, and businesses must look ahead in this manner as well. this outbreak is past the point of prevention, and the response must now focus on minimizing the effects as people get sick. one of the most effective ways to think about resilience is to recall every cliché you have ever heard about weak links and apply systems thinking. what happens if your child's school closes and you are forced to quarantine at home with them? do you have backup, and can you work remotely? what about vendors, contractors, and suppliers that you depend on-is there adequate redundancy in your supply chain? does your organization already have an experienced virtual meetings culture? are people used to video conferencing? are the necessary technology toolsets already in their hands? have the newest, least experienced, and/or most struggling colleagues been given what they need to do their part? an addictive quality of humanitarian response actions is the clarity of purpose. there are no endless meetings discussing long-term strategies, and every decision is not accompanied by weeks of handwringing. priorities are often set on a daily basis. everyone knows their tasks and where they fit in. extraneous activity is eliminated as a necessity. think through priorities with a clear mindset and eliminate the nonessential to free resources for the things that must happen and that need the most help. in biodefense strategies, the shift from containment to mitigation initiates the direct-response phase, a shift from prevention to action. just as the airlines ll open access always instruct passengers to place the oxygen mask on themselves before helping others, people must ensure that they themselves and their organizations are secure before attempting to help others. there are many ways that data scientists can be of significant help, and their own communities may be the best place to start. for data scientists who can code, look for opportunities to do that. the same is true for analysts; statisticians; high-content data experts; data policy, governance and regulatory experts; payor and reimbursement policy experts; and all other domains. people looking to help should lead with their strengths but also remember that each of us is more than just the sum of our parts. during the ebola outbreak of - , i was setting up technology at a remote clinic in sierra leone. the clinic had not been opened for patients as the water supply had not been inspected or certified. my undergraduate degree happens to be chemical engineering, and i was happy i could help. be imaginative. with respect to matching specific expertise and problems, telehealth has great promise and is being touted as a solution, but the current availability is spotty geographically, and rapid implementation will need expert guidance. specifically, regulatory, geolocation, and reimbursement expertise are needed for telehealth to reach its full potential during this outbreak. for those with expertise in research and epidemiology, you are likely already in high-demand and activated. if not, as the response to coronavirus will be managed primarily within communities, contacting your state, town, county, district board of health, or equivalent may yield instant opportunity to be of assistance. if you work in biomedical product research and development, your company is likely concerned and developing plans to ensure the resilience of clinical trial sites and participants. by definition, patients in clinical trials all have underlying health conditions, and many have health conditions that will put them at high risk of serious effects if infected with the coronavirus. shortages of drugs and drug manufacture ingredients are already putting the clinical supply chain at risk. trial deviations and the need to adapt trial designs are all possibilities that will require biostatistical and data science support. the list goes on and on. communications strategies must be resilient, and facts must be clear and substantiated. synthetic and in silico modeling of response, outbreaks, supply chain, transmission, and almost every other aspect must be available when and where needed. the tools of digital disease detection are reasonably strong, but if you have ideas that will make them stronger, contact groups such as healthmap (https://healthmap.org/en/) and share your ideas. misinformation campaigns must be countered. campaigns such as ''flatten the curve'' that are making the rounds on social media are based upon solid facts and can be very useful in educating all of us in the importance of slowing the transmission of the virus regardless of how far it has already gone. further, anything that can be done to educate patients and caregivers in ways that preserve precious resources such as coronavirus test kits is important. cyberchondria is a real thing, and many people will be seeking care with minor symptoms and low risk of actual infection. for coders and epidemiologists, there are already very strong and dedicated platforms for building, modifying, and deploying mobile applications for coronavirus tracking and response use cases. a toolset that i have personally used over the years for rapid development and deployment is commcare by dimagi, and they have already built a toolkit and guide specifically for covid- outbreak response. another excellent toolset that has been crowdsourced specifically for coronavirus response is the coronavirus tech handbook. these are not the only toolsets; there are others. pick the one that fits your immediate needs best and get to work. the important thing is not to waste precious time building things from scratch in outbreak settings when time is of the essence. reach for something that works and get it into the hands of those that need it. of course, these are just examples, and the actual opportunities are countless. use your professional and social networks to find the right opportunity to help. if you are not the best fit, use your network to matchmake between people and problems. every solution counts. one fascinating attribute of infectiousdisease outbreaks is that it can be chal-lenging to determine when or how they will end. in the - ebola outbreak in west africa, the epidemic ended quite abruptly and somewhat unexpectedly, and the response went rapidly from start-up mode to shut-down mode. my last trip during that outbreak focused upon rebuilding local infrastructure to enable the local health systems to get back to full operation, including the possibility of an ebola-infected patient presenting and seeking care. health systems being overrun by infectious-disease outbreaks is not limited to lowand middle-income countries (lmics). in the us, we have seen seasonal flu outbreaks severe enough that hospitals have had to erect tents to manage the overflow of patients. unavoidably, healthcare workers will be infected. clinics will be low on staff and supplies, and some clinics will be forced to close and transfer their burden elsewhere. re-establishing services and adequate staffing levels and helping institutions through the fragile state of re-opening and expansion is the third domain of necessity and opportunity. after truly catastrophic events, communications must be re-established and be clear and authoritative. schools and places of business will need to be ready to be re-opened. travel and transportation infrastructure must be re-established, and all of these activities must be supported through fragile initial states. this may require new data systems. during the rebuilding period after ebola, we were tasked with readying clinics for traditional services but also to be prepared for the possibility of an ebola-infected person to show up seeking care. specially designed triage applications that walked newly trained health workers through algorithmically guided case definition steps were an essential element of rebuilding the healthcare system. another essential element of active response is following through on projects that are likely to be needed during the next outbreak or pandemic. in the modern world, it is amazing how fast priorities change and people move on. following the rapid shutdown of the ebola response in west africa, many loose ends were dropped only to be resurrected years later for the ebola response in the democratic republic of congo (drc). had teams been given the resourcing to finish, package, and archive complete solutions, months could have been saved at the beginning of the drc outbreak. just as the strategic national stockpile in the us ensures the supply of potentially lifesaving drugs and medical supplies, we need a technology and data stockpile equivalent that is properly maintained and ready to go when the next outbreak strikes. too much time is wasted in the earliest parts of an outbreak when the curve is furthest from being flat. this is also a good time to simply ''show up'' physically to lend whatever type of assistance your local clinic or public health service needs. at this point, the risks of infection should be greatly decreased, and many hands make light work. whether it is coding, data analysis, swinging a hammer or paintbrush, doing a supply run, or helping document and publish data and/or care and research findings, this should be a time of not just recovery but of improvement. it is well known that strong, resilient health systems are and will always be the first line of defense against epidemics. the goal of this reconstruction pause should not be to return them to prior capability but to make them stronger and ready for the next outbreak. it may be here soon enough. lastly, first and foremost before considering or acting on anything suggested above, take care of yourself, your family, your livelihood, and your community first. the fewer of us who get sick, the more of us who are available to help others before, during, and after the acute phases of this outbreak and the essential rebuilding phases that follow. a primer on biodefense data science and technology for pandemic preparedness, duke university's forge https:// forge homeland security presidential directive/hspd- about the biodefense policy landscape analysis toll v we don't need another national biodefense strategy. modern war institute at west point frequently asked questions about biosafety and covid- commcare for covid- outbreak response about the author eric perakslis is a research, informatics, technology, and r&d leader with more than years of direct experience in information technology, informatics, research, healthcare, government regulation, biotechnology, and pharmaceuticals discovery and development. he received his phd in chemical and biochemical engineering from drexel university. he became cio and chief scientist (informatics) at the fda in before moving to harvard medical school to serve as executive director at the center for biomedical informatics. he currently is senior vp key: cord- - a u uux authors: moss, ronald b title: prospects for control of emerging infectious diseases with plasmid dna vaccines date: - - journal: j immune based ther vaccines doi: . / - - - sha: doc_id: cord_uid: a u uux experiments almost years ago demonstrated that injections of a sequence of dna encoding part of a pathogen could stimulate immunity. it was soon realized that "dna vaccination" had numerous potential advantages over conventional vaccine approaches including inherent safety and a more rapid production time. these and other attributes make dna vaccines ideal for development against emerging pathogens. recent advances in optimizing various aspects of dna vaccination have accelerated this approach from concept to reality in contemporary human trials. although not yet licensed for human use, several dna vaccines have now been approved for animal health indications. the rapid manufacturing capabilities of dna vaccines may be particularly important for emerging infectious diseases including the current novel h n influenza a pandemic, where pre-existing immunity is limited. because of recent advances in dna vaccination, this approach has the potential to be a powerful new weapon in protecting against emerging and potentially pandemic human pathogens. throughout recorded history, infectious diseases have plagued human existence. one effective approach to limiting these diseases has been vaccination. for example, in a recent report by roush and colleagues at the u.s. centers for disease control and prevention (cdc), ever since the introduction of vaccines the incidence of infectious diseases like diphtheria, mumps, pertussis, tetanus, hepatitis a and b, haemophilus influenza and varicella zoster has declined by more than % in the u.s [ ] . furthermore, after the introduction of vaccines, large scale transmission of measles, rubella, and polio has been eliminated in the u.s., while smallpox has been eradicated worldwide. however, new emerging infectious pathogens such as hiv (human immunodeficiency virus), sars coronavirus (severe acute respiratory syndrome virus), and highly pathogenic avian influenza (h n ) viruses have adapted strategies to rapidly change their genetic compositions. as the influenza pandemic of (h n ) killed approximately to million people worldwide, massive disease and death is similarly feared from newly emerging pathogens. in addition, the current novel swine derived h n pandemic further exemplifies the need for a rapid and effective vaccine against emerging pathogens [ ] . thus a vaccination strategy to control emerging diseases will require a more effective and rapid response than available from conventional approaches such as live-attenuated vaccines, inactivated vaccines, or protein subunit vaccines. plasmid dna vaccines, as reviewed in this article, may be an option to effectively combat current emerging infectious diseases. almost years ago, malone and felgner at vical incorporated, and wolff and colleagues at the university of wis-consin, demonstrated that mrna and closed loops of double-stranded dna (plasmids) injected into muscle tissue could be taken up by cells at the administration site (transfection) resulting in the production (expression) of proteins not normally made by the host cell [ ] . it was soon realized that this approach could be utilized for both gene therapy as well as vaccine applications, and thus the field of dna vaccines was born. shortly after these original observations, many groups including those led by liu and colleagues at merck research laboratories [ ] , weiner and colleagues at university of pennsylvania [ ] , johnston and colleagues at university of texas [ ] , robinson and colleagues at university of massachusetts [ ] , and hoffman and colleagues at naval medical research center [ ] , demonstrated that immunization with dna could result in the production of foreign proteins or antigens that stimulate the immune system resulting in protection from or amelioration of infectious diseases in animal models. development in this area has greatly advanced over the years and human clinical trials of dna vaccines have now been conducted against various infectious pathogens including the malaria parasite, dengue viruses, cytomegalovirus (cmv), ebola virus, seasonal influenza viruses, avian or pandemic influenza viruses, west nile virus (wmv), sars coronavirus, hepatitis b virus, and hiv. the precise mechanism of the induction of immunity after pdna vaccination is complex [ ] and multi-factorial ( figure ). it is thought that after immunization, transfected muscle cells may produce antigen or foreign proteins that then directly stimulate b cells of the immune system, which in turn produce antibodies. transfected muscle cells could possibly transfer the antigen to so-called antigen presenting cells (as demonstrated by cross priming) which then transport the proteins via distinct pathways (the mhc i for cd +t cells or mhc ii for cd +t cells) that result in the display of different processed fragments of expressed proteins (antigens). finally, direct transfection of antigen presenting cells (such as dendritic cells) with subsequent processing and display of mhc-antigen complexes may also occur. because the process of antigen production by host cells after dna vaccination mimics the production of antigens during a natural infection, the resulting immune response is thought to be similar to the type induced by pathogens. indeed, dna vaccination generates antigens in their native form and with similar structure and function to antigens generated after natural infection. a plasmid used in dna vaccination ( figure ) contains a gene encoding an antigen of the target pathogen (immunogen gene). expression of the protein antigen is "turned on" in the host cell by a promoter, and "turned off" by a terminator (a polyadenylation signal sequence, generally referred to aspoly-a). other genes such as the bacterial origin of replication sequence and an antibiotic resistance gene are incorporated for manufacturing purposes. the resulting plasmid is a stable, self-contained unit that can be manufactured by consistent and scalable bacterial fermentation and purification processes. production of dna vaccines starts with e. coli cells which are transformed with the plasmid of interest. these cells are grown and stored frozen in a stock of vials called a master cell bank. growth of the e. coli is typically done via a fermentation process similar to that used in the manufacturing of certain alcoholic beverages. the recovery process then requires lysis of the cells, in order to release the plasmid retained within the e. coli cells. dna is then purified using various chromatographic methods. dna vaccination has many advantages compared with conventional vaccine approaches (appendix ) particularly in the setting of protecting against potentially lethal emerging infectious diseases. protecting against a particular pathogen may require immunity to more than one component of the organism and may require stimulation of different components of the host immune system. traditionally, most preventive vaccines have relied on antibodies as the main correlate of protection, components that prevent infection or disease. however, t cells play an important role in controlling disease for established infection. conventional vaccines based on whole pathogens typically induce immune responses against a number of irrelevant components of the organism. subunit protein vaccines target individual components of the pathogen and usually only stimulate antibodies. dna vaccines can accommodate a combination of different genes that code for different antigens from one or more different pathogens. this can result in the generation of broad immunity to multiple protein antigens. dna vaccines have also been observed to stimulate both antibody and t cell arms of the immune system including those that are specialized to kill viruses or cancer cells (via cytotoxic or killer t cells). a significant advantage, especially for emerging pathogens, is that dna vaccines do not require the handling of potentially deadly infectious agents. in addition they have a significantly shorter production time, something paramounrt with an ongoing pandemic. overall, a well-tolerated safety profile has been observed in human clinical trials of dna vaccination [ ] . early in the development, there were numerous theoretical safety concerns regarding dna vaccines. in particular, there were concerns about the fate of the injected genes and the potential for insertion into the host dna possibly result- ing in the uncontrolled stimulation of other genes such as those that may cause cancer. these concerns have dissipated over the years based on thousands of human subjects who have undergone dna vaccination or plasmidbased gene therapy. furthermore, in pre-clinical safety studies in animals, the potential for plasmid integration into the host genome has been shown to be negligible and several orders of magnitude below the spontaneous mutation rate that occurs naturally in mammalian genes [ ] . there were also early concerns regarding the induction of autoimmune reactions, as dna may be considered as a self antigen to the host. increased rates of autoimmunity or anti-dna antibodies such as those observed in systemic lupus erythematosis have not been observed in clinical trials of dna vaccination [ ] . interestingly, as plasmid dna vaccines are noninfectious, significant immune responses are not developed against the plasmid itself. therefore only specific immune responses to the expressed antigen are stimulated with this approach. in contrast, with viral vectors such as adenoviruses, pre-existing and post vaccination immunity to the vector itself can be generated thereby limiting the pathogen specific immune response in contrast, dna vaccination can be repeated without diminishing the specific immune response. this may be a pertinent beneficial aspect of dna immunization, particularly in light of recent clinical trials in humans using viral vector-based vaccines, such as the common cold adenovirus, where immunity to the vector itself has been raised as a potential safety issue [ , ] . in summary, the potential risks of dna vaccines appear to be minimal based on safety data from human clinical trials in thousands of subjects. over the years, much progress has been made in optimizing dna vaccine immunogenicity. recent progress has targeted many different aspects of dna vaccination which has successfully resulted in improved immune responses (appendix ). with regard to the plasmid itself, significant advances have been made in optimizing the genetic sequence of the encoding gene as well as other related components. coadministration of the plasmid that encodes the pathogen along with other genes that encode for immune stimulating substances such as cytokines or chemokines has also been used to further enhance the immune response of certain dna vaccines [ ] . in some cases, once various components of the plasmid have been optimized, the encoded protein may be sufficiently immunogenic without the need for additional components (unformulated or "naked" dna) such as adjuvants. one of the earliest trials in humans of an unformulated or "naked" dna vaccine was one that targeted the malaria parasite. wang and colleagues at the naval medical research institute immunized subjects with a plasmid that encoded naturally occurring forms of malaria proteins [ ] . in this trial, more than half of the subjects were shown to have cells that can kill or lyse malaria infected cells (cytotoxic t cell or killer cells). in a more recent clinical trial, a dna vaccine optimized for human expression and encoding modified forms of west nile virus proteins was studied by martin and colleagues at the national institute of health (nih) and demonstrated that the vaccine stimulated antibodies that inhibited the virus (neutralizing antibodies) in all individuals receiving the vaccination regimen [ ] . this unformulated dna vaccine appears to induce a similar level of immune responses to those observed in vaccinated horses protected from wnv. in addition, a recent clinical trial by these same nih researchers tested an optimized but unformulated dna vaccine for sars coronavirus and demonstrated neutralizing antibodies in all subjects who received three doses of the vaccine [ ] . another area of research to enhance the immune response to dna vaccines is related to the route of administration. although intramuscular injection is the predominant route of vaccine delivery, other routes of administration have also been studied such as intradermal delivery. it is conceivable that intradermal delivery, which is a more superficial injection, may result in better transfection of antigen presenting cells, particularly a type of cell called dendritic cells. the mode of delivery may also be a pertinent factor in eliciting the proper immune response after dna immunization. needle and syringe is the predominant method to deliver both dna vaccines as well as conventional vaccines. however, roy and colleagues at powdermed (now pfizer incorporated) have used dna precipitated onto gold particles which are driven into to skin with a blast of pressurized gas, and called this approach "particle-mediated epidermal delivery" (pmed). in one clinical study by roy and collaborators using pmed, individuals exhibited potent antibody responses to a hepatitis b dna vaccine [ ] . in another study, drape and colleagues at pow-dermed also observed strong antibody responses to the influenza virus dna vaccine with the pmed approach [ ] . another approach for the delivery of dna vaccines is the use of needle-free devices. needle-free injection of dna vaccines has been utilized in numerous clinical studies and appears to be well-tolerated and may have some advantages of further augmenting the immune response to dna vaccination. for example, nabel and colleagues at the nih injected individuals intramuscularly with a sixplasmid unformulated dna vaccine for hiv (env a, env b, env c, subtype b gag, pol, and nef) with the needle-free device (biojector ® ) and observed hiv specific t-cell immune responses in over % of individuals [ ] . similarly, this same group at the nih completed a recent study of an ebola dna vaccine also using this same needle-free device. they demonstrated that ebola-specific antibody and cd + t-cell immune responses were elicited in all individuals who received the three-dose vaccination regimen [ ] . another novel mode of enhancing dna vaccines has been a more invasive technique called electroporation. this method involves administration of brief electrical pulses of various voltages, after injection of a dna vaccine, in order to enhance the uptake of dna, presumably through the formation of transient pores in the muscle cell membrane. many groups have shown encouraging results using electroporation in animals. one of the first groups to use this technique, ulmer and colleagues, at chiron corporation, demonstrated that antibody and t-cell responses to the hiv protein gag encoded in a dna vaccine were enhanced by electroporation in rhesus macaques [ ] . more recently, studies by pavlakis and colleagues at the nih have also noted that dna potency measured by t-cell responses to the hiv protein gag was augmented in nonhuman primates using electroporation [ ] . although encouraging in the strong magnitude of responses generated in animals, the wide clinical applicability of electroporation in humans in relation to tolerability remains to be determined. some researchers have used plasmid dna in what has been called a heterologous "prime-boost" vaccination approach. this method involves delivery of one or more plasmid dna vaccine priming doses followed by a boost with a viral vector (such as adenovirus) which codes for the same antigens. in the prime-boost setting, dna vaccination plays an important role in priming different types of t-cells (cd + and cd + t-cells) specific for various proteins. in studies, for example, by nabel and colleagues at the nih, dna vaccination with plasmids encoding hiv antigens followed by a boost with an adenoviral vector carrying the same hiv gene sequences, resulted in stronger t-cell responses compared with adenoviral vector or dna vaccine alone [ ] . similarly, pantaleo and colleagues at the university of lausanne have shown that a dna vaccine for hiv followed by a boost with a poxvirus vector (nyvac) resulted in stronger immune responses compared to immunization with pox vector alone [ ] . in another prime-boost study, jacobson and colleagues, at the university of california at san francisco, immunized subjects with a dna vaccine for cmv who were then boosted with a live-attenuated cmv virus (towne strain). faster and stronger virus-specific t-cell responses were observed in the group of subjects that received the dna and towne strain compared with a group of subjects that received the towne strain alone [ ] . an area of potentially paramount importance for dna vaccines is formulations and adjuvants. adjuvants are common to most licensed vaccines and are included to potentiate the immune responses elicited by vaccination. for dna vaccines, various delivery systems and adjuvants have been tested. one of the earliest promising adjuvants for plasmid dna vaccines was poly-lactide coglycolide (plg), cationic microparticles. ulmer and colleagues at chiron corporation evaluated hiv dna vaccines formulated with plg microparticles and found strong antibody and t-cell responses in macaques [ ] . poloxamers represent another class of adjuvants tested with dna vaccines. some poloxamers are nonionic block copolymers, and when combined with a cationic surfactant, bond with dna to form small particles. a study by wloch and colleagues at vical incorporated examined dna immunizations of human volunteers with cytomegalovirus (cmv) plasmids formulated with a specific poloxamer adjuvant. in that study cmv-specific t-cell responses were detected in a majority of cmv sero-negative individuals who were vaccinated [ ] . vaxfectin ® is another example of a delivery system and adjuvant for dna vaccines that has been recently tested in humans. vaxfectin ® is a cationic lipid-based adjuvant that bears a positive charge that binds electrostatically to negatively charged dna. studies in animals also demonstrated that vaxfectin ® -adjuvanted dna vaccines can be protective against lethal viral challenges. for example, webby and colleagues at st. jude children's research hospital and vical incorporated, immunized ferrets with three plasmids containing dna components of the h n pandemic influenza virus formulated with vaxfectin ® [ ] . after one or two immunizations, all animals were completely protected from lethal pandemic influenza virus challenge, while unvaccinated control animals died. similarly, griffin and colleagues at johns hopkins immunized juvenile and infant rhesus macaques by intramuscular and intradermal routes with measles antigen encoding plasmids formulated with vaxfectin ® [ ] . all of the vaccinated monkeys developed strong and durable neutralizing antibodies and they were challenged with high doses of measles virus after one year. all of the unvaccinated control animals developed viremia and became ill with rashes in contrast to the vaccinated ani-mals which remained healthy and had no detectable virus levels. lastly, the first human clinical trial of a dna vaccine formulated with vaxfectin ® has been completed with plasmids that encode pandemic influenza virus antigens (h n ). the preliminary results reported from this trial by smith and colleagues at vical incorporated suggest that vaccination with h dna plasmids formulated with this adjuvant were well-tolerated and stimulated strong h antibody responses in up to % of subjects [ ] . notably, the antibody response rate and safety profile observed in this trial are comparable to most conventional proteinbased vaccines for pandemic influenza. although dna vaccines have yet to be approved for human use, three have already been approved for animal health. as noted in appendix , the first dna vaccine approved for animal health was one that protected horses against wnv. wnv is a mosquito-borne virus, which causes encephalitis or inflammation in the brain of infected animals and humans. prior to approval of a vaccine, approximately one-third of the horses infected with wnv would die or be euthanized. the wnv dna vaccine, developed by fort dodge laboratories, was approved by the u.s department of agriculture (usda) in after the demonstration of safety and efficacy [ ] . a dna vaccine has also been recently approved to prevent a fatal viral disease that afflicts salmon, called infectious hematopoietic necrosis virus. in mid- , an epidemic occurred in atlantic salmon killing up to % of the fish. scientists at aqua health, a unit of novartis in canada, conducted a field trial by immunizing millions of salmons with a single dose of dna vaccine encoding for a protein of the virus [ ] . the vaccine was approved in based on the results of this trial that demonstrated that the vaccine, called apex-ihn, protected the fish from death without adverse effects. additionally, a therapeutic dna vaccine designed to treat dogs with skin cancer (melanoma) was granted conditional approval in . this vaccine was developed through a collaboration of memorial sloan-kettering cancer center (mskcc) and merial ltd. canine melanoma is an aggressive form of cancer. dogs with melanomas that have gone beyond initial stages typically have a lifespan of one to five months with conventional therapies. in addition, the cancer is often resistant to chemotherapy. in a study of the dna vaccine conducted by mskcc, many dogs who received the vaccinations lived beyond the average month survival [ ] . based on the significantly extended survival, the usda gave this dna vaccine conditional approval in . this is the first therapeutic vaccine approved by the u.s. government for the treatment of cancer in animals or humans. perhaps most relevant to emerging infectious diseases, dna vaccines have the distinct advantage of a rapid development time, are non-infectious, and have a well-defined manufacturing process. dna vaccines contain no infectious components and can be produced safely without the handling of hazardous infectious agents. furthermore, there is a well-defined analytical process for the manufacturing of all dna vaccines, which is universally applicable to any dna vaccine. vaccination is an important component of a response to potential pandemics such as avian influenza. according to the world health organization (who), since november , approximately cases of human infection with highly pathogenic avian influenza a (h n ) have been reported worldwide [ ] . pandemic (h n ) influenza virus has evolved into at least distinct clades or subclades. as noted earlier, the emergence of triple-reassortment swine influenza with limited cross reactivity antibody responses after vaccination with seasonal influenza vaccines suggests the need to rapidly produce new vaccines to this particular emerging virus [ ] . the manufacturing time of conventional protein-based vaccines may be excessive, as they typically require growth in egg or cell cultures, which involve a relatively slow production time. dna vaccines, in contrast, have estimated vaccine production times that can be months earlier, as only the dna sequence is required and the manufacturing process is standard (figure ) [ ] . dna vaccines therefore have a unique advantage of large scale production for human use in a relatively streamlined period of time. in the case of potentially fatal emerging pathogens, reducing the production time of an effective vaccine may be critical in preventing spread of infection and death. in summary, based on recent advances in enhancing protective immune responses and a well-tolerated safety profile in humans, plasmid dna vaccines have the potential to become an integral part of the arsenal dedicated to enhancing human health by preventing diseases through immunization in our ever changing microbial environment. the author declares that they have no competing interests. ability to immunize against multiple antigens and/or pathogens vaccine-preventable disease table working group: historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states implications of the emergence of a novel h influenza virus direct gene transfer into mouse muscle in vivo heterologous protection against influenza by injection of dna encoding a viral protein gene inoculation generates immune responses against humanimmunodeficiency virus type genetic immunization is a simple method for eliciting an immune response protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid dna plasmid dna malaria vaccine: the potential for genomic integration after intramuscular injection dna vaccination: antigen presentation and the induction of immunity preclinical and clinical safety studies on dna vaccines. huamn vaccines ensuring safety of dna vaccines karlsson hedestam gb: dna vaccines: recent developments and future possibilities human versus hiv: round defeat in aids vaccine development human immunodeficiency virus type vaccine development:recent advances in the cytotoxic t-iymphocyte platform "spotty business dna vaccines: ready for prime time? hoffman : induction of antigen -specific cytotoxic t lymphocytes in humans by a malaria dna vaccine a west nile virus dna vaccine induces neutralizing antibody in healthy adults during a phase clinical trial a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial induction of antigen-specific cd + t cells, t helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis b virus dna vaccine epidermal dna vaccines for influenza is immunogenic in humans study team: safety and immunogenicity of a gag-pol candidate hiv- dna vaccine administered by a needle-free device in hiv- -seronegative subjects a dna vaccine for ebolavirus is safe and immunogenic in a phase i clinical trial ulmer lb: enhancement of dna vaccine potency in rhesus macaques by electroporation increased immune responses in rhesus macaques by dna vaccination combined with electroporation vrc study team: phase i clinical evaluation ofa six-plasmid multiclade hiv-l dna candidate vaccine pantaleo giuseppe: an hiv- clade c dna prime, nyvac boost vaccine regimen induces reliable, polyfunctional, and long-lasting t cell responses a cmv dna vaccine primes for memory immune responses to live-attenuated cmv (towne strain) induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus dna vaccines safety and immunogenicity of a bivalent cytomegalovirus dna vaccine in healthy adultsubjects plasmid dna-based vaccines protect mice and ferrets against lethal challenge with anietharnl / (h nl) influenza virus use of vaxfectin adjuvant with dna vaccine encoding the measles virus hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques against measles virus vaxfectin-formulated pandemic influenza dna vaccines: preliminary clinical results. paper presented at: ibc life sciences' next generation vaccines; national harbor fort dodge animal health care products mmwr rapid-response vaccines-does dna offer a solution the author would like to thank my colleagues at vical incorporated including vijay samant, alain rolland, alan engbring, jenny chaplin, and larry smith for their helpful critical comments. key: cord- -qwpya ox authors: adibe, bryant; perticone, kathryn; hebert, charles title: creating wellness in a pandemic: a practical framework for health systems responding to covid- date: - - journal: nejm catal innov care deliv doi: . /cat. . sha: doc_id: cord_uid: qwpya ox caring for covid- patients poses a high risk of short- and long-term psychological distress to hospital employees. rush university system for health has developed a simple, practical framework for prioritizing their well-being. the psychological impact of the covid- pandemic on frontline health care workers has yet to be fully understood. as the national conversation moves to discussions of reopening and a return to a new normal, the importance of mental health and well-being is more relevant now than ever. existing disaster models predict an impending period of disillusionment in our future, characterized by high stress, physical exhaustion, burnout and substance misuse as the adrenaline, camaraderie, and broad community support of the past few months begin to fade. infectious disease outbreaks pose unique challenges to health care workers compared with other disasters, arising both from the nature of the disease and from the need to protect themselves from infection with measures such as quarantine and the use of personal protective equipment. undesirable effects on psychological well-being among frontline health care workers have already been documented, including increased risk of depression, anxiety, substance misuse, and sleep disturbances. , we sought to preempt these issues by thoughtfully designing a proactive organizational approach to supporting the mental health and well-being of our frontline staff. the institution's comprehensive plan for staff wellness first took into account concerns for the attainment of basic physiologic needs and the promotion of physical and emotional safety; as well as the basic needs of their immediate families. , institution-wide measures to address these issues included the creation of additional on-site childcare, transportation assistance, and alternative lodging. early in our evolving response to the covid- pandemic, the office of the chief wellness officer commissioned a special wellness task force devoted to coordinating the institution's efforts, as part of an overarching command center structure. representation on the wellness task force included the office of the chief wellness officer, chaplains, social work, nursing (psychiatric nurse liaisons), psychiatry and behavioral sciences. the task force used its collective expertise to develop four key mitigation strategies, described in detail below, to reinforce staff wellness throughout the crisis: wellness rounds, a wellness consult service, an advanced mental health intervention program known as wellness plus, and a central wellness resource hub with wellness rooms on frontline floors. the task force also created an interdisciplinary wellness response team to serve as the primary workforce supporting the covid- staff well-being efforts. volunteers were recruited from redeployed staff representing each of the task force departments, many of whom were available as a result of lower overall non-covid- hospital census, yielding some - individuals with consistent availability. if the needs of the hospital extended beyond the capabilities of the group, as would be the case if additional icu units needed to be opened, up to additional providers were available. two operational leads were designated to develop consistent staffing ratios and schedules, as well as to ensure quality control for the training and on-boarding of new wellness response team members. each team member was given a resource tool kit that included an algorithm for the triage and assessment of employees in need, an escalation pathway for rare but serious scenarios, such as an employee at risk of self-harm; as well as a list of all institutional wellness resources available for staff use. to ensure ease of communication among the group and to provide rapid responses to emerging issues, all wellness response team members were given access to the cisco webex teams platform. this portal functioned as the primary tool for quick group discussions, notifications, follow-up of urgent cases, and the sharing of resources and best practices. the initiatives outlined below were created to promote mental health and well-being for staff during the covid- pandemic. while supportive rounding is not a new concept, distinguishing features of our approach include its formal structure, consistency, interdisciplinary composition, and empowerment of the participating group to address urgent issues through a real-time feedback loop with the highest levels of organizational leadership. these characteristics create a dynamic and agile framework for organizational decision-making. members of the wellness response team were divided into unit-specific teams targeting areas of the medical center with the heaviest covid- clinical burden. five standing teams were created, with an additional "flex" team that covered general medical (non-covid- ) floors and a night team that covered all floors three times a week, from pm to midnight. each team has a physician leader, a psychologist, a nurse (often a psychiatric nurse liaison), a chaplain, and a licensed clinical social worker. each team rounds on the same locations at the same time every day, to create familiarity and a sense of rapport with the clinical teams. wellness response team members were preferentially assigned to floors where they have established relationships, promoting rapid assimilation into the units. because mornings are busy for clinical staff on patient units, the wellness rounds take place in the afternoon. each day the wellness response team huddles at : pm for a regular briefing, and the rounds begin at pm. these briefings include updates from the chief wellness officer regarding the latest developments from our covid- command center and the latest talking points on emerging issues. team members also share their key findings from rounds the day before. we established a consult service (figure ) where any clinical unit or individual can connect directly with a member of the wellness response team for evaluation, triage, and recommendations to improve mental health and well-being. clinicians are familiar with the "consult" model, and this approach helps us normalize the concept of wellness by incorporating it more formally into the clinical environment. group sessions are made available for entire units, departments, or clinical teams in need. all individual consults are anonymous and are not added to a staff member's medical record. we created an immediate, targeted response to individual employees in a mental health crisis. through a pre-determined escalation algorithm (figure ) , any member of the wellness response team can trigger the wellness plus pathway. when triggered, the individual is escorted to one of the unit-level wellness rooms or the central wellness resource hub (see below) where an experienced clinician (typically a physician or other prescriber) completes a thorough mental health assessment, including identifying an immediate therapeutic intervention and appropriate followup. we established a centrally-located wellness resource hub, managed by psychologists and licensed clinical social workers, where any staff member can receive confidential, on-site counseling support, escape busy clinical areas, process their emotions, and relax. to facilitate respite, on entering the hub staff are greeted by calming music, a -foot projection of nature scenes, and available lounge chairs evenly spaced -feet apart. for frontline staff unable to leave the unit, a network of five wellness rooms were launched throughout the medical center, with a special focus on high-volume covid- intensive care units. these rooms include healthy snacks, resources for self-care and written prompts on the walls to encourage reflection. although social workers were not assigned to individual wellness rooms, we encouraged regular use of the wellness resource hub for staff with more personalized needs. we faced several hurdles in implementing this initiative. • stigma attached to mental health services. the wellness response team was heavily weighted with mental health professionals, and some staff were reluctant to interact with them initially because they were concerned for their privacy and thought the team's function was to identify mental illness. to address this, we made sure that teams included a mix of disciplines--chaplains, nurses, and social workers--and that their on-boarding emphasized a consistent message of wellness. if an employee specifically requested a mental health evaluation, or was demonstrating poor coping strategies that interfered with patient care duties, team members were encouraged to default to their professional judgement. in these cases, the framework of the wellness response team provided rapid linkage to a mental health practitioner for prompt assessment via the wellness plus pathway. • integrating the wellness response team into the daily routine. in the initial phases of rolling out this program, wellness response team members were often met with skepticism and, at times, even confusion. clinical teams were often busy, did not want to be interrupted, or were otherwise reluctant to talk. this was overcome through an emphasis on consistency; each unit had a designated team that rounded at the same time each day. the teams were encouraged to engage only when clinicians were available and interested. as the initiative continued, wellness response team members became identified experts in resources for employee wellbeing, as well as a low barrier access point to receive support. over time, the teams experienced an increase in staff appreciation as well as anticipation of their visits. • measuring impact. we faced a dilemma regarding how to effectively measure impact of the initiative because we did not want to overburden clinicians with a new assessment or survey while they were grappling with the stress of an evolving disaster. in place of an initial assessment, we adapted an emotional well-being screening tool, originally developed for the identification of acute and chronic stress disorders, including ptsd, in our military veteran population, for use in front line healthcare workers. as of this writing, the tool is under irb review; once complete we plan to disseminate it broadly throughout our community, with a particular focus on clinical units with the heaviest covid- case load. the assessment will provide valuable information regarding our organization's current state, and we also hope to perform a comparative analysis against a comparable outside institution with no such wellness infrastructure in place. further, the screening tool includes an assessment of burnout prevalence, which we can compare against existing internal data. across the board, the initiatives have been incredibly well-received since their launch. increasingly, we found staff to be more at ease contributing concerns, thoughts, and feelings that they faced when interacting with covid- patients. over time, staff have moved from the "i'm fine" position to being more forthright about their distress and anxieties. centrally recurring themes include the following: ( ) moral distress around patient deaths, resource allocation, and absolute scarcity, ( ) personal safety, ( ) economic insecurity, ( ) social and family life disruption, ( ) stigmatization of health care workers, and ( ) sense of powerlessness. over time, staff have moved from the "i'm fine" position to being more forthright about their distress and anxieties." calls to the wellness consult service and escalations to wellness plus varied in scope, but steadily increased over time. these ranged from practitioners whose levels of anxiety made them apprehensive about providing needed care to covid- patients, to requests from managers elevating concerns about employee well-being. over a four-week period utilization of the wellness resource hub increased from - people per day to or more per day with a total to date of more than people. the majority of visitors are daytime employees encompassing a wide range of departments and functions in the hospital. as the number of visitors increased, we established back-up staffing from wellness response team members for additional immediate support in the wellness resource hub. unit-level wellness room use was not tracked, but anecdotal evidence suggests a similar trend of increasing use over time. in order for an initiative like this to work, having a senior-level executive champion is critical. in our hospital system, senior-level leadership was provided by the chief wellness officer. however, this need not be the case, and institutions may appoint a different executive leader for such efforts, particularly one who does not have competing responsibilities within the overall pandemic response and has the ability to oversee an interdisciplinary team and convey emerging concerns to appropriate channels among hospital decision-makers. we believe that without clear leadership, the initiative will breakdown over time; we found that the daily huddles with the chief wellness officer re-energized the team and helped it focus on its mission. covid- has presented unique challenges to health systems across the globe. the impact of this pandemic on the psychological well-being of frontline health care workers is expected to be widespread. as we continue to reflect on our experiences thus far and understand more about this evolving situation and its broad impacts, we are progressing to the next phase of our institutional wellness response. while wellness rounds have taken place in one form or another throughout our health system since their inception at rumc, the comprehensive framework detailed here will be implemented more formally at each site going forward, including the identification of an executive sponsor to ensure local support. as rush looks to resume normal clinical operations, we plan to continue this infrastructure for the next - months, albeit with a less frequent rounding schedule (likely - times per week), as redeployed staff return to their original roles. most importantly, we will allow the data obtained from the emotional well-being screening tool to inform our next phase of targeted interventions. adapted from zunin & myers as cited in training manual for mental health and human service workers in major disasters. us department of health and human services, substance abuse and mental health services administration, center for mental health services mental health and the covid- pandemic survey of stress reactions among health care workers involved with the sars outbreak alcohol abuse/dependence symptoms among hospital employees exposed to a sars outbreak creating wellness in a pandemic: a practical guide for health systems responding to covid- managing mental health challenges faced by healthcare workers during covid- pandemic national child traumatic stress network and national center for ptsd) key: cord- -szqrjxq authors: campbell, margaret c; inman, j jeffrey; kirmani, amna; price, linda l title: in times of trouble: a framework for understanding consumers’ responses to threats date: - - journal: j consum res doi: . /jcr/ucaa sha: doc_id: cord_uid: szqrjxq the covid- pandemic and the accompanying economic downturn have dramatically impacted the lives of consumers around the world. from a conceptual perspective, such health and economic threats can severely disrupt consumers’ sense of ontological security and elicit adaptive responses by both consumers and marketers. given the opportune timing, this issue of the journal of consumer research is focused on articles that address questions of consumers’ responses to external threats. the purpose of this introduction is to provide an organizing “conceptual tapestry” to connect the articles appearing in the issue. this framework is provided as a tool to help researchers structure their particular projects within the broader landscape of consumer threat response and to present some potential directions for future research. in conjunction with these articles, we hope that this conceptual framework will provide a point of departure for researchers seeking to enhance the understanding of how consumers and markets collectively respond over the short term and long term to threats that disrupt consumers’ routines, lives, or even the fabric of society. t he year is unfolding in unprecedented ways. first, the covid- pandemic spread incredibly quickly and threatened global health. to slow the spread, country after country went into lockdown, effectively closing large portions of their economies-and thus the global economy-for many weeks. economic shuttering posed a threat to consumers' income and quality of life. at the same time, stay-at-home and physical distancing orders cut consumers off from social interactions and rituals, including celebrating events like weddings, graduations, and funerals, practicing religion, or even sharing family weekend suppers or going out to a restaurant and movie for date night, threatening social interactions. lack of information, misinformation, and conflicting information threatened consumers' ability to understand, plan, and cope with the health, economic, and social threats. the presence of all of these threats has created shifts in consumers' marketplace and socio-cultural behavior. indeed, threat-induced disruptions have ricocheted around the structure of consumers' lives with potential and actual impacts on consumers' wellbeing. the world is experiencing health, economic, social, and information disruption and unrest that few of us have seen in our lifetimes. how do consumers respond and adapt to sudden and widespread threats such as these? what are the most common responses? are they universal, or are there key boundary conditions and moderators that come to bear? are responses and adaptations ephemeral such that consumers revert to their pre-threat behavior once the threat has passed, or do some of the responses and adaptations "stick," resulting in longer-term changes? if so, what factors induce ephemeral versus enduring adaptations to threats? how do consumers' responses impact the market and reflect back to influence disruption? we believe that the field of consumer research is uniquely positioned to shed light on questions related to responses to threats, including those that have arisen due to current circumstances. our field has a long history of examining and explicating consumer response to threats ranging from threats to physical health (botti, orfali, and iyengar ; mittal and griskevicius ; pavia and mason ) , financial health (mittal and griskevicius ) , social lives and personal identities (lee, kim, and vohs ; thompson, henry, and bardhi ; weinberger and wallendorf ) , daily routines and practices (phipps and ozanne ) , and general well-being (de mello, macinnis, and stewart ) . notwithstanding, we argue that much more can be done in examining the processes underlying how consumers respond to threats as well as the effects of threats, particularly large-scale ones, on consumer behavior. as all of us sheltered at home and continued to carry out roles as consumers, family members, friends, educators, researchers, and editors, we identified several manuscripts addressing questions of consumers' responses to external threats that were either accepted or conditionally accepted at the journal of consumer research. given this opportunity for the journal of consumer research to highlight some of what our field has to offer on the topic of consumer threat response, we elected to focus this issue around this important topic. we hope that the articles in this issue will help lay the groundwork for the surge in research that will follow the covid- pandemic, the accompanying deep economic recession, as well as some of the unusual, quasi-experimental aspects of the current response, such as physical distancing rules and the politicization of information. our times are defined by myriad threats from natural disasters, global migration, misinformation, and rapid economic, institutional, and technological changes that continuously disrupt and alter consumers' lives (bauman ; giddens ; bardhi and eckhardt ) . we believe that consumer research on threats goes beyond contemporary circumstances and can help consumers, public policy officials, and firms prepare for and respond to other, different, threats that will occur in the future. the purpose of this introduction to the jcr issue on consumer threat response is twofold. first, we seek to set the stage for the articles that appear in this issue. these articles address responses to economic threats (coskuner-balli ; mittal, griskevicius, and haws , this issue; wilroy, meloy, and carlson , this issue) and health threats (galoni, carpenter, and rao , this issue; huang and sengupta , this issue; scott, rozin, and small , this issue) , including the threat of mortality (dunn, white, and dahl , this issue) . second, we seek to provide a broad foundation for future research on this important, timely, and timeless topic. to be clear, we do not claim to provide a "grand theory" of consumer threat response. rather, drawing on prior research, we present a general "tapestry" of threats, resulting disruptions, responses, and potential mediators and moderators. our goal is not to provide a comprehensive review of existing literature, but rather a guide to researchers seeking to increase our collective understanding of how consumers and markets together respond over short and long durations to threats that disrupt their very being. our framework is provided as a tool to help researchers structure their particular projects within the broader landscape of consumer threat response and to present some potential directions for future research. to provide context for the articles in this issue, we present our conceptual tapestry first. our goal is to help generate more research and greater understanding of how consumers respond to external threats. by external threats, we mean both the actual or potential occurrence of events with negative repercussions for consumer well-being. similar to environmental stressors (mittal et al. , this issue) , external threats can cause harm to consumers. the impacts of threats can occur at every level from individual, to local, national, or global. whereas actual threats occur when consumers encounter negative events, potential threats manifest when consumers think negative events might occur. we provide a conceptual framework for thinking about how and when threats affect consumer responses; we also highlight that threats can lead to marketplace adaptations (see figure ). we propose that threats can lead to disruptions that adversely impact consumers' ontological security; that is, they potentially shatter consumers' sense of order, meaning and social frameworks. the resulting ontological insecurity can lead to a variety of consumer responses, from short-term affective and psychological (e.g., fear), to longer-term psychological (e.g., anxiety or depression), to behavioral (e.g., increased purchase and consumption of alcohol). the marketplace likewise responds to the disruptions and to consumers' responses introduction and, importantly, both consumer and market responses can cycle back, impacting the level of threat and disruption. common categories of threats include economic, health, social, informational, and environmental (i.e., physical world), though there are likely others. economic threats can damage a consumer's financial situation; anticipating that unemployment rates will increase would be a potential threat, while losing one's job would be an actual threat. health threats negatively impact a consumer's own, or close others', physical health, such as the potential threat of getting sick from a contagious disease, or the actual threat of a loved one's cancer diagnosis. social threats potentially or actually hurt our place and role in society or society itself. a potential social threat would include worry about terrorism or social unrest, while an actual social threat might entail facing racial discrimination or losing connection to your family. informational threats affect a consumer's ability to learn, know and understand. a potential informational threat is the possibility that a consumer's local newspaper will go out of business whereas an actual threat arises when two governmental agencies provide conflicting information on the efficacy of the same drug. potential or actual environmental threats arise from aspects of the environment that impact consumer welfare. a potential environmental threat is that climate change would lead to decreased food supply, whereas lead-tainted drinking water is an actual environmental threat. as shown in the figure, threat leads to disruption, the extent of threat moderates this relationship, and disruption then has implications for consumer and market response. we propose that the extent of threat is influenced by severity, scope and psychological distance. severity refers to the "size" of the threat in terms of potential harm to wellbeing; spraining your ankle presents a much milder threat than discovering a malignant tumor. scope refers to the "size" of the threat in terms of the number of people, communities, and geographic areas that the threat can or does impact, as well as its duration; one person temporarily being furloughed is much smaller scope than % of the employees in a town permanently losing their jobs when a plant closes. psychological distance refers to how removed or close a threat seems to the person perceiving the threat (trope and liberman ) and affects felt involvement (celsi and olson ) . psychological distance can arise from social, spatial, temporal, or experiential distance (trope and liberman ) ; each of these can create a sense of a small, moderate or large gap between the self and the threat. a prediction that a hurricane will hit your town produces smaller psychological distance from the threat as compared to a prediction that a hurricane will hit the next town, or the next state, over. greater severity, greater scope, or smaller psychological distance all arguably increase the likelihood and extent to which threat results in the disruption of consumer's normal or expected course of actions. threats such as pandemics, recessions, and social unrest can be shared by large groups of consumers or felt individually, such as one person's job loss or divorce. either an individual or a large-scale threat can disrupt a consumer's life. considerable literature across behavioral health, social sciences and the humanities has examined external threats and consequences (carver, scheier, and weintraub ; perry and quarantelli ; wisner et al. ) . a full review is beyond the scope of this article. however, by a framework of consumer responses to threats focusing on consumers' and markets' experiences and responses to disruptions caused by threats, our approach has broad applicability for consumer research not only related to the current experienced threats, but to a range of threats, from a neighborhood or school disrupted by a horrific event, a family experiencing the death of a child from cancer, or a consumer living with diabetes. finally, the extent of threat increases when different categories of threats happen at the same time. when multiple threats co-occur, interactions among them can magnify resultant disruptions in an ongoing cycle. for instance, what began as a health threat (the covid- pandemic) broadened into economic, informational and social threats. the pandemic led to stay-at-home and physical distancing directives, which brought on the economic threat of recession and financial loss. the effect of the health and economic threats were exacerbated by the abundance of misinformation about causes, cures, and outcomes. finally, the pandemic also created a social threat of losing connection with one's friends and family, leading consumers and marketers to scramble to adapt to an uncertain environment. arguably, the fear and frustration of the health, economic and social threats may have contributed to the outrage and social unrest of protests about black lives matter. obviously, threats can severely disrupt consumers' lives. in the next section, we discuss the conceptual linkage between actual and perceived threats and the nature of the disruptions that they can create. threats can disrupt many normal or expected courses of action. a family member's medical diagnosis, an unexpected expense, layoffs at a company, a hurricane, / , or the fukushima nuclear disaster can interrupt consumers' certainties and routines (giddens ) . the degree of disruption, which is the extent to which it interrupts the norms, beliefs and practices that make up everyday life, depends on the extent of threat. covid- has proven to be a highly infectious, fast-moving and lethal disease, making it a severe threat with broad scope. as it has moved across the globe, it has caused much disruption, interrupting consumers' norms, beliefs, practices and routines. consumers' lives consist of countless, often taken-forgranted norms, beliefs and routines that comprise consumers' experience of the world. these norms, beliefs and routines are conditioned around consumers' prior experiences and what they are able to anticipate based on those experiences. norms regarding others' choices and behaviors guide how consumers behave as well as how they expect others around them to behave (goldstein, cialdini, and griskevicius ) ; norms are collectively defined, socially enforced and vary widely depending on the collective. for example, consumers have norms around institutional roles, rights to safety, privacy, and access to life-saving health care, to name a few, and these norms differ across communities and countries. consumers also have countless taken-for-granted beliefs about what is real, true and appropriate, and these beliefs shape expectations and responses. for example, many consumers expect their local stores to be stocked with their favorite brands and products, they do not expect to lose their jobs suddenly, and they expect to send their children to school during the academic year. norms and beliefs come together in the everyday practices and routines that make up consumers' lives. for example, consumer routines may include taking two buses to buy groceries; eating dinner with extended family, including multiple generations; socializing with friends and family; attending religious services; shaking hands, kissing cheeks, or hugging when meeting others; setting rules (e.g., amount of screen time for their children); and casually interacting with colleagues at work. gauging the effect of threat on disruption is essential for understanding consumers' experiences. for example, rural residents who are typically "physically distanced" in their daily lives may feel the virus threat is remote until someone in their community falls ill. however, those same rural ranchers and farmers who were largely undisrupted by the health threat of the virus may feel very disrupted by economic threats. not surprisingly, data suggests extreme inequality both in deaths directly attributable to the threat and in the disruptions caused by covid- (cdc ). disruption to consumers' norms, beliefs, routines and practices caused by a threat affects ontological security, a term coined by psychologist robert laing to reflect the degree to which consumers feel their world, and role within it, is secure and predictable (cannon, goldsmith, and roux ; laing laing / . routines provide consumers with "a protective cocoon" and a "firewall against chaos," (giddens, , ) . when consumers' normal and anticipated lives are disrupted, they experience insecurity, uncertainty and anxiety. as phipps and ozanne ( ) write, "it's unsettling when a storm brings down electrical lines and there is no light, warmth or entertainment. . . .it is unsettling to walk into a grocery store and see shelves laid bare (p. )." disruptions can shake consumers' confidence in "the continuity of their self-identity and the constancy of their social and material environments of action" (giddens , ) resulting in ontological insecurity stemming from perceived lack of order, meaning and continuity. ontological insecurity is not the absence of security, but its diminishment, and consumers move along a continuum between ontological security and insecurity (banham introduction ; bondi ) . for example, bondi ( ) describes the everyday experiences of insecurity, along with the testimonies of those living in highly precarious conditions such as refugees and asylum seekers. despite insecurity, they still retain a sense of self-worth and a "faith in the possibility of a beneficent environment," thereby harnessing hope (bondi , ; macinnis and de mello ) . further, ontological security is "not simply a matter of selfpreservation or self-interest," but relies on "the well-being of others as well," (banham , ) . when consumers' ontological security is disturbed, they have a significant emotional investment in reestablishing trust that the world is predictable (garfinkel ) . the extent to which a consumer experiences ontological insecurity is likely to influence how the consumer responds to threat-induced disruption. as discussed, next, consumers are likely to respond to feelings of insecurity and uncertainty in a variety of affective, cognitive and behavioral ways. there are important questions of how consumers will respond to the ontological insecurity elicited by the current threats, what changes will be short-term, what changes will be more lasting, and what effects marketing, government, and other institutional responses and actions will have. the term "adaptive" broadly refers to the responses both consumers and markets make to "get along" in altered and uncertain circumstances. adaptation is the process of change by which consumers and markets respond to the present environment. while adaptations vary along a continuum from those that enhance consumer and market functioning to those that do not, in the short term it is difficult to make that evaluation, hence our use of the term "adaptive." ideally, adaptive responses will help consumers and markets become better suited to the altered environment, but this will not always be the case. in this section, we outline some possible consumer responses, briefly describe the vital role that market responses have from the perspective of consumers, and outline the important interplays that over time can contribute in positive or negative ways to the extent of threat and disruption. although we categorize responses as affective, cognitive, and behavioral, these responses are highly related. to start with, ontological (in)security is an emotional process, and emotional response is key (banham ) . consumers feeling insecure and uncertain may respond with a full gambit of strong emotional reactions such as anxiety, fear, stress, sadness, grief, anger, frustration, or even depression (carver, scheier, and weintraub ) . in the context of a health threat, affective responses include the fear of contamination and disgust with seeing symptomatic others (galoni et al. , this issue; rozin, haidt, and mccauley ; tybur et al. ) . research shows that the behavioral immune system (neuberg, kenrick, and schaller ) motivates people to avoid potential contamination, which in a consumer context would imply the avoidance of crowds (e.g., reducing attendance at movies, theaters, and sporting events). economic threats may evoke anxiety (lowe, loveland, and krishna ) regarding the effect on the household's financial precarity (meuris and leana ) and retirement savings. social threats, such as social unrest or terrorism, also lead to anxiety, outrage at social injustice, and fear of death. threats, disruption, and ontological uncertainty likewise impact consumers' cognitions. these include loss of control (beck, rahinel, and bleier ; van bergen and laran ); self-regulation (van bergen and laran ); helplessness (chaxel ) ; loneliness (su, wan, and jiang ) ; need for connection (rindfleisch, burroughs, and wong ) ; and mortality salience (ferraro, shiv, and bettman ) . for instance, mortality salience, that is, the accessibility of thoughts about death, may lead to various outcomes relevant to consumers, including increased indulgence (ferraro et al. ) , increased purchase and consumption (mandel and smeesters ) , and materialism (rindfleisch et al. ). uncertainty (shen, hsee, and talloen ) is another example of a cognitive response to threat. whereas a general sense of uncertainty may lead to ontological insecurity, threats can lead to specific uncertainty about product availability, planning horizon, and even the veracity of available information (e.g., concerns about what news is fake/exaggerated and what is valid). interestingly, uncertainty leads to the use of affective inputs (faraji-rad and pham ), suggesting that consumers during the pandemic may be more affectively driven. these affective and cognitive responses can impact a variety of behavioral responses, either as mediators that lead from perceived insecurity to behavioral responses or as moderators of other processes. threats evoke both approach and avoidance coping strategies (han, duhachek, and rucker ) . feelings of fear and uncertainty could spark consumer behaviors such as stockpiling riskmitigating products such as antiseptic wipes during a health threat or switching from national brands to private label brands during an economic downturn (lamey et al. ) . when consumers feel a loss of control or are stressed and anxious, they may seek out brands, products, and services to help. some consumers are likely to prefer logos and products that are themselves bounded to compensate for the feelings of loss of control (cutright ). in addition, melumad and pham ( ) show that consumers turn to their smartphone when feeling stressed and the personal nature, portability, and haptic gratification offer stress relief and psychological comfort. marcoux ( ) uncovers how new yorkers used souvenirs both to remember and forget the / terrorist attacks on the twin towers in new york in . beck et al. ( ) suggest that consumers experiencing low personal control are more likely to frequent leading brands as a way of reasserting agency. similarly, materialistic individuals are likely to respond to insecurity by forming strong connections with brands (rindfleisch et al. ). consumers trying to cope with times of trouble may also break from their usual choices and behaviors. when faced with life-threatening trade-offs, such as deciding whether to take a child off life support, consumers may prefer to relinquish control and let someone else decide for them (botti et al. ). wood ( ) finds that, while consumers predict they will want comfort foods to minimize the uncertainty and discomfort of a new or dynamic environment, they may instead "break habitual cues that favor old favorites" and promote a "change mindset" leading to variety seeking and new product acceptance (p. ). phipps and ozanne ( ) show that some consumers whose norms, beliefs, routines, and practices were disrupted by the worst and longest drought in australian history developed trajectories of new practices to reassert ontological security and that some of these continued even after the threat eased. other research suggests that an event that creates a disjuncture between before and after may promote a fresh start mindset (price et al. ) . consumers may respond with new choices and goals such as a gym membership, variety seeking, and being more willing to forgive past grievances such as an unsatisfactory service encounter (dai, milkman, and riis ; gu et al. ; price et al. ) . in a six-country study investigating consumers' view of the world under covid- , % said that their views had changed and % reported that the pandemic had changed the products and services they considered important (carufel ) . hence, while consumers may turn to old favorites in some contexts, consumers may trust different products and services during times of threat. technologies have taken an unprecedented role during this pandemic and are likely to play a paradoxical role in consumers' behavioral responses (mick and fournier, ; kozinets ) . reluctant consumers have felt forced to take work, family life, and leisure online. consumers have struggled and sometimes succeeded in reassembling their rituals, routines, work, and family practices in technology-mediated spaces, sometimes using these spaces in response to need for connection, and sometimes for work. they have attended funerals, weddings, graduations, religious services, classes, and countless meetings online. consumers' responses to technology are mixed. while technology-mediated spaces afford the opportunity to continue everyday practices, it is difficult to fully convert face-to-face encounters to online ones (epp, schau, and price ) . consumers who went to an external workplace prior to the covid- threats and disruptions respond both negatively and positively to working at home. while they note disruption and difficulties (especially if co-working and also educating children in the same space), many expect and desire the option of continuing some work from home as part of the longer term (carufel ) . working at home also means changes in consumption practices on the way to and from work and changes in leisure consumption. concurrent with the excessive presence of tech-mediated practices in consumers' daily life is the reinvigoration of low-tech intimate family rituals, such as doing puzzles, playing boardgames, and family outdoor excursions (allwood ) . while anxiety and instability may lead to negative emotions and cognitions, they can also spark innovative and creative responses. when we are no longer operating in our taken-for-granted world, we pay more attention to what we are doing and consequently may do things differently. not only are consumers more likely to try new options, but also their altered perceptions of risk, lowered expectations, and lessened fear of failure as they cope with constraints and ill-defined problems may help creative solutions flourish (lamberton and wood ; moreau and dahl ; moreau and engeset ) . consistent with tversky and kahneman's ( ) research on prospect theory, when things are dire consumers are more willing to take risks to improve the situation. an explosion of creativity may be a covid- pandemic "silver lining" (lamberton and wood ) . with a focus on problem-solving and not performance, fear of failure loses its grip. consumers may experiment with something they have always been afraid to try (e.g., homemade sour dough bread; home improvements; new exercise routines; new hobbies; and new technologies). examples abound of how disruptions caused by the covid- pandemic have led to creative and imaginative individual and collective consumer responses such as: drive by birthday parties and graduations; co-constituted choirs and concerts; passover dinners via zoom; homemade, creative face masks; and consumer barter systems, with neighbors posting exchanges for products such as coffee for toilet paper (barrett ) . perhaps consumers will want to go back to their prior habits and routines, if and when things return to normal, but there is also reason to believe that some of these new behaviors will take hold and be part of their post-pandemic new lives. thus, ontological insecurity can be "the mother of invention," in both short-and long-term consumer responses (phipps and ozanne ). markets also respond to disruptions caused by threats; these responses both guide and are guided by consumer responses. given that our focus is primarily on consumers, we briefly describe the vital role that market responses introduction have from the perspective of consumers. for consumers, markets are sites of constancy in a social and material world, spatial contexts where day-to-day routines are performed, sites where consumers feel they have agency and control over caring for their families and expressing their identities. although undertheorized, we suggest that, like homes, schools, and other significant sites, markets play a central role in consumers' ontological security (dupuis and thorns ) . as such, when markets experience disruptions, market responses are highly consequential for consumers. threats are likely to expose weaknesses in market systems as they stress test the boundary conditions of our marketing infrastructure. the pandemic has overwhelmed healthcare systems and put essential workers, not just in health care but sanitation, transportation, delivery, farm, food processing, grocery, services, and utilities at risk. global supply chains in many product categories have also been put at risk, making it difficult to bring products to market. these market responses, in turn, have led to consumer responses, such as panic buying and hoarding. this, in turn, has increased pressure on markets. market responses to the pandemic have varied from negligent to highly effective. some grocery stores installed plexiglass shields at checkout stands, limited store hours to facilitate restocking and cleaning, set aside hours for consumers over or , limited access and supply of high demand items, created store flows to facilitate social distancing, and made adjustments as circumstances evolved. home delivery was stretched to capacity and was viewed as both a life saver and a necessary evil. amazon, uber, and instacart came under severe criticism for poor work conditions and poor service. at the same time, consumers who long resisted ordering online, particularly those who were elderly, saw home delivery as a vital lifeline. market organizations also responded with a surge of creativity. education and exercise classes quickly went online, and restaurants offered curbside and take-out, in some cases flipping their business model to provide meals for employees and/or "soup" kitchens. netflix offered new ways to watch movies together while physically distanced, and peloton sold many bikes for in-home zoom sessions. meal kit companies went from struggling to keep customers to struggling to service both existing and new demand, modifying the number of recipes and ingredients offered per week to be able to successfully respond. these responses help establish norms and beliefs and create, reimagine, or restore routines to offset ontological insecurity. as illustrated in the figure, the consequences of a largescale threat on consumer and market disruption depend on the interplay of consumer, market, and governmental responses, situated within broader, global macro-forces. we posit that these responses interact, recursively impacting the extent of threat and disruption. for example, initial scarcity (e.g., no hand sanitizer or masks) was worsened by panicked consumers who hoarded supplies and also led some consumers to creatively figure out how to make products for themselves and others. some businesses, such as bakeries and restaurants, stocked scarce items, such as toilet paper, hand sanitizer, and eggs, to attract customers, while other companies switched their production to make scarce items or innovated to create processes for extending the utility of scare items, such as cleaning n- masks. the responses of consumers and markets increased the supply of masks and hand sanitizer, likely reducing the extent of threat by decreasing the level of contagion and, thus, lowering disruption for many consumers. however, the interplay of consumers and markets may sometimes increase the extent of threat. for example, meat-packing plants with serious covid- outbreaks shut down and slowed supplies to markets. consumers responded by stockpiling meat, further increasing shortages. empty meat counters created political pressure for processing plants to reopen, which many did, even though they had inadequate procedures in place for employee protection; in this case, the interacting responses increased threat of contagion. in short, different forces within the market are intertwined, making the entire system more complex. in the next section, we describe some potential moderators of the relationships among disruption, ontological security, and consumer and market adaptive responses. although there is an endless list of potential moderators, including many of the affective and behavioral responses we have already described, we focus on a few that seem particularly promising. not only are there significant differences across consumers in the amount of disruption experienced by threat, but also there are many moderators that affect short-term and long-term consumer responses. as shown in the figure, these factors could moderate relationships among different variables, for example, the extent to which disruption leads to ontological insecurity, as well as the impact of ontological security on consumers' responses. in addition, the factors will moderate the extent and manner in which consumers respond to marketplace changes, and vice versa. this is not intended as an exhaustive list of relevant constructs; rather, its purpose is to provide a conceptual jumpstart to researchers seeking to expand our theoretical understanding of the linkages among threats and responses. big picture factors, such as socioeconomic status, racial and economic inequality, access to basic health care, shopping options and platforms, digital access, and numerous other psychological, social, institutional, political, and cultural factors, will moderate the influence of disruption on ontological insecurity and/or consumer responses. for example, inequality in digital access has emerged as a major influence on consumers' responses, and the success of their coping, during the covid- crisis (beaunoyer, dup er e, and guitton ). many of the affective and cognitive responses mentioned in the previous section, such as mortality salience, uncertainty, scarcity, and loss of control, are also likely to serve as potential moderators. for instance, scarcity (real or perceived) is a particularly pertinent construct in multiple threat contexts (cannon et al. ; roux, goldsmith, and bonezzi ) . in the health threat context, a prime example is the shortage of toilet paper, paper towels, antiseptic wipes, and home baking items (e.g., yeast) widely seen under the covid- lockdown. spikes in demand led to stockpiling, hoarding, and price gouging. photos of empty shelves and shopping carts loaded with toilet paper were quite common in the early days of the covid- lockdown. interestingly, such behavior may elicit guilt (duke and amir ) in consumers after hearing reports that many older households had no supplies because of hoarding. consumers could assuage such guilt feelings by donating their excess supply to local shelters or returning some of the items. interestingly, prior work suggests that consumers become more self-interested when resources are scarce (roux et al. ) . however, people also come together during times of external threat; there are many examples of consumers helping during the covid- pandemic, such as making and distributing meals for first responders, helping out elderly neighbors, and making homemade masks and giving them away (giving new meaning to the term "household production function"). in addition, many individual difference variables could influence the effects of threat-induced ontological security on how consumers respond. these include agencycommunion focus (bakan ; yang and aggarwal ); political ideology (jung et al. ; ordabayeva and fernandes ) ; religiosity (kurt, inman, and gino ; mathras et al. ) ; regulatory focus (zhou and pham ) ; risk aversion (holt and laury ) ; and mindset (gollwitzer and bayer ) . for example, consumers with a greater communion focus compared to agency focus would theoretically be more inclined to endure greater personal hardships (e.g., extended lockdown, wearing a mask) to mitigate the risk of infection by others. a consumer with a growth mindset (murphy and dweck ) when faced with a health threat may be more inclined to take action to counter the threat than a consumer with a fixed mindset. similarly, more promotion-focused consumers may be more likely to take risks as compared to more prevention focused consumers (zhou and pham ) and more risk-averse consumers should be more likely to take action to avoid personal threat than less riskaverse consumers. as seen in news reports, political ideology and religiosity also influence how severe a consumer considers the threat, where the consumer seeks information about it, whom the consumer believes, and how the consumer responds. for instance, political ideology may lead personal freedom-oriented consumers to resist impositions on their ability to return to "normal" consumer behaviors, while social-focused consumers may prefer to continue to curtail their pre-threat consumer behaviors to reduce the potential of spreading the virus. in addition, the extent of threat may be perceived differently by those who are highly religious compared to those who are not. highly religious consumers (regardless of the specific religion) may view the covid- pandemic as god's will and thus have a more fatalistic view of how to respond to it. they may be more likely to engage in behaviors that increase the likelihood of threat from the pandemic. as mentioned earlier, the constructs listed in this section are meant to provide conceptual kindling for research on consumer response to threats and the accompanying disruptions. obviously, there are many other potential constructs that could be pertinent to such research. we now present a summary of each article in the issue and then close with a discussion of high-potential directions for future research. the articles in this issue investigate consumer responses to economic and health threats. consumers' overall wellbeing is affected by their economic well-being (netemeyer et al., ) . as maslow ( ) pointed out more than years ago, the ability to satisfy physiological needs for ourselves and our loved ones is foundational to motivation and well-being. thus, economic threats such as reduction in hours, pay, or complete loss of job are likely to create disruption and insecurity and impact consumer well-being. three articles in this issue focus on consumers' responses to economic threats. the international monetary fund labeled "the great lockdown" as the worst recession since franklin d. roosevelt's us presidency. economists and consumers alike wonder what will happen next and whether the "the american dream" is still possible, especially in the face of rising inequity and massive financial precariousness. the american dream intertwines consumption with being a good citizen. individuals pursuing personal wants through consumption are perceived to serve the general good (cohen ; giesler and veresiu ) . coskuner-balli ( , this issue) look at archival data and textual analysis of presidential speeches of four us presidents (reagan, clinton, bush, and obama) over a time period that introduction included a series of economic recessions. she examines the strategies these four presidents used during times of recession to mobilize the national mythology of the american dream and call on the shared responsibility of the citizenconsumer to attain economic goals. in the aftermath of recessions, their speeches put more emphasis on the consumer for three of the four economic recessions ( , and ) , but not (which also included the september attacks). she found differences in how the presidents linked the concept of the consumer to the socioeconomic issues at that time. for example, in reagan's speeches, the consumer is more closely linked to terms such as economy and freedom, whereas in clinton's speeches the consumer is linked to products and workers. nevertheless, all of these presidents mobilize rhetoric to manage the citizen-consumer in support of economic goals. coskuner-balli's article shows how the moral citizenconsumer subject is called on to address economic problems. the combined threats of a pandemic and recession make this an especially interesting time to investigate the interplay of political rhetoric and the responsible citizenconsumer. in a different vein, mittal et al. ( , this issue) examine the interactive effect of childhood economic environment and current threat on subjective life expectancy (i.e., a consumer's estimate of how long s/he will live). they find that the extent to which a consumer was rich or poor during childhood affects subjective life expectancy, but only when there is a current external stressor (i.e., threat). in the presence of an external threat such as economic uncertainty, consumers whose childhoods were poor are likely to become pessimistic, leading to lower subjective life expectancy. subjective life expectancy affects consumer-relevant variables, such as how much money to save for retirement, whether to buy long-term care insurance, and how much to spend on life insurance. the findings of this article suggest that economic recessions, pandemics, and terror threats can affect subjective life expectancy for some consumers, leading to different financial and health decisions than they might make otherwise, as well as potentially impacting their mental health. a particularly relevant question in terms of what to expect after the covid- pandemic abates is consumer spending patterns once a budget contraction is lifted (e.g., once the economy recovers from recession). in their research, wilroy et al. ( , this issue ) explore this important question. prior research has found that a contracting economy or personal budget causes consumers to reduce spending on nonessential items (dargay ) and to reduce the overall number of different items they buy (carlson et al. ) . interestingly, this research has ignored the downstream effect that a budget contraction has on consumers' future choices, given that the budget has been restored. wilroy et al. find that a budget contraction forces consumers to reevaluate what is really important to their well-being. as a result, consumers learn that many "essentials" are not as essential as they previously supposed and they update their preferences. this, in turn, leads to an enduring reduction in the variety of products they choose post-contraction. these findings have interesting implications for consumers' spending patterns as the economy recovers. some product categories may experience a v-shaped recovery where revenues revert to pre-disruption levels, while others may experience a reverse j-shaped pattern where some categories' revenues (or brands' revenues) will only partially recover. future research using an event study paradigm is needed to assess the types of product categories and brands that suffer lasting effects versus those that fully recover. the other four articles in this issue examine health threats. two of these address the specific question of how the threat of contagious disease influences consumers' product choices. galoni et al. ( , this issue) focus on consumers' emotional responses to the specific health threat of potential exposure to contagious disease and the impact of such emotional responses on consumption. noting that the prior literature has shown that potential exposure to contagious disease leads to feelings of disgust, galoni et al. ( , this issue) propose that such exposure can additionally evoke feelings of fear. the authors propose that, unlike the avoidant response elicited by disgust, the joint impact of the mixed emotional responses of fear and disgust lead both to avoidance and approach; consumers avoid the actual threat but seek familiar products. exposure to the threat of contagion leads to mixed emotional responses of disgust and fear, which appear to increase consumers' desire for comfort, which they can gain from the purchase of familiar products. this research increases our conceptual understanding of how consumers are likely to respond to a health threat while also providing managerial insight into the specific products likely to be impacted by a societal threat. huang and sengupta ( , this issue) take a different approach to the same basic question of how the threat of contagious disease could impact consumers' product choices. taking an evolutionary approach, huang and sengupta focus on the impact of disease threat on the motive to avoid other people. according to the evolutionary perspective, self-protection and disease avoidance are two fundamental motives for reproductive fitness (griskevicius and kenrick ) . building on this literature, huang and sengupta propose that environmental factors that impact awareness of the potential for contagion influence consumers' product preferences. specifically, this research provides evidence that salience of disease contagion increases motivation to avoid others, which increases the relative preference for atypical versus typical products. because typical products are associated with others, when consumers want to avoid others, preferences for atypical, versus typical, products increase. both galoni et al. and huang and sengupta provide insight into how increases in awareness of disease contagion could impact consumers' consumption choices. interestingly, while galoni et al. provide the evidence of increased preference for familiar products, huang and sengupta show the evidence of decreased preference for typical products. while these may seem contradictory, galoni et al. are focused on preferences for things that are familiar, and thus comforting, to the individual consumer. huang and sengupta, on the other hand, do not examine individual preferences based on prior brand experience, but rather look at preferences for products that are typical, and therefore associated with more people in general. an interesting area for future research would be to explore factors that elicit one versus the other of these processes. another article relevant to health threat concerns how consumers view natural versus synthetic products as a source of disease prevention. scott et al. ( , this issue) find that consumers have lay theories that natural products are safer but less potent than synthetic products. for instance, an herbal medicine or natural shampoo will be perceived as safer, but less powerful than medicine or shampoo that includes chemicals. as a result of this lay theory, consumers are more likely to prefer natural products for prevention purposes but to prefer synthetic products for curative purposes. one interesting question is whether these results may be moderated by the severity of the health threat. for instance, to prevent getting covid- , some consumers who did not have the disease took medicines with strong chemicals because of misinformation about the medicine's benefits; in some cases, this proved fatal. once again, this shows how multiple threats can compound each other. the salience and severity of the health threat may move consumers' relative preference for natural versus synthetic products in both the curative and prevention phases. a core aspect of many health threats is that they may make one's mortality salient; the ultimate threat to health is death. prior work shows that mortality salience makes consumers more materialistic, at least in some circumstances (ferraro et al. ; rindfleisch et al. ). in contrast, dunn et al. ( , this issue) demonstrate that mortality salience can lead to more prosocial donation behavior when it has high transcendence potential. specifically, reminders of one's mortality can result in greater donations of self-relevant possessions as a way of achieving transcendence (i.e., continuing to live beyond death). in other words, consumers may give away a prized possession, such as a beloved book, so that they live on through that possession. an interesting question for future research might be to consider conditions under which the recipient will value this possession. this would be the case when the reminder of the dead person evokes a positive memory (e.g., a beloved family member's clothing) versus a negative memory (e.g., clothing of a beloved family member who was infected with covid- ). a disease prevention perspective suggests that the latter might lead to both fear of contamination and disgust. as shown by the articles in this introduction to this focused issue, consumer research has examined a variety of questions apropos to understanding and predicting how consumers respond to external threats to their well-being. while existing research can inform academics, marketers, and public policy decision-makers, many questions remain unexplored and unknown. in this section, we outline some promising areas for future research, structuring ideas around elements of our framework: disruption, extent of threat, and consumer adaptive responses. one particular disruption of the covid- pandemic has been the physical distancing experienced by millions of consumers globally. stay-at-home regulations have affected daily consumption decisions, including whether and when to shop and how to spend leisure time. consumers struggle to make sense of what is happening, restore practices, align new practices, and purchase and consume products (humphreys and thompson ; phipps and ozanne ; thompson ) . while staying at home has limited interaction with people outside of the household, it has also required extended, close interaction with members of the household, typically families or house mates. an important research question is how families negotiate these interactions, particularly in cultures that are characterized by an independent self-construal (markus and kitayama ) . an historical analysis of articles published in the journal of consumer research highlights that research on family decision-making has declined over time (wang et al. ) . only a handful of articles have examined family interactions in the last two decades price , ; thomas and epp ) . the covid- pandemic raises interesting questions about consumer decision-making that can have negative consequences for others within-and outside-the family unit. for example, research could examine the extent to which the pandemic has affected consumers' self-construal (markus and kitayama ) . to the extent that consumers consider the impact of their decisions on others, consumers with a more independent self-construal (e.g., those in many western nations) may be (temporarily) shifting toward a more interdependent one (e.g., those in asian countries). a consumer accustomed to making decisions based on her own welfare introduction (e.g., going to the gym or restaurant) may now take into account the effect this might have on vulnerable family members, such as parents or grandparents. although these may be temporary shifts primed by the pandemic, they may also have long-term effects on consumer behavior. future research can lead to better understanding of how making choices for the self extends to different choosing-for-others contexts and how to prompt consumers to make choices that are good for others (liu, dallas, and fitzsimons ) . another issue has to do with the effect of stay-at-home orders on family consumption, such as leisure, as well as decision-making. because families spend all their time in the same home (e.g., young children are home instead of at school; college students are home; parents are working from home), their coping strategies may have changed. there may be differences in roles, expectations, and decision-making. thomas and epp ( ) describe how difficult it is to implement a new set of family routines even with considerable forethought and planning because of the "messiness of doing." this is likely to be exponentially more difficult when faced with big, unforeseen disruptions to everyday family life. on the one hand, some consumers may increase consumption of joint leisure activities (e.g., playing board games, watching netflix together); however, others might become more isolated, avoiding social contact by escaping into online media and games. some of this will likely be moderated by socioeconomic status, such as the size of the home, the safety of the neighborhood, and access to the internet. in this respect, the pandemic experience of poorer consumers may be vastly different from those of richer ones, especially since the former also experience stronger economic threat. threats can also cause major disruption to consumers' mental budgets (thaler ) . this can be due to an effect on the overall amount available for expenditures or consumers' allocation of the budget across mental accounts (heath and soll ) . for example, a threat of job loss or furlough poses a disruption of household income, implying a reduction in expenditures. alternatively, having to stayat-home has precluded away-from-home dining for many consumers, so the "entertainment account" may be reallocated elsewhere. interestingly, this reallocation may contribute to post-threat stickiness of changes in consumer spending habits. how threats impact consumers' mental budget calculus in the short term and long term is an intriguing and important question for future research. since the pandemic is a society-level experience unlike others in the recent past, it raises the question of how consumers deal with communal threats. the covid- pandemic affects many consumers worldwide. how do consumers react to widespread threats versus local ones? much consumer research provides deep insight into how consumers respond to a threat to themselves or their ingroup, such as the decision as to whether to continue an infant's life support (botti et al. ). while some research does explore aspects of larger scale threats (winterich et al. ), many questions remain. as one example, does the extent to which others are likewise impacted change how a consumer responds to her or his own negative event? many employees (including faculty) have received pay reductions; how do responses depend upon whether others are experiencing the same cut? what types of social comparison occur? as another example, given that an effective coping response can be for a consumer to draw upon her or his social network, how will or should consumers cope when the threat is also being faced by their social network or in-group? this is particularly true when members of the social network may perceive the extent of threat differently. for instance, the extent of threat perceived by consumers of different political ideologies is different, partly based on the information that they believe. research shows that consumers of conservative media, such as fox news, saw the perceived threat from covid- as less severe than did consumers of liberal media, such as the new york times (jamieson and albarracin ). more research is needed regarding the role of misinformation in exacerbating differences in the perceived extent of threat. relatedly, researchers (hsee and rottenstreich ; schley, de langhe, and long ) find that many people tend to exhibit scope insensitivity, the phenomenon whereby valuation judgments exhibit strong insensitivity to the magnitude or scope of the object(s) being valued. for example, hsee and rottenstreich ( ) report that participants were unwilling to pay more for a set of used madonna cds than for a set of five. chang and pham ( ) show that scope insensitivity is more likely to occur for decisions that are psychologically closer to the decision-maker. does this mean that psychological distance moderates consumer sensitivity to the scope of an imminent threat? or is this effect countered by the personal implications of the imminent threat? another area with research potential involves the tensions or trade-offs that consumers confront in the face of multiple threats. how do consumers balance conflicting forces from multiple threats? for instance, although the health guidelines say not to reuse disposable face masks and other health-related goods to mitigate health threat, both product scarcity and economic scarcity may lead consumers to reuse such goods; this is particularly likely for consumers with lower income or in poor countries where resource scarcity is high. what might be different moderators of this relationship? although the impact of multiple threats is under-researched, it is likely to reveal additive as well as multiplicative effects. another question the pandemic has brought to the fore is understanding which types of consumer and marketplace responses will be ephemeral versus lasting. for instance, due to the shutting down of movie theaters, some consumers subscribed to a streaming service for the first time. are these new customers likely to stay? two steams of literature seem relevant here. first, the literature on new product adoption suggests that this may depend on the cost and convenience of the new technology, the availability of substitutes, and relative advantage (rogers (rogers / . streaming services are expecting many new customers to remain even after movie theaters open because of the convenience and low cost of such services. the other relevant literature concerns the formation of habits, defined broadly as dispositions to behave in a certain way. habits develop over time in in stable environments, when repeated behavior becomes automatic (ouellette and wood ) . habits can also develop through conscious deliberation, which may be the case of the pandemic. this suggests that the length of time that consumers engage in threat-induced behavior is likely to affect the extent to which it becomes a habit. however, the pandemic environment is anything but stable, and it is unclear whether consumers see their changed behavior as a necessity or as something desirable. an event study paradigm could be useful in this regard. consumers' spending changes during the coronavirus crisis should be assessed and then compared to postcrisis spending, along with conceptually relevant moderators. as with significant threats of the past, such as the great depression, the psychological effects of the pandemic are likely to be long-lasting. however, whether or when such threats change the traits and orientations of adult consumers is unclear. van bergen and laran ( ) find that loss of control can increase self-regulatory behavior, which is a response learned in childhood. however, as shown by mittal et al. ( , this issue) , the effects of external threats on adult consumer behavior are influenced by childhood experience; such interactive effects offer another area for future research. a longitudinal study of today's younger generation in terms of their future consumer behavior would be a challenging, but potentially rewarding, research endeavor. the pandemic has had a significant impact on the sharing economy, including car, clothing, and home rental. the sharing economy is characterized by the temporary use of resources, mediation by platforms, and crowdsourced supply (eckhardt et al. ; scaraboto ) . a major threat to the sharing economy is the health threat. given fear of contagion, consumers are reluctant to use products touched by other people. research questions include the extent to which consumers are likely to reengage in the sharing economy. what are characteristics of products and services that might survive the crisis? presumably, tangible products that require physical contact, such as clothes and homes, may be less likely to be shared than intangible products, such as ideas and data. in terms of tangible products, are products that are closer to the self (e.g., clothing) likely to be shared less than those that might be more distant (e.g., bicycles)? the answers to these questions are complicated because the sharing economy is also affected by the economic threat. economic recession may drive consumers toward increased participation in the sharing economy because renting houses, clothing, and bicycles is cheaper than purchasing them. in the case of airbnb and uber, however, non-purchase alternatives, such as hotels and taxis, may be preferred. consumers are more likely to trust well-known brands, such as marriott and hilton, who have greater reputation costs than do individual property owners. in short, the interplay between health and economic threats raises interesting questions for research. for example, while consumers with access to personal vehicles may discontinue the use of bike-sharing programs because of safety concerns, consumers who typically use mass transit may increase the use of bike sharing, viewing it as safer. during the pandemic, many commuters purchased bikes to shift from mass transit to bicycling, creating scarcity in bikes available for purchase, which could also contribute to higher levels of bike sharing. the environmental impact of shifting consumer mobilities may also be quite consequential and worthy of further investigation. the changed environment during the covid- pandemic raises many questions about possible extensions of existing findings in the literature. for instance, the notion of busyness is likely to have a different meaning during a stay-at-home environment. bellezza, paharia, and keinan ( ) find that being busy at work is considered a status symbol; however, in the work from home age, how is busyness defined? is status conferred by being able to manage both work and household duties, since these often intermingle? what kinds of identity do people have around being (or not being) an "essential" worker, and how does this affect consumption? relatedly, time-money trade-offs (monga, may, and bagchi ) change during economic threats, as consumers have more time and less money. do these trade-offs have different implications under economic threat? what about when combined with health threat? as another example, atasoy and morewedge ( ) find that digital goods are valued less than physical goods. is this true in times of widespread threat, when the importance of digital goods is higher because they are consumers' lifeline to the world? recent research has identified a therapeutic role of consumption servicescapes, focusing on how they "heal" consumers through rituals of therapeutic relations, release, and renewal (higgins and hamilton ) . when and how are servicescapes likely to provide this therapeutic role for consumers experiencing disruption following contagion or economic threats? introduction overall, the threat framework gives rise to a plethora of questions about new processes and possible boundaries to a variety of interesting consumer behaviors, as well as the dynamic nature of threat-induced disruptions leading to responses that recursively influence the extent of threat and disruption. we have argued that threat, moderated by the size, scope, and psychological distance of the threat, leads to disruption in consumers' lives, which can create ontological insecurity. we posit that the resulting combination of consumer and market responses can then impact the extent of threat and disruption. consumers' emotional, cognitive, and behavioral reactions depend not only on the severity of their ontological insecurity but also on many other factors including individual differences in beliefs, personality, and background and differences in material, social, and economic circumstances. our hope is that our conceptual framework will guide researchers seeking to enhance the understanding of how consumers and markets collectively respond over the short term and long term to threats that disrupt consumers' routines, lives, or the fabric of society. our aspiration is that this article will spark the talented scholars in our field to accept the challenge of increasing our understanding of the conceptual drivers and practical implications of this critically important topic. introduction introduction board games and puzzles sell out during covid- shutdown digital goods are valued less than physical goods the duality of human existence emotion, vulnerability, ontology: operationalising 'ontological security' for qualitative environmental sociology liquid consumption still can't buy toilet paper? you can barter for it liquid modernity covid- and digital inequalities: reciprocal impacts and mitigation strategies company worth keeping: personal control and preferences for brand leaders conspicuous consumption of time: when busyness and lack of leisure time become a status symbol feeling insecure: a personal account in a psychoanalytic voice tragic choices: autonomy and emotional responses to medical decisions a self-regulatory model of resource scarcity the budget contraction effect: how contracting budgets lead to less varied choice how covid- has changed consumers' entire view of the world-and their own country assessing coping strategies: a theoretically based approach covid- in racial and ethnic minority groups the role of involvement in attention and comprehension processes affective boundaries of scope insensitivity why, when, and how personal control impacts information processing: a framework a consumers' republic: the politics of mass consumption in postwar america citizen-consumers wanted: revitalizing the american dream in the face of economic recessions the beauty of boundaries: when and why we seek structure in consumption the fresh start effect: temporal landmarks motivate aspirational behavior the effect of income on car ownership: evidence of asymmetry threats to hope: effects on reasoning about product information guilt dynamics: consequences of temporally separating decisions and actions a little piece of me: when mortality reminders lead to giving to others home, home ownership and the search for ontological security marketing in the sharing economy the role of brands and mediating technologies in assembling long-distance family practices family identity: a framework of identity interplay in consumption practices the storied life of singularized objects: forces of agency and network transformation uncertainty increases the reliance on affect in decisions let us eat and drink, for tomorrow we shall die: effects of mortality salience and self-esteem on self-regulation in consumer choice afraid and disgusted: consumer choices under the threat of contagious disease modernity and self-identity: self and society in the late modern age creating the responsible consumer: moralistic governance regimes and consumer subjectivity a room with a viewpoint: using social norms to motivate environmental conservation in hotels deliberative versus implemental mindsets in the control of action fundamental motives: how evolutionary needs influence consumer behavior turning the page: the impact of choice closure on satisfaction distinct threats, common remedies: how consumers cope with psychological threat mental budgeting and consumer decisions therapeutic servicescapes and market-mediated performances of emotional suffering risk aversion and incentive effects music, pandas, and muggers: on the affective psychology of value the influence of disease cues on preference for typical versus atypical products branding disaster: reestablishing trust through the ideological containment of systemic risk anxieties the relation between media consumption and misinformation at the outset of the sars-cov- pandemic in the us blue and red voices: effects of political ideology on consumers' complaining and disputing behavior consuming technocultures: an extended jcr curation religious shoppers spend less money the divided self: an existential study in sanity and madness why a crisis frees us of our old mental models how business cycles contribute to private-label success: evidence from the united states and europe stereotype threat in the marketplace: consumer anxiety and purchase intentions a framework for understanding consumer choices for others a quiet disquiet: anxiety and risk avoidance due to nonconscious auditory priming the concept of hope and its relevance to product evaluation and choice the sweet escape: effects of mortality salience on consumption quantities for high-and low-self-esteem consumers souvenirs to forget culture and the self: implications for cognition, emotion, and motivation a theory of human motivation the effects of religion on consumer behavior: a conceptual framework and research agenda the smartphone as a pacifying technology the price of financial precarity: organizational costs of employees' financial concerns paradoxes of technology: consumer cognizance, emotions, and coping strategies silver spoons and platinum plans: how childhood environment affects adult health care decision from cradle to grave: how childhood and current environments impact consumers' subjective life expectancy and decision-making eliciting time versus money: time scarcity underlies asymmetric wage rates designing the solution: the influence of constraints on consumer creativity the downstream consequences of problem-solving mindsets: how playing with lego influences creativity mindsets shape consumer behavior how am i doing? perceived financial well-being, its potential antecedents, and its relation to overall well-being human threat management systems: self-protection and disease avoidance better or different? how political ideology shapes preferences for differentiation in the social hierarchy habit and intention in everyday life: the multiple processes by which past behavior predicts future behavior the reflexive relationship between consumer behavior and adaptive coping what is a disaster?: new answers to old questions routines disrupted: reestablishing security through practice alignment the fresh start mindset: transforming consumers' lives the safety of objects: materialism, existential insecurity, and brand connection diffusion of innovations on the psychology of scarcity: when reminders of resource scarcity promote selfish (and generous) behavior disgust selling, sharing, and everything in between: the hybrid economies of collaborative networks system is not scope insensitive: a new, dual-process account of subjective value consumers prefer 'natural' more for preventatives than for curatives the fun and function of uncertainty: uncertain incentives reinforce repetition decisions filling an empty self: the impact of social exclusion on consumer preference for visual density mental accounting and consumer choice the best laid plans: why new parents fail to habituate practices consumer risk perceptions in a community of reflexive doubt theorizing reactive reflexivity: lifestyle displacement and discordant performances of taste construal-level theory of psychological distance advances in prospect theory: cumulative representation of uncertainty disgust: evolved function and structure loss of control and self-regulation: the role of childhood lessons a historical analysis intracommunity gifting at the intersection of contemporary moral and market economies preference refinement after a budget contraction donation behavior toward in-groups and out-groups: the role of gender and moral identity at risk: natural hazards, people's vulnerability, and disasters the comfort food fallacy: avoiding old favorites in times of change no small matter: how company size affects consumer expectations and evaluations promotion and prevention across mental accounts: when financial products dictate consumers and investment goals key: cord- -me ajoyb authors: schountz, tony; prescott, joseph title: hantavirus immunology of rodent reservoirs: current status and future directions date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: me ajoyb hantaviruses are hosted by rodents, insectivores and bats. several rodent-borne hantaviruses cause two diseases that share many features in humans, hemorrhagic fever with renal syndrome in eurasia or hantavirus cardiopulmonary syndrome in the americas. it is thought that the immune response plays a significant contributory role in these diseases. however, in reservoir hosts that have been closely examined, little or no pathology occurs and infection is persistent despite evidence of adaptive immune responses. because most hantavirus reservoirs are not model organisms, it is difficult to conduct meaningful experiments that might shed light on how the viruses evade sterilizing immune responses and why immunopathology does not occur. despite these limitations, recent advances in instrumentation and bioinformatics will have a dramatic impact on understanding reservoir host responses to hantaviruses by employing a systems biology approach to identify important pathways that mediate virus/reservoir relationships. hantaviruses (family bunyaviridae, genus hantavirus) are negative-stranded, trisegmented viruses that cause about , disease cases annually, with case fatality rates of . %- %, depending on the virus [ ] . the viral gene segments encode four or five polypeptides. the large (l) segment encodes the rna-dependent rna polymerase (rdrp), the medium (m) segment encodes a precursor that is posttranslationally cleaved into gn and gc glycoproteins, and the small (s) segment encodes the nucleocapsid (n) protein. some hantaviruses encode a second nonstructural polypeptide (nss) downstream from the n start site in frame [ , ] . little is known about the immunomodulatory abilities of these proteins; however, there is evidence the n, gn and nss may alter host cellular responses during infection. more than hantavirus species have been classified, and many more unclassified hantaviruses have been identified. they are hosted by several species of rodents (rodentia), insectivores (insectivora), and bats (chiroptera) [ ] . little work has been conducted to understand hantavirus infections of insectivores or bats; however, much is known about the ecology of rodent-borne hantaviruses because of their impact on human health (table ) . a central problem of hantavirus/reservoir host research is the lack of reagents and methods for experimentally examining the immune response. recent experimental work on the immunology of rodent reservoirs, summarized in an exceptional review by easterbrook and klein [ ] , has begun to clarify these issues. the immune response is energetically expensive for wild animals, thus the findings of experimental studies will be critical for understanding the ecoimmunology of reservoir hosts of hantaviruses [ , ] , and experiments using wild rodents in natural or semi-natural environments [ , ] will be required to validate laboratory findings. rodentia is the largest mammalian order and is comprised of about , species [ ] . only a few dozen species have been identified as susceptible hosts, and most hantaviruses are hosted by a single rodent species [ ] . in each hantavirus/rodent reservoir relationship, infection results in two prominent features: no conspicuous pathology and persistent infection [ ] [ ] [ ] . the earliest report of experimental infection of a reservoir host with its hantavirus was by lee et al. [ ] that described infection of striped field mice (apodemus agrarius) with hantaan virus. inoculated mice developed chronic infection with transient viremia, and shed virus principally in urine, saliva and, to a lesser extent, feces, despite the production of neutralizing antibodies. currently, three laboratory infection systems have been developed to study hantavirus infections of reservoir hosts: seoul virus (seov) infection of the norway rat (rattus norvegicus), puumala virus (puuv) infection of the bank vole (myodes glareolus), and sin nombre virus (snv) infection of the deer mouse (peromyscus maniculatus) [ , , ] . in humans, these viruses are etiologic agents of hemorrhagic fever with renal syndrome (hfrs; seov, puuv) and hantavirus cardiopulmonary syndrome (hcps; snv) [ ]. these diseases share many pathologic similarities and because little damage to the endothelium occurs during infection, it is thought that the inflammatory immune response contributes to pathogenesis [ , ] . because the norway rat is a model organism with many specific reagents, including monoclonal antibodies to immune markers, significant progress has been made toward understanding the reservoir host immune response to seov [ ] [ ] [ ] . fewer methods and reagents are available for bank voles and deer mice. despite these limitations, emerging technologies will be useful for understanding why rodent reservoirs are infected without disease and why they are unable to clear infection. a significant limitation of hfrs research is that none of the hfrs-causing hantaviruses cause disease in animal models. however, two new world hantaviruses cause an hcps-like disease in syrian golden hamsters (mesocricetus auratus): andes virus (andv) and maporal virus (mapv) [ , ] . andv causes most hcps cases in south america; however, no human cases of disease have been associated with mapv. andv is hosted by the long-tailed pygmy rice rat (oligoryzomys longicaudatus) and mapv is hosted by the delicate pygmy rice rat (oligoryzomys delicatus) [ , ] . andv is considered an animal biosafety level- pathogen in most nations, whereas mapv is considered an absl- virus [ ] . the natural route of transmission among reservoir rodents is thought to be principally through aerosols and/or biting [ ] . however, experiments have been equivocal in clarifying transmission mechanisms. weanling bank voles caged with infected individuals lead to transmission as early as days post exposure [ ] . similar experiments examining snv transmission between deer mice have been less informative; however, in artificial enclosure experiments, transmission appeared to be facilitated by deer mice with higher amounts of viremia and wounding [ ] and males likely play a dominant role in transmission in natural populations [ , , ] . in bank voles, offspring of puuv-infected dams were less likely to be infected after exiting the nest because of protective maternal antibody [ ] . although it is thought horizontal transmission is most important, work by hutchinson et al. [ ] showed that vertical transmission occurred among cotton rats (sigmodon hispidus) infected with black creek canal virus, so it is possible that both routes may influence transmission at the population level. the route of transmission has important ramifications in terms of the host immune response where, presumably, a mucosal response occurs with aerosol transmission and a localized response at a bite site. experimental data have also shown that patterns of the expression of genes related to the immune response are different in infected males and females [ ] , and it is likely these differences have important roles in hantavirus ecology. spillover to other rodent species also occurs [ ] [ ] [ ] [ ] , but it is unknown if the rodents remain infected. recent work has shown that deer mice are experimentally susceptible to andv; however, virus is cleared several weeks after infection [ ] . a pronounced th /tfh gene expression profile occurs, including il- pathway activation, that does not appear to be substantially activated in snv-infected deer mice [ , ] . this system provides an opportunity to identify viral and reservoir host factors that are important for sterilizing immunity that clears infection. the principal target cells of infection in rodents (and humans) are the microvasculature endothelial cells of many tissues [ ] . experimental intramuscular infection of deer mice with snv resulted in detectable virus in the lungs as few as two days later [ ] . many organs appeared infected, although infection was limited to the vasculature within those tissues. two infectious outcomes occur in experimentally infected deer mice; a disseminated infection of three or more organs, or a restricted infection of the lungs and heart [ ] . the relevance of these two patterns to transmission efficiency is unknown. the levels of viral rna vary dramatically in infected deer mice, with most having modest to moderate levels of rna at the peak of infection. however, some deer mice have significantly greater amounts of viral rna, suggesting these deer mice may produce substantially more virus than others, and it is possible they transmit virus more efficiently (e.g., "supershedders") [ ] . this also occurs in semi-natural transmission experiments [ ] and suggests certain individuals may have a prominent role in population-level transmission of hantaviruses. most serological assays for detecting antibody responses in hantavirus reservoirs use virus neutralization, elisa or strip immunoblotting [ , , [ ] [ ] [ ] . while some of these assays are igg-specific, others use antiserum to whole igg, including the light chains. since light chains are shared by all immunoglobulins, these detection antibodies are not igg-specific. moreover, no assays are in place for detecting iga, which should be prominent in mucosal infections. igm assays have been problematic despite the availability of anti-igm capture antisera that are cross-reactive with igm from at least one hantavirus reservoir species [ ] . some immunoglobulins have isotypes with specific effector activities, such as complement fixation or antibody-dependent cell cytotoxicity. laboratory house mice have four igg isotypes; igg , igg a, igg b and igg . it is likely that reservoirs also have immunoglobulin isotypes with distinct effector functions and which might predominate during hantavirus infections. these reagent deficiencies are a current obstacle for assessing antibody responses in rodent reservoir hosts. despite these limitations, many field studies have been conducted examining antibody responses in natural and semi-natural hantavirus infections of rodent reservoirs [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in experimentally-infected deer mice, snv nucleocapsid-specific antibodies can be detected in serum as early as days post infection, and neutralizing antibody can be detected after three weeks post infection [ ] . similarly, experimentally-infected bank voles produce puuv-specific antibodies two to three weeks after inoculation [ ] and rats experimentally infected with seov also produce igg within two weeks post inoculation [ ] . the presence of igg in these naturally and experimentally infected reservoirs is an indicator of class switching and affinity maturation, events that are mediated by t cells [ ] . thus, rodents mount adaptive t cell/b cell immune responses to their reservoir hantaviruses; however, it appears to be inadequate for virus clearance. while inflammatory signatures are present [ , , ] , the magnitude of these signals appears to be modest relative to expression levels found in a syrian hamster pathology model of hcps [ ] . it is noteworthy that immunization of rodent reservoirs with homologous nucleocapsid antigen or plasmids encoding the antigen protects from subsequent challenge [ , ] , suggesting infection can be prevented in reservoir hosts. several hantavirus proteins have been implicated in modulation of the host cell's antiviral defenses ( table ). the gn glycoproteins of pathogenic new world hantaviruses and seov possess an immunoreceptor tyrosine activation motif (itam) in the cytoplasmic tail that binds to fyn tyrosine kinase, and the itam may also interact with lyn, syk, and zap- kinases found in lymphocytes [ , ] , although there is no evidence that lymphocytes are susceptible to hantaviruses. the itam may also promote polyubiquitination of the gn polypeptide to facilitate its degradation [ ] ; however, it is unclear how it impacts the host response to infection. presumably, the itam interferes with the antiviral response of an infected cell since the motif is cytoplasmic. the gn protein may also alter the rig-i pathway that leads to irf phosphorylation and subsequent ifnβ expression [ ] . the nucleocapsid may also antagonize the expression of ifnβ by binding to importin-α and interfering with nf-κb nuclear transport, which is required for ifnβ expression [ ] [ ] [ ] . additionally, both caspase and granzyme b are targets of the nucleocapsid of some hantaviruses [ ] and both are essential components of ctl-mediated apoptosis. the lack of damage to the endothelium of infected rodent reservoirs suggests this may be an important mechanism of preventing viral clearance. the nucleocapsid of andv, but not other hantaviruses, also inhibits autophosphorylation and activation of tbk , an enzyme that activates irf and nf-κb and induction of type i interferon gene expression [ ] . recently, putative nonstructural nss sequences have been identified in some hantaviruses [ , ] . this sequence is in an alternative reading frame of the nucleocapsid transcript. in other bunyaviruses, nss has anti-interferon activity [ ] [ ] [ ] ; however, its role in hantavirus infections is less well characterized. importantly, these studies have been conducted with cells from nonreservoir hosts where they, presumably, are not optimized for manipulating the immune response in a manner that benefits the virus but without host pathology. future studies should examine the roles of these proteins in cells from reservoir hosts. the presence of high-titer igg antibodies during hantavirus infections of reservoir hosts indicates both t cell and b cell responses occur because t cells induce class switching and affinity maturation of antibodies produced by antigen-specific b cells. in experimental infections of rats with seov and deer mice with snv, early infection results in subtle inflammatory signatures, but a regulatory t cell (treg) response predominates at persistence (figure ) [ , ] . treg responses are critical for suppressing inflammation [ , ] ; however, inflammation is a prominent feature of hantavirus disease in humans [ ] and hamsters [ , ] . in other viral diseases, the occurrence of a treg response is associated with persistent infection because these cells, while suppressing inflammation, also prevent virus clearance [ , ] . for reservoirs of hantaviruses, the treg response may limit inflammatory immunopathology to an otherwise innocuous infection, but it may also impair virus clearance. how this relationship evolved is unknown, but considering the presence of hantavirus proteins with immunomodulatory activities, it suggests the viruses may manipulate the host response to favor persistence; a treg response may prevent sterilizing immunity, thus allow virus to remain in a population. it may also explain the ecoimmunology and ecology of hantavirus infections of reservoir hosts, thus studies assessing the energetics of inflammatory and anti-inflammatory immune responses should be performed. much of the work examining reservoir responses to hantaviruses has been conducted in rats infected with seov and deer mice infected with snv or andv. using cdna arrays, klein et al. [ ] identified nearly genes that were differentially expressed in male and female rats infected with seov. many immune-associated transcription factors, proinflammatory, antiviral, t cell and ig family member genes were significantly higher in females, which may reduce transmission from females. in deer mice infected with snv, early expression signatures of a mixed th /th /treg response were present in virus-specific cd + t cells, including ifnγ, il , il and tgfβ, before transitioning to a treg-like response at persistence [ ] . the expression of immune response genes differed in the spleen, where signatures of inflammation occurred within two days, peaking by days to before subsiding, and lungs, where little immune gene expression occurred [ ] . some deer mice produced nucleocapsid-specific igg by day while others had igg around day or later, and neutralizing antibody was not detected until after weeks. . during acute infection, snv elicits a modest inflammatory response that initially limits, but does not clear, virus. within a few weeks, the response transitions to a regulatory response that may allow episodic recrudescence of virus that can be shed. in deer mice infected with snv, early expression signatures of a mixed th /th /treg response were present in virus-specific cd + t cells, including ifnγ, il , il and tgfβ, before transitioning to a treg-like response at persistence [ ] . the expression of immune response genes differed in the spleen, where signatures of inflammation occurred within two days, peaking by days to before subsiding, and lungs, where little immune gene expression occurred [ ] . some deer mice produced nucleocapsid-specific igg by day while others had igg around day or later, and neutralizing antibody was not detected until after weeks. assessment of cytotoxic t cell responses of reservoir hosts has not been reported. most assays that assess ctl functions require susceptible syngeneic target cells, which have been difficult to obtain with reservoir hosts. susceptible primary cell lines from reservoir hosts have been produced [ , ] , but these are typically obtained from embryonic fibroblasts, thus matching of mhc alleles for use in ctl assays is difficult. many zoonotic viruses antagonize the innate immune response in human cells, and their pathogenic potential often correlates with their abilities to inhibit the innate response in vitro ( [ ] [ ] [ ] [ ] for review). pathogenic hantaviruses inhibit antiviral responses despite high levels of replication, whereas nonpathogenic viruses are efficiently recognized and elicit innate responses that limit replication [ , ] . this antagonistic capacity must have evolved in the reservoir hosts of these hantaviruses because humans are dead-end hosts. to date, few studies have addressed the interactions between hantaviruses and their rodent hosts in vitro. this is partially due to the unavailability of cell lines, and the reagents and techniques to generate primary cell cultures from the various reservoirs of hantaviruses. the norway rat/seov system is the most tractable for studying virus/reservoir interactions. inoculation of rat lung-derived endothelial cells with seov resulted in virus replication, but little or no induction of cytokines or chemokines, suggesting seov can efficiently antagonize antiviral responses [ ] . despite this, endothelial cells increased their expression of the protein pd-l , which correlated with the ability of these cells to induce treg cell activity. in addition, antigen presenting cells isolated from norway rats and infected with seov in vitro were resistant to stimulation, suggesting that virus infection inhibits the normal signaling activities of these cells [ ] . thus antagonism of the innate immune response likely allows for viral replication, while at the same time promotes an anti-inflammatory response that limits immunopathology. similar studies have been performed using bank vole cells infected with puuv [ ] . embryonic fibroblasts inoculated with puuv did not express increased amounts of ifnβ or mx , although non-related viruses were able to induce up-regulation of these genes. this suggests, as with seov, puuv efficiently antagonizes host innate responses in its natural reservoir. the syrian golden hamster develops an hcps-like disease when infected with andv or mapv [ , ] . hamsters inoculated with andv mount an inflammatory response, as measured by elevated mrna encoding pro-inflammatory mediators, prior to succumbing to the disease [ ] . infection also results in the activation of the adaptive immune response, characterized by antigen-specific proliferation of t cells and the generation of virus-specific antibodies [ , ] . in contrast, snv, which is highly pathogenic in humans, replicates in hamsters, but does not cause disease and is cleared by the immune response [ ] . passaging of snv in hamsters results in a virus that is able to replicate efficiently and cause a persistent infection similar to what is seen in the rodent reservoir, yet still does not cause disease [ ] . examination of immune responses elicited by andv (pathogenic in hamsters) and snv (non-pathogenic in hamsters) showed that passaged snv evoked a stronger adaptive immune response than did andv; however, andv infection induced a much stronger innate immune response at late time points, despite both viruses replicating to similar levels. depletion of t cells did not alter the outcome of infection [ , ] ; thus, these data suggest that, at least in the hamster model, the activation of the t cell-mediated immune response is not responsible for immunopathogenesis, and perhaps the innate immune response, either elicited from infected endothelial cells, macrophages and/or neutrophils, might contribute to disease. infectious diseases and the immune response are complex processes that are challenging to study, even with substantial reagent resources and mature methodologies. many difficulties exist for studying hantavirus infections of their reservoir hosts, including the lack of molecular and immunological reagents, methods for experimental investigation, and that few reservoir species have been colonized for laboratory use. in addition, pathogenic hantaviruses require bsl- and/or absl- containment, which presents logistical hurdles for examining the reservoir host/hantavirus relationship. despite these limitations, novel instrumentation, particularly those for transcriptome profiling (e.g., rna-seq) and metabolomics, and development of molecular and cellular methods, provide an opportunity to rapidly develop the tools necessary for examining reservoir host responses using a systems biology approach. the key feature of these new technologies is that they are species-independent in the data they generate, but they require significant computational and bioinformatics resources. infection triggers a cascading host response that is highly orchestrated by the vertebrate immune system. many genes are modulated (expressed or repressed) during the course of infection and identification of mrna and noncoding rnas can be used to identify the mechanisms that control, or fail to control, disease. moreover, some infectious diseases, including hantavirus disease, have substantial immunopathologic components. the quantitative assessment of the transcriptional landscape (patterns of gene expression) can be used to profile the host responses in infected and uninfected animals of the same species, or a disease model species to reservoir host species to identify mechanisms of susceptibility or resistance. rna-seq is one such method for profiling transcriptional landscapes [ ] . the depth of coverage and costs of rna-seq have improved dramatically in the last few years, and it is likely to become less expensive. however, the computational resources necessary for using rna-seq for studying host responses is substantial, often requiring hundreds of gigabytes of ram and multicore, multiprocessor systems typically found in servers running a linux operating system [ ] . this depth is often necessary to detect rnas that occur in extremely low abundance because their proteins are highly potent (e.g., cytokines). even then, it is possible that differentially expressed genes may not be detected and other, more sensitive methods, such as real-time pcr, may be required to validate pathway signatures. despite these requirements, bioinformatics tools for rna-seq are now widely available, many of which are free. a typical first step of differential gene expression profiling is the de novo assembly of all rna-seq samples from an experiment, which represents the totality of expressed genes from the experiment. there are several de novo assemblers available, including the trinity suite [ ] and oases [ ] . each of these packages has advantages and disadvantages, thus it is important to understand how each performs assemblies, particularly isoforms that may have specific activities. included in the trinity package is rsem [ ] that estimates transcript abundances, including isoforms, in experimental samples by counting reads from replicates against the de novo assembly. an important feature of rsem is that it does not require an annotated genome; it determines the abundance of transcripts from the unannotated assembly, identifies differentially expressed transcripts, and provides a % credibility interval for each gene. additional tools, such as deseq and edger [ , ] provide statistical evaluation of differential gene expression between samples and provide quantitative (higher/lower) and qualitative (on/off) data. the differentially expressed transcripts are subsequently identified by other means (e.g., blast) and can then be mapped to pathways (such as reactome or kegg) [ , ] to visualize [ ] where viruses may influence the host response and identify mechanistic targets of hantaviruses. in recent years, micrornas (mirna) have been identified that are important regulators of antiviral responses. activation of tlr/rig-i pathways leads to the expression of several mirnas [ ] that likely are important in lymphocyte functions [ ] . because hantavirus gn targets rig-i [ ] , it is possible that these mirnas are dysregulated, which could provide the virus an advantage over the host cell response. while mirna expression has been evaluated in hantavirus-infected human endothelial cells, epithelial cells and macrophages [ , ] , no work has been conducted to examine the role of mirnas in reservoir host cells infected with hantaviruses. considering the importance of mirna in host responses, it is likely they play an instrumental role in the immunological events leading to persistent infection of the reservoir hosts. the use of rna-seq can identify global mirna expression [ ] and clarify their regulatory roles in infected reservoirs. metabolic products can provide substantial information about the interactions of viruses and infected host cells, and the how immune system responds to infection [ , ] . the field of metabolomics is young but potentially informative for understanding hantavirus/reservoir host interactions. many viruses metabolically remodel the host cell to optimize infection. because metabolic products (e.g., carbohydrates, lipids, prostaglandins, etc.) are identical or highly similar between vertebrate species, this approach may help identify which enzymes, by virtue of their metabolic products, may be targeted by hantaviruses. while there are no reports of metabolic assessment of hantavirus infections, rift valley fever virus (rvfv) modulates the activity of adenosine ' monophosphate-activated protein kinase (ampk) in infected cells. this enzyme is a regulator of several metabolic pathways, including enhancement of catabolic pathways such as autophagy and atp production, but it represses anabolic pathways, such as lipid biosynthesis. infection of cells by several viruses, including rvfv, results in activation of ampk and restriction of viral replication, suggesting an antiviral role for this enzyme [ ] . other studies have revealed metabolic pathway targeting by viruses [ ] [ ] [ ] , thus efforts to examine how hantaviruses may manipulate the metabolomes of infected cells could lead to the identification of therapeutic targets for treating hantavirus disease. the use of monoclonal antibodies has been particularly challenging for hantavirus/reservoir research. identification of cell surface markers could shed light on what cells respond during hantavirus infection of reservoir hosts. while cell surface antigens tend to be more divergent, intracellular proteins, such as antiviral proteins, tend to be more conserved, particularly phosphoepitopes. thus, it is likely that many available antibodies to house mouse (mus musculus) or norway rat antiviral proteins will be cross-reactive with orthologs from hantavirus rodent reservoir species. without information as to which proteins may be of interest, screening of antibodies is a daunting and expensive task since most likely will not be informative. however, combined with rna-seq and metabolomic data, it is likely that target proteins and pathways can be identified so that investigators can focus their efforts and resources. in addition, software tools can help predict whether antibodies may be cross-reactive to proteins from reservoir hosts [ ] . the use of cytokines or neutralizing cytokine antibodies to perturb the host response during infection has identified many mechanisms that contribute to susceptibility or resistance. for example, administration of ifnγ to laboratory mice facilitates clearance of lcmv, whereas antibody that neutralizes ifnγ impairs ctl responses and clearance, thus leading to persistent infection [ , ] . depletion of immune cell subsets with antibodies can also determine the roles of those cells during infection. this approach revealed a critical role for cd + t cells for sustaining ctl responses to lcmv [ ] . other than the norway rat, an array of cytokines and antibodies for experimental manipulation of the host response of hantavirus reservoirs is substantially limited. thus, it is difficult to determine the mechanisms controlling the host response of reservoirs. some cytokines are broadly cross-reactive and can be used for studying reservoir responses. recombinant house mouse gm-csf and human il- stimulate deer mouse cells [ ] , and it is likely that other commercially-available cytokines can also be used. identification of cross-reactive cytokines should be a high priority of the hantavirus community. with genome and transcriptome sequencing, many cytokine genes can be rapidly cloned into expression vectors that are codon-optimized for the expression system of choice using de novo synthesis services (e.g., geneart, life technologies). moreover, some antibodies may be cross-reactive with reservoir species' orthologs [ ] . anti-mouse cd (clone gk . ) and anti-rat cd β (clone ) antibodies can be used to deplete cd + and cd + t cells from hamsters, respectively [ , ] , and they may also be useful for reservoir host studies to examine the roles of these cells. finally, it is difficult to assess ctl activity in reservoir hosts. most colonies are established with wild rodents that are highly polymorphic. this limits use of traditional ctl assays that require mhc class i-matched target cells. the generation of highly inbred strains is challenging and may result in alleles that do not represent the natural biology of hantavirus infection, thus it may not be desirable to generate highly inbred rodents. it is possible to establish mhc homozygotes with controlled breeding and screening of littermates for the same haplotypes. even then, the generation of susceptible cell lines can be problematic. while endothelial cell lines can be generated with retroviral transformation, it is possible the cells may have activated antiviral pathways that could alter in vitro ctl responses. the use of growth factors for expanding endothelial cells in culture may be more attractive. until methods are established for generating syngeneic, susceptible target cells, assessment of ctl responses in reservoir hosts will be difficult. the immunological relationships between hantaviruses and their rodent reservoir hosts are complex. infection typically leads to disseminated infection within a few days but without conspicuous signs of disease. expression of immune genes can be detected in as little as a few days that suggests innate and adaptive immune activation, but the magnitude of expression is substantially less than in hantavirus pathology models. the lack of reagents and methodologies for studying hantavirus reservoirs, most of which are not model organisms, presents a significant challenge. however, new technologies have recently emerged that are cost effective and species-independent, but which generate large amounts of data that require substantial computational and bioinformatics support for data reduction. with these tools, it should be possible to accelerate research to understand the relationships of hantaviruses and their reservoir hosts. work was provided by the department of microbiology, immunology and pathology, colorado state university (ts), nih grant ai (ts), and intramural program of niaid, nih (jp). t.s. and j.p. reviewed the literature and wrote the manuscript. the authors declare no conflict of interest. hantaviruses: a global disease problem tula and puumala hantavirus nss orfs are functional and the products inhibit activation of the interferon-beta promoter the andes hantavirus nss protein is expressed from the viral small mrna by a leaky scanning mechanism phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents immunological mechanisms mediating hantavirus persistence in rodent reservoirs long-term patterns of immune investment by wild deer mice infected with sin nombre virus refining 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serologic analysis of black creek canal virus and its association with human disease and sigmodon hispidus infection cocirculation of multiple hantaviruses in texas, with characterization of the small (s) genome of a previously undescribed virus of cotton rats (sigmodon hispidus) population, environmental, and community effects on local bank vole (myodes glareolus) puumala virus infection in an area with low human incidence. vector borne zoonotic dis social status does not predict responses to seoul virus infection or reproductive success among male norway rats t cells and follicular dendritic cells in germinal center b-cell formation and selection regulatory t cell-like responses in deer mice persistently infected with sin nombre virus pathogenesis and host response in syrian hamsters following intranasal infection with andes virus genetic vaccines protect against sin nombre hantavirus challenge in the deer mouse (peromyscus maniculatus) yeast-expressed puumala hantavirus nucleocapsid 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nucleocapsid protein gene tula hantavirus nss protein accumulates in the perinuclear area in infected and transfected cells la crosse bunyavirus nonstructural protein nss serves to suppress the type i interferon system of mammalian hosts nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase bunyamwera bunyavirus nonstructural protein nss counteracts the induction of alpha/beta interferon natural regulatory t cells in infectious disease regulatory t cells in virus infections hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease t cells are not required for pathogenesis in the syrian hamster model of hantavirus pulmonary syndrome the role of virus-induced regulatory t cells in immunopathology regulatory cells and infectious agents: detentes cordiale and contraire differential expression of immunoregulatory genes in male and female norway rats following infection with seoul virus a model system for in vitro studies of bank vole borne viruses sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon nipah and hendra virus interactions with the innate immune system antiviral escape strategies developed by bunyaviruses pathogenic for humans the pathogenesis of influenza virus infections: the contributions of virus and host factors pathogenic and nonpathogenic hantaviruses differentially regulate endothelial cell responses differential antiviral response of endothelial cells after infection with pathogenic and nonpathogenic hantaviruses seoul virus suppresses nf-kappab-mediated inflammatory responses of antigen presenting cells from norway rats the adaptive immune response does not influence hantavirus disease or persistence in the syrian hamster temporal analysis of andes virus and sin nombre virus infection of syrian hamsters hamster-adapted sin nombre virus causes disseminated infection and efficiently replicates in pulmonary endothelial cells without signs of disease field guide to next-generation dna sequencers de novo transcript sequence reconstruction from rna-seq using the trinity platform for reference generation and analysis oases: robust de novo rna-seq assembly across the dynamic range of expression levels accurate transcript quantification from rna-seq data with or without a reference genome differential expression analysis for sequence count data a bioconductor package for differential expression analysis of digital gene expression data the reactome pathway knowledgebase kegg: kyoto encyclopedia of genes and genomes cytoscape: a software environment for integrated models of biomolecular interaction networks micrornas in the regulation of tlr and rig-i pathways potential roles for short rnas in lymphocytes andes virus regulation of cellular micrornas contributes to hantavirus-induced endothelial cell permeability hantaviruses induce cell type-and viral species-specific host microrna expression signatures computational analysis of noncoding rnas lipids at the interface of virus-host interactions multifaceted roles for lipids in viral infection amp-activated kinase restricts rift valley fever virus infection by inhibiting fatty acid synthesis dengue virus infection perturbs lipid homeostasis in infected mosquito cells metabolic effects of influenza virus infection in cultured animal cells: intra-and extracellular metabolite profiling viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection modulation by gamma interferon of antiviral cell-mediated immune responses in vivo mechanism of recovery from acute virus infection. viii. treatment of lymphocytic choriomeningitis virus-infected mice with anti-interferon-gamma monoclonal antibody blocks generation of virus-specific cytotoxic t lymphocytes and virus elimination mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that lyt- + t lymphocytes mediate clearance of virus and regulate the antiviral antibody response generation of competent bone marrow-derived antigen presenting cells from the deer mouse (peromyscus maniculatus) discrimination of peromyscus maniculatus leukocytes by flow cytometry the authors thank brian hjelle, charles h. calisher, rushika perera and heinz feldmann for helpful comments, support and advice leading to the preparation of this manuscript. support for this key: cord- - m bsrw authors: shaw, alan r.; feinberg, mark b. title: vaccines date: - - journal: clinical immunology doi: . /b - - - - . - sha: doc_id: cord_uid: m bsrw nan vaccines represent one of the most effective and cost-effective medical and public health achievements of all time. worldwide, vaccination programs are currently estimated to save over million lives each year. in addition to having such a major beneficial impact on vaccine-preventable disease morbidity and mortality, the direct and indirect impacts of vaccination programs translate into economic savings of many billions of dollars each year. in what is considered to be one of the most significant medical successes of all time, a collaborative and comprehensive vaccination campaign against smallpox resulted in the global eradication of the disease in . similarly, efforts to eradicate poliomyelitis have made tremendous progress in reducing the global disease burden, and will hopefully soon overcome certain residual societal and programmatic obstacles to provide the second successful example of elimination of a major health threat by vaccination. concerted global efforts to provide measles vaccine have resulted in the control and elimination of the disease in many countries, including substantial reductions in mortality in a number of developing countries where the residual disease burden is greatest. these and other examples provide clear evidence of the power of vaccines in favorably manipulating host immunity to confer dramatic public health benefits, at both the individual and population level. as vaccines are administered to healthy individuals (often to entire age cohorts or populations), to prevent diseases caused by infectious agents to which they might be exposed in the future, they differ in important ways from pharmacologic agents that are used to treat individuals in whom a disease process is already manifest (or who display predispositions to disease). for this reason, vaccines are unique in the way that they impact on societies and in the way that societal commitment to vaccination determines their ultimate impact. as a result, vaccination efforts provide an informative window on challenges that need to be successfully navigated at the interface between scientific opportunity and societal capacity and commitment. indeed, current limitations in realizing the full global potential of available vaccines relate more to existing inadequacies in health care financing and infrastructure (especially as they are manifest in developing countries), and the relative value that societies place on disease prevention, than they do to any inherent biological limitations of vaccines themselves. fortunately, recent acceleration of new vaccine introductions in developing countries through public and private initiatives to build immunization infrastructure and provide funding of vaccine purchase offers hope that vaccines will one day be equitably available to all who need them. the importance of vaccines extends beyond their use as public health tools to include their role as drivers of immunologic discovery. the history of vaccine development is rich with immunologic insights that emerged from careful observations of how diseases spread in populations and how such spread differs in disease-naïve and experienced populations, as well as of how innovative experimental approaches revealed fundamental aspects of immune system function. the general concept of immunity induced by prior exposure to a disease (including its specificity and potential lifelong duration) was appreciated by the ancient greeks. use of the word 'immunity' itself dates to the th century when it was applied to describe the relative susceptibility and resistance of populations to plague. the subsequent successes of edward jenner and louis pasteur in the development of effective smallpox and fowl cholera immunization strategies, respectively, provided a foundation for modern immunology; pasteur himself coined the term 'vaccine' in recognition of jenner's use of vaccinia virus. jenner's smallpox immunization studies also provided early experimental support for the concept of immune memory. pasteur's efforts provided the first demonstration of the attenuation of pathogens by their propagation in culture (or by passage in nonnatural animal hosts), while robert koch demonstrated that killed pathogens could also engender immunity. the discovery of bacterial exotoxins by emile roux and alexandre yersin facilitated the discovery of antibodies and their potential use in passive immunotherapy with antitoxin antibodies by emil von behring and shibasaburo kitasato. these discoveries enabled the development of active immunization against diphtheria and tetanus using toxin-antitoxin mixtures. paul ehrlich's development of accurate methods for antibody quantitation made passive immunotherapy and active toxin-antitoxin immunization far more reliable and effective, and provided a stimulus for significant advances in immunologic theory. in each of these instances, vaccine development illuminated central mechanisms of immune system biology. vaccine development today has transitioned from an approach that was once largely empirical to one that is based on the hypothesis-driven application of techniques in molecular biology and immunology. evidence for this synergy can be seen in recent studies of vaccine-elicited immune responses to illuminate primary and memory t-and b-cell responses in humans, as well as the strong discovery stimulus provided by ongoing efforts to develop new vaccines for major infectious diseases for which vaccines are not currently available. vaccine development today faces a number of significant challenges. there exist tremendous public health needs to address major well-known pandemic diseases, including acquired immunodeficiency syndrome (aids), tuberculosis, and malaria, for which no vaccines currently exist and for which natural immunity does not provide a helpful guide for vaccine development. furthermore, there exists a need to confront effectively newly emerging and re-emerging diseases, ranging from the well-known, but constantly changing, threats from influenza pandemics to the appearance of previously unknown zoonotic infections such as the coronavirus that causes severe acute respiratory syndrome (sars). with changes in population density, mobility, and social constructs, along with alterations in the global climate, ecological circumstances, and the proximity of humans to animal reservoirs for previously confined infectious agents, the concept of new infectious agents entering human populations and spreading rapidly around the world is no longer novel. in confronting prevalent or newly emerging diseases, vaccines are looked to as the most promising line of defense. however, the speed at which new infectious disease threats have been shown to emerge and spread, and the fact that the pathogens that now need to be confronted may display tremendous genetic variability (e.g., human immunodeficiency virus (hiv)) or an identity that cannot be predicted in advance (e.g., avian influenza or agents like sars) places unprecedented demands on the vaccine development process. in addition to these new challenges, there remain unmet needs in the derivation of vaccines that can achieve the greatest public health benefit. these needs include the development of new ways to achieve more effective vaccine-elicited immune responses in neonates whose immune systems are immature (or are impacted by maternal antibodies) (chapter ) and in the elderly whose immune system function may be waning as a result of immune senescence (chapter ). fortunately, the scientific foundation provided by basic and applied immunology and the use of new methods for pathogen identification, antigen discovery, vaccine production, adjuvant development, and novel vector derivation afford important opportunities for vaccine development and additionally present the possibility of improving on natural immunity. success in vaccine development will be predicated on continuing the historical synergy between advances in vaccine technology and basic immunologic discovery. toward that end, this chapter focuses on preventive vaccines for infectious diseases and how they are developed. although current routine vaccine recommendations are reviewed, given the active state of new vaccine introduction and evolving vaccine recommendations, as well as differences in recommendations in different countries, readers are encouraged to refer to up-to-date national resources for the most current information. while vaccine approaches are being actively explored to modify beneficially malignant and immunologic diseases (autoimmunity and allergy), these are beyond the scope of the current discussion. n impact of vaccination programs n unlike other medical interventions, vaccines confer benefits to both individuals and populations. , while individuals may be protected from infection or disease by vaccine-induced immune responses, decreasing the number of susceptible hosts in a population also helps break the chain of transmission that pathogens require to spread and persist in human populations by induction of 'herd immunity. ' the benefits of herd immunity depend on achieving sufficiently high immunization rates in a population to impact pathogen transmission dynamics (including the potential for extinction of ongoing interhost transmission). the requisite level of vaccination coverage of a population needed to compromise pathogen spread significantly varies between pathogens, and is influenced both by vaccine efficacy (and its duration) and by the reproductive characteristics and infectiousness of the pathogen. analysis of the impact of vaccination programs in the usa provides an example of the beneficial impact of vaccines when used routinely and when high coverage levels are achieved. as shown in tables . and . , vaccination programs in the usa dramatically decreased the annual morbidity of many vaccine-preventable diseases. in many instances, the disease burden from several vaccine-preventable diseases of childhood has been reduced by over % since vaccine introduction (e.g., diphtheria, tetanus, measles, mumps, rubella, and polio). the somewhat lower rate of decline of pertussis (the annual morbidity of which has been reduced by a nonetheless impressive %) relates to the limited duration of vaccine-induced immunity, which is estimated to wane within - years after childhood vaccination. it is anticipated that recent availability of pertussis booster vaccines for use in adolescents and adults will lead to significant further declines in pertussis morbidity. even for diseases targeted by vaccines that have been in widespread use for less time (< years), impressive decreases in disease morbidity have been seen (e.g., varicella, hepatitis a, and pneumococcal disease). in a notable recent demonstration of the population benefits of vaccines, introduction of the -valent pneumococcal conjugate vaccine resulted in a decrease of % in disease morbidity in children under years of age within the first years of its introduction. interestingly, the rate of meningitis and bloodstream infections caused by antibiotic-resistant streptococcus pneumoniae also fell by % in this age group. in a striking related finding illustrating how vaccines can impact pathogen transmission dynamics, rates of antibiotic-resistant pneumococcal infections also declined by % in individuals over the age of who had not received the vaccine. thus, direct protection by vaccination of children who represent a reservoir of infection provided, via herd immunity, significant indirect benefits to those who did not themselves receive the vaccine. in addition to their benefits in preventing disease morbidity and mortality, routine vaccination programs are also impressively cost-effective. evaluation in the usa of the impact of ten vaccines routinely given as part of the childhood immunization schedule (diphtheria, tetanus, pertussis, haemophilus influenzae b (hib), polio; measles, mumps, rubella, hepatitis b and varicella) found that more than million cases of disease and more than deaths were averted over the lifetime of the immunized birth cohort of children. when the cost of the vaccination program was compared to the economic impact of diseases prevented, these vaccines alone are estimated to save nearly $ billion each year. when including indirect economic benefits (such as the time parents take off from work to care for sick children), the annual savings to society exceed $ billion. when preventive services were ranked based on clinically preventable disease burden and cost-effectiveness, childhood immunization received the highest score. progress in the development of new vaccines accelerated significantly towards the end of the th century, with the development of vaccines against diseases that were not previously preventable by vaccination, but also with the development of improved versions of existing vaccines. thus, the number of diseases that can be prevented by vaccines included in the us centers for disease control and prevention's (cdc) routine childhood and adolescent immunization schedules grew from seven in to in (table . and fig. . ) . moreover, in the past several years, new vaccines have been introduced for adolescents and young adults (e.g., pertussis booster (tdap), meningococcal conjugate, and human papillomavirus (hpv) vaccines), and older adults (e.g., tdap and zoster vaccines) have shown that the value of vaccines extends across the human lifespan (figs . and . ). new combination vaccines have been developed to increase the simplicity and acceptability of vaccination regimens, as well as to improve overall compliance with the recommended series of vaccines. such combinations include either those that contain multiple inactivated or recombinant antigens (such as a combination diphtheria, pertussis, tetanus, hib, and hepatitis b vaccine) or multiple live attenuated viruses (such as a combination measles, mumps, rubella, and varicella vaccine (mmrv)). the development of a combination vaccine is often more complicated than simply combining individual antigens, for when antigens are administered in combination, immunologic interference is sometimes seen. this necessitates titration of antigen combinations (and in the case of combinations of inactivated and/or recombinant antigens, adjuvant selection) to achieve immune responses that are not inferior to each of the antigens administered individually. despite their readily demonstrable public health impact, the value of vaccines is often not appreciated, for when vaccine programs are successful the diseases that they cause become less prevalent and may disappear. however, to prevent resurgence of an infectious disease that has been brought under control, vaccination programs need to be continued. the difficulties facing current efforts to eradicate poliomyelitis have demonstrated that failure to maintain high immunization coverage rates can lead to prompt re-emergence and spread of the disease. even in developed countries, maintenance of strong immunization programs with high degree of coverage is needed where infectious diseases can travel with remarkable speed -and do so even before the extent of spread is evident. n principles of immunization n the terms vaccination and immunization are often used interchangeably. however, vaccination specifically refers to efforts to induce protective immune responses by administration of a vaccine, whereas immunization more generically refers to interventions -either active or passive -that seek to confer immune protection. active immunization describes the induction of immune responses by administration of a specific antigen or antigens, while passive immunization involves the administration of exogenous immunologically active substances (historically, antibodies present in sera obtained from immune individuals or animals) to confer temporary protection from an infectious pathogen or toxin. although the approaches for passive immunization waned in the later half of the th century, the advent and increasing robustness of monoclonal antibody technology have led to a resurgence of interest in passive immunization. vaccines seek to engender immune responses similar to those that confer immunity to re-infection in individuals who experience (and survive) natural infection with a given pathogen. in lieu of formal demonstration of a specific type of antibody or cellular immune response that contributes to prevention or accelerated clearance of an infection, most often vaccine efficacy is demonstrated first in the course of a placebo-controlled trial. in some instances, specific immune effector mechanisms, such as a specific level or type of antibody response, can be identified that correlate with immune protection. in this case, the 'correlate of immunity' provides a benchmark against which similar vaccines can be compared. in the case of most inactivated vaccines, subunit vaccines, and recombinant vaccines that produce antibody responses, but generally meager cd t-cell responses, it is likely that humoral immune responses are the primary or sole protective immune mechanism. in the case of live attenuated vaccines that induce both cellular and humoral immune responses against the pathogen, it is likely that both arms of the immune system act in concert to confer immunity. however, the actual mechanisms of immune protection induced by either a natural infection or a vaccine are generally not understood in detail for many infectious diseases. similarly, although vaccines depend on the induction of immunologic memory, the magnitude, character, and duration of immune memory differ between vaccines, as can the actual mechanism of immune protection. for certain vaccines, such as those that protect against bacterial diseases induced via production of toxins (e.g., diphtheria or tetanus), protection induced by toxoid-based vaccines is clearly dependent on persistent antibody (igg) and memory b-cell responses, ensuring that sufficient antitoxin antibodies are present at the time of toxin exposure to inactivate and clear the toxin. in other cases, such as long-lived protection against hepatitis b, if sufficient levels of antibodies are achieved in the initial immunization period, even hosts who may with time lose detectable levels of antibody responses remain protected. in this instance, given the relatively long incubation period of hepatitis b, memory antiviral b-cell responses induced by the vaccine can be activated, facilitating neutralization and clearance of the infection before clinical disease is manifest. although it is popularly believed that vaccines confer protection by inducing 'sterilizing immunity' -wherein an infectious agent is blocked from even infecting one cell in an exposed host -this is clearly not the case for a number of vaccines. for example, the inactivated poliovirus and live attenuated rotavirus vaccines do not prevent some degree of local replication of their pathogenic counterparts in the gastrointestinal tract of exposed hosts. however, they are both effective in preventing clinical disease. in the case of poliovirus vaccine, this is mediated by elicitation of antibody responses that block dissemination of the infection to the central nervous system; while in the case of rotavirus, as yet unidentified immune effectors limit local virus replication so that significant gastrointestinal damage does not occur following infection. , the major types of vaccines licensed for use include live attenuated organisms, killed or inactivated organisms, subunit vaccines consisting of purified (or partially purified) components of an organism, and subunit vaccines produced by recombinant dna technologies. the use of live attenuated vaccines dates back to the early work of jenner and pasteur on smallpox and fowl cholera vaccines, respectively. , the fundamental concept of live attenuated vaccines is to mimic the effective host immune responses that follow natural infections. most live attenuated vaccines currently in use were derived by propagation of initially pathogenic organisms in culture on cells from different (nonhuman) species, or at nonphysiologic temperatures, for prolonged periods. driving pathogen evolution in culture to select for variants adapted to growth in heterologous cell types ex vivo often leads to the derivation of pathogen variants that grow poorly in vivo in humans and are unable to cause clinical symptoms. vaccines developed via this approach include those used to prevent a number of viral and bacterial infections, including yellow fever, measles, mumps, rubella, polio (the 'sabin vaccine'), varicella-zoster (used both for the prevention of chickenpox and shingles) and rotavirus (one version of the available vaccines), tuberculosis, and cholera. more recent technologies being applied to live attenuated vaccine development include the application of reverse genetic strategies ( fig. . ) and those involving genetic reassortment with attenuated viral variants, as have been used to develop polyvalent live attenuated vaccines against influenza and rotavirus ( fig. . ) . , the live attenuated vaccines currently in use are highly efficacious (> %) and protection is frequently durable. the efficacy of many live attenuated vaccines likely reflects the ability of the attenuated vaccine to replicate within vaccinated hosts, and to expose the immune system to pathogen-derived antigens in a manner that closely resembles the nature, location, and effects of natural infection. because live attenuated vaccines replicate within immunized individuals, they can induce both cellular (cd and cd ) and humoral (b-cell) effector responses and immunologic memory. in addition, as the live attenuated vaccines likely activate the host innate system in a manner similar to their pathogenic parents, they provide inherent adjuvant effects in augmenting adaptive immune responses. a key consideration in the development of any live attenuated vaccine relates to the relative balance between the ability to induce sufficient immune responses in vivo to confer protection (often associated with level of preserved replicative ability in vivo), and the ability to cause symptoms (which may also relate to the extent of in vivo replication). as such, an effective but also safe and well-tolerated vaccine needs to strike a specific balance between level of attenuation and level of immunogenicity. in addition, depending on the nature and number of genetic mutations responsible for the attenuated phenotype, a potential risk of reversion to a pathogenic form exists for certain vaccines. for most live attenuated vaccines, this has not been observed to be a problem in clinical practice -likely because the attenuating mutations are sufficiently numerous or genetically stable. one vaccine where reversion to pathogenic form was seen involved specific components of the live attenuated oral poliovirus vaccine (opv; the 'sabin vaccine'). in this instance, vaccine reversion to wild-type was shown to lead rarely to cases of paralytic polio (approximately one case per million doses administered). based on these observations and the elimination of endogenous polio transmission in many developed countries, the inactivated polio vaccine (ipv; the 'salk vaccine') was substituted for opv. however, in light of a favorable cost-benefit ratio, high degree of efficacy, and ease of administration, opv continues to be the mainstay of polio vaccination efforts in developing countries. the use of physical or chemical methods to kill or otherwise inactivate a pathogenic organism represents a second major approach to vaccine production. , in most cases, treatment with chemical agents such as β-propiolactone and formaldehyde is used to eliminate pathogen infectivity. while this approach has the benefit of presenting most of a pathogen's antigenic repertoire to the immune system of the immunized host, it can only be used in instances where the inactivated pathogen does not possess constituents that would confer significant toxicity. vaccines based on killed pathogens are believed to exert their protective effects via elicitation of pathogen-neutralizing antibodies and the induction of memory b-cell responses (likely in concert with cd t-cell memory). however, because inactivated pathogens cannot accomplish de novo synthesis of pathogen-derived gene products in antigen-presenting cells (apcs), they do not typically induce cd t-cell responses (chapter ). in addition, killed vaccines are generally less immunogenic than live attenuated vaccines. as a result, they are commonly administered with an adjuvant (most often alum: see section on adjuvants, below) to augment their immunogenicity. a number of viral and bacterial vaccines currently in use are killed/inactivated vaccines, including whole-cell bordetella pertussis vaccine and the influenza virus, rabies virus, and hepatitis a virus vaccines. a number of bacteria produce toxins that represent the major pathogenic components responsible for disease in infected humans. examples include corynebacterium diphtheriae and clostridium tetani. detoxified versions of these toxins are referred to as 'toxoids,' and represent the purified components of vaccines preventing diphtheria and tetanus, respectively. toxoids have historically been produced by chemical inactivation of toxins, but more recently, genetic inactivation via targeted mutagenesis has been employed. the acellular pertussis vaccine is also a purified subunit vaccine composed of a defined set of protein constituents prepared from cultured bordetella pertussis. the mechanism of immune protection conferred by purified subunit vaccines is the antibody response elicited by vaccination. antibodies directed against the capsular polysaccharides present on encapsulated bacteria also confer protective immunity in a number of important instances by inducing antibodies that exert opsonophagocytic effects (promoting phagocytosis of antibody-coated bacteria) and, in some instances, bactericidal effects. initial successful vaccine efforts against streptococcus pneumoniae and neisseria meningitidis utilized purified preparations of capsular polysaccharides. although such purified polysaccharides can induce protective levels of antibody responses in adults, they are poorly immunogenic in children under years of age (as a function of the relative immaturity of their immune systems). in addition, t-independent antibody responses elicited by purified capsular polysaccharides are less durable than those that are produced in the presence of cd t-cell help. as a means of both augmenting antibody responses against polysaccharide antigens in young children and facilitating their persistence, the development of conjugate vaccines represented an important advance. in this approach, purified polysaccharides are chemically conjugated to a carrier protein (such as diphtheria toxoid or an outer-membrane protein complex (ompc) derived from n. meningitidis). the carrier protein augments cd t-cell helper responses to the polysaccharide antigens, and enables elicitation of durable protective antibody responses even in young children. polysaccharideconjugate vaccines have been produced that protect against haemophilus influenzae b, streptococcus pneumoniae, and n. meningitidis infections. if two such segmented viruses with different genetic characteristics are used to infect one cell, the progeny viruses from this mixed infection will carry a range of mixtures of the genes of the two parent viruses. using either genetic or immunologic screening methods, reassorted viruses carrying the precise gene composition of interest can be selected. this approach has recently been employed to generate live attenuated vaccines against rotavirus and influenza virus. the strategy for generation of the pentavalent bovine-human reassortment rotavirus vaccine is shown above. rotaviruses have a segmented double-stranded rna genome comprising independent rna elements. the outer shell of the virus comprises two proteins vp and vp that are involved in cell binding and entry and that specify the viral serotype (p type for vp and g type for vp ). vp and vp also represent the targets of virus-neutralizing antibodies. the pentavalent bovine-human rotavirus vaccine was generated by a 'modified jennerian' approach in which the bovine rotavirus wc (which is attenuated in humans as a result of host range restriction) serves as the gene donor for the backbone on to which gene segments encoding four common human rotavirus g types (g - ) as well as one very common p type (p ) (derived from individual rotavirus isolates) were reassorted via a process of cell co-infection and subsequent selection of the recombinant viruses with the desired composition of bovine and human gene segments. an analogous genetic reassortment approach has also been used to generate live attenuated influenza vaccines. in this instance, three attenuated 'cold-adapted' viral strains (two a types and one type b) are used in co-infections in tissue culture with recent circulating wild-type influenza strains to derive vaccine strains that include the two relevant hemagglutinin (ha) and neuraminidase (na)-encoding gene segments admixed with the six 'backbone' genes from the attenuated master donor virus for use in annual influenza vaccines. the first recombinant vaccine developed, the recombinant hepatitis b surface antigen (hbsag) prepared in yeast, was developed in hopes of avoiding safety concerns related to the plasma-derived hbsag vaccine. the knowledge that immune sera could provide protection by passive immunization of naïve hosts, and that purified inactivated plasmaderived hbsag vaccine could elicit protective antibodies, laid the groundwork for development of this recombinant vaccine. the recombinant vaccine, when combined with adjuvant (alum), elicits favorable immune responses, is highly efficacious and is well tolerated -all features that recombinant vaccines are now expected to deliver. the second recombinant vaccine developed targeted prevention of borrelia burgdorferi infection (the cause of lyme disease), and was based on a purified recombinant version of the ospa protein. this vaccine, although conferring some degree of efficacy, faced implementation challenges, and was not widely embraced. as a result, it was withdrawn from the market. more recently, recombinant technology-derived purified subunit vaccines have been developed that consist of virus-like particles (vlps) that self-assemble when the l protein of hpv is produced in isolation of other viral proteins ( fig. . ). the l protein is the target of virusneutralizing antibodies and vaccines consisting of a mixture of types and (the cause of ~ % of cases of cervical cancer) and and (the cause of ~ % of cases of genital warts) or of hpv types and alone have been shown to be highly efficacious and well tolerated. interestingly, hpv vlps induce antibody responses that exceed those that follow natural hpv infections. in light of these successes, and the power and versatility of recombinant antigen production methods, a major proportion of new vaccine development efforts involves the use of protein subunit vaccines produced by recombinant technologies. vaccines produced by this method are those that depend largely or exclusively on the induction of antibodies against individual or a selected subset of pathogen proteins. because a number of proteins produced in isolation by recombinant methods have been observed to elicit lower immune responses than do natural infections or live attenuated vaccines, the development and use of adjuvants to optimize recombinant vaccine immunogenicity represent an important parallel area for future exploration. n vaccine development and evaluation n as a necessary prelude to clinical evaluation of candidate vaccines in humans, extensive preclinical research and development activities are undertaken to establish that the vaccine candidate has the desired properties. toward this end, a number of key issues need to be addressed. first, animal studies must show that the vaccine candidate raises the desired type and magnitude of immune response against the infectious agent. second, the vaccine needs to protect animals against death or disease in an appropriate challenge model, when feasible. ideally, in the course of these studies, a specific type or level of immune response, referred to as a correlate of immune protection, can be identified. third, the vaccine should be relatively free of serious discernible toxicities and side effects in animals when administered by the route intended for humans. fourth, it is necessary to demonstrate that the vaccine can be produced in a consistent manner by a process that is consistent with the current good manufacturing practices (cgmp) process by which the first clinical trial materials will be produced (www.fda.gov/cber/gdlns/indcgmp.pdf ). bioengineered l proteins ( ) l pentamer self-assembled virus-like particle in specific instances, vlps can be produced via a process of self-assembly of individual viral capsid proteins produced by recombinant dna methods in cell culture systems. this approach has a number of attractive aspects, including the ability to produce vlps that accurately display conformationally correct epitopes recognized by neutralizing antibodies and the absence of pathogen-derived nucleic acids. in addition, recombinant vlps have been employed to derive safe and effective vaccines for pathogens, such as hepatitis b virus (hbv) and human papillomavirus (hpv), that cannot be grown in culture (and are thus refractory to standard vaccine approaches of attenuation or inactivation). the generation of the vlps that comprises newly developed hpv vaccines is shown. the hpv l proteins (which represent the major capsid protein and target of virus-neutralizing, protective antibodies), derived from hpv types of interest (e.g., types , , , and ) are produced via recombinant methods. under appropriate conditions, individual bioengineered l proteins first self-assemble into pentamers, and then into vlps that are comprised of pentamers and that are almost identical, both morphologically and antigenically, to infectious hpv virus particles. vlps prepared from individual hpv types are then combined with specific adjuvants to prepare the final vaccine products. even before preclinical studies are completed, vaccine developers typically begin an initial dialog with regulatory authorities (such as the food and drug administration (fda) or the european medicines agency (emea)) to set expectations about what will be necessary and sufficient for advancement to clinical studies in humans (www.fda.gov/cber/ genetherapy/isct sh.pdf ). phase i studies primarily focus on detailed assessment of the safety and tolerability of a vaccine, but evaluation of its immunogenicity is also frequently conducted. generally, a phase i study includes fewer than healthy volunteers divided unequally between those who receive vaccine or placebo ( or vaccinees per placebo recipient). phase i studies typically employ escalating doses of the candidate vaccine, with a dose range progressively increasing in steps of three-to fivefold often being used. blood samples are taken at prescribed intervals and analyzed for laboratory evidence of potential toxicity, as well as for evidence of vaccine-elicited immune responses. a phase i study is considered successful if it demonstrates that the candidate vaccine is well tolerated or identifies any immediate safety concerns that will need to be closely monitored in potential future clinical studies. ideally, phase i studies also provide an initial indication of the optimal dose level and number of doses required. a phase ii study typically includes several hundred to a few thousand volunteers (randomized between vaccine and placebo) and can assume two general design types. phase iia studies provide additional safety data on a larger number of individuals of the intended age who receive the intended vaccine dose (who are more representative of the general population intended for vaccine use than the very healthy individuals included in the phase i study), as well as provide additional data on vaccine immunogenicity. even larger phase iib studies can provide additional data on vaccine safety and immunogenicity in subjects generally representative of those for whom the vaccine might be recommended, but importantly, also provide the first opportunity to address to answer the question, 'does this vaccine work in humans?' the size of a phase iib study needed to detect a signal of vaccine efficacy depends on the attack rate of the infection being targeted by the vaccine. the development of new vaccines depends on the convergence of public health need, biological plausibility, and practical feasibility. vaccine development programs are influenced by multiple considerations, including: >> what are the major unmet medical and public health needs today? >> what is known about the natural history and pathogenic mechanisms of the infection of interest? >> is immunity to a given antigen associated with protection against disease following re-exposure in the context of natural infection? >> if natural immunity capable of preventing re-infection follows an initial infection with the pathogen, can a specific host immune effector mechanism (e.g., antibody, cd t-cell) be identified as the likely agent (or 'correlate') of immune protection? if so, can a threshold level of this specific immune correlate needed for protection from re-infection be defined? >> can the pathogen be grown in culture? if so, does the pathogen cause such a life-threatening disease that an attenuated version of the virus would face an impossible barrier for demonstration of safety? >> can a specific antigen (or antigens) be identified that represents the target of protective host immune responses? >> if the protective immune response is mediated by antibodies, can the target antigen (be it a protein or polysaccharide) be produced in scalable quantities in a form that mimics its native structure so that it can effectively elicit antibody responses that can block the key functional role(s) of the target molecule in the pathogen lifecycle or otherwise lead to the clearance of an incipient pathogen infection? >> having chosen an antigen and presentation system, what is the best way to produce it on a large scale? choices will be limited by the nature of the antigen and delivery system, but definition of an optimal system for producing the vaccine (prokaryotes like escherichia coli, or diverse eukaryotic hosts including yeast, insect cells, plants, or cultured plant cells, mammalian cells) is a central consideration. >> what is the most effective way to present the antigens of the pathogen of interest to the immune system? modern molecular biology and biochemistry have provided numerous options for vaccine immunogen presentation, including recombinant proteins (and recombinant virus-like particles (vlps)), synthetic proteins, protein-polysaccharide conjugates, and gene delivery systems (recombinant viral vectors, or dna vaccines) >> is the antigen of interest sufficiently immunogenic on its own, or is augmentation of the desired immune response by conjugation to a specific carrier or addition of an adjuvant necessary to elicit a sufficient and sufficiently durable immune response in individuals in the target population for vaccination? >> what types of potential safety concerns can be anticipated for the vaccine in question? >> what is the attack rate of the infection in the general population? if the infection occurs relatively rarely in an overall population, can a subset of the population be identified that has a higher risk of infection so as to accelerate the achievement of statistically significant protection? is this subset sufficiently similar to the rest of the population to enable extrapolation of the clinical results to the broader target population as a whole? >> what tests to evaluate vaccine immunogenicity will need to carried out on clinical samples obtained from participants in the clinical trials? will measurement of antibody titers, t-cell responses, pathogen presence and quantity, pathogen serotype, and any other parameter peculiar to the disease in question represent the primary criteria for vaccine effect? development and validation of theses tests represent an essential component for the feasibility and success of a vaccine clinical study phase ii studies also present the first opportunity to identify a potential laboratory immunological correlate of protection from disease -if nature and prior experience have not already done so. in order to do so, the placebo recipients in the phase ii trial must experience a sufficient number of cases of disease while vaccine recipients need to exhibit significant evidence of decreased risk of infection or disease. in addition, immunological measurements in the vaccinees need to capture the relevant protective immune responses (e.g., the type and level of antibody and/or cellular immune response that predict protection) and measure them with sufficient precision and reliability. if laboratory measurements of immunity correlate with vaccine protection, subsequent refinements of the vaccine, its adjuvant, its manufacturing process, or its regimen may be assessed by simple immunogenicity studies, rather than repeating efficacy studies. once efficacy is established for a vaccine, it is very difficult to carry out a double-blinded, placebo-controlled efficacy study. vaccines that have been shown to be immunogenic and well tolerated in phase ii studies can then advance to pivotal phase iii studies required for vaccine licensure by regulatory authorities. phase iii studies are intended to expand further the safety database in a larger number of individuals (who are representative of the specific populations for which the vaccine will ultimately be used), establish definitive evidence of protective efficacy, and to establish clinical consistency of the vaccine made by the process run in the facility intended for licensure and commercialization (www.fda.gov/cber/genetherapy/ isct jcr.htm). typically, phase iii studies include or more subjects in a blinded, placebo-controlled design. this size trial allows the identification of less frequent safety events. it also provides an opportunity to capture data on health care utilization, cost, and impact of the vaccine on these parameters. as a new vaccine will ultimately be included in a vaccine program where multiple vaccines may be administered at the same time, it is also necessary to conduct concomitantuse studies. the developer of the new vaccine must show that the new vaccine does not impact on the immunogenicity of the existing vaccines, and that the existing vaccines do not impact on the immunogenicity of the new vaccine. in contrast to drugs, where licensure by the fda is the primary determinant of how a new product is implemented in medical practice, vaccine use in the usa includes an additional process that evaluates how best to employ a new vaccine to optimize its implementation and public health impact. the us cdc has responsibility for making recommendations about the use of licensed vaccines, and it relies on its advisory committee on immunization practices (acip) for guidance. the acip considers several aspects in addition to a vaccine's safety and efficacy, including the anticipated costeffectiveness and practical feasibility of potential alternative vaccine deployment strategies and consideration of how a new vaccine may be successfully implemented in clinical practice to achieve the greatest public health impact. once the cdc has received, reviewed, and accepted the recommendation of the acip, the recommendation is published in its final official form in morbidity and mortality weekly report (mmwr; www.cdc.gov/nip/publications/acip-list.htm). the recommended immunization schedule for children and adolescents ( fig. . ) is updated on an annual basis and can be accessed at www.cdc. gov/nip/recs/child-schedule.htm. the recommended adult immunization schedule (fig. . ) is also updated on an annual basis and can be accessed at www.cdc.gov/nip/recs/adult-schedule.htm. the recommended adult immunization schedule includes information concerning use in special populations (such as health care workers and pregnant women) and individuals with specific conditions associated with altered or impaired immune function (such as individuals with congenital and acquired immunodeficiency syndromes, recipients of immunosuppressive therapies, malignancies, asplenia, liver disease, and renal disease). readers are encouraged to check to ensure that they are following current recommendations. pregnancy registries currently exist for four vaccines in the usa. health care professionals are encouraged to report exposures of pregnant women to the appropriate registry: hbv vaccine ( - - ), hpv vaccine ( - - ), meningococcal vaccine ( - - ), and varicella vaccine ( - - ). n vaccine safety n unlike drugs that are utilized to treat individuals suffering from a given disease state, vaccines are administered to normal, healthy infants, adolescents, and adults. consequently, standards for the safety and tolerability of vaccines are set at a very high level. when developing a new vaccine, a graded process of clinical studies is employed that involves increasingly larger numbers of volunteers and that typically progresses from individuals who are selected to be free of any identifiable health problems to those who are selected to be representative of the overall population for whom the vaccine is being developed. if phase i studies reveal no evidence of safety concerns and the desired evidence of immunogenicity, a major focus of the series of larger randomized double-blind, phase ii placebo-controlled studies that are then conducted is to explore the safety and tolerability of a vaccine in increasingly vulnerable populations (such as those who may have identified pre-existing health problems or asymptomatic abnormalities detected on screening laboratory studies). reflecting the importance of documenting the safety of a new vaccine, phase iii studies to assess the safety and efficacy of a new vaccine now typically involve large numbers of volunteers. indeed, as a result of needing to provide evidence for safety, it is now common to have the size of the phase iii trial be significantly larger than would be necessary to document vaccine efficacy. the ability of a study to identify an increased risk of any given adverse event with sufficient statistical power is directly related to the size of the population in the study. as a general rule, a study of - subjects is needed to measure the risk of an event that happens in one out of individuals. for one in , - subjects are needed. even in studies of this size, very rare events may not be identified, and if a specific safety concern exists substantially larger trials may be needed. the recent experience with the development of rotavirus vaccines provides an illustrative example of the importance placed on documenting vaccine safety. rotavirus is an important cause of serious gastroenteritis in infants and young children, and the associated diarrhea and vomiting can lead to life-threatening dehydration. in developing countries where health care resources and effective rehydration options are limited, over infants die of rotavirus gastroenteritis each year. given the global importance of rotavirus gastroenteritis, the first licensure of an orally administered rotavirus vaccine in was a very welcome advance. however, as the vaccine entered routine pediatric practice, it was recognized that a low, but increased incidence of intestinal intussusception was seen after the first and second doses (with about one case of intussusception seen per vaccinees.) upon recognition of this association, the vaccine was withdrawn from the market. with the evident public health need for a safe and effective rotavirus vaccine, it was hoped that alternative rotavirus vaccines then in development (both oral vaccines based either on a combination of bovine-human reassortant viruses (fig. . ) or an attenuated human rotavirus strain) might differ from the first licensed rotavirus vaccine and not result in an increased rate of intussusception. however, to demonstrate that these alternative rotavirus vaccines were safe, and that an increased risk of intussusception was not inherent to rotavirus vaccines as a class, very large-scale safety studies were required. toward this end, the safety of each of these vaccines was evaluated in studies involving about infants -just to evaluate whether the rate of intussusception in vaccinees was discernibly increased compared to the normal background rates seen in the placebo recipients. , fortunately, both vaccines were found to be well tolerated and no increase in intussusception was observed in vaccine as compared to placebo recipients. in light of the documented efficacy of these vaccines determined in earlier and significantly smaller phase iii trials, both have now been licensed in a number of countries. however, even with the large phase iii studies conducted for these newer rotavirus vaccines, they will still be studied in large postlicensure active surveillance safety studies and closely monitored in active and passive vaccine safety surveillance systems (see below). following vaccine licensure, safety is tracked via a number of means, including both active and passive surveillance studies of adverse events. active surveillance includes phase iv postmarketing studies of vaccine safety in larger populations in real-world use. formal postmarketing studies can include tens of thousands of individuals or more. an alternative type of postmarketing safety study is carried out by the us fda and the cdc within the context of the vaccine adverse event reporting system (vaers) database (www.vaers.hhs.gov or by telephone: - - ). the vaers database accepts spontaneous reports of adverse experiences from health care providers, patients, parents, vaccine manufacturers, and other sources. the best use of the vaers database is to identify signals in a population that may appear following the introduction of a new vaccine. a newer vaccine safety surveillance system, known as the vaccine safety database (vsd), has been developed by the cdc in cooperation with seven large health maintenance organizations (hmos) around the usa. the vsd contains the complete medical records of all the members from the participating hmos, and the information used to populate the database is entered by health care professionals using relatively consistent terminology, improving the quality, uniformity, and usefulness of the data. particularly important is that the vsd construct allows comprehensive epidemiological analyses to determine if the incidence rate of a specific adverse event is higher among vaccinees than nonvaccinees. in addition to vaers and the vsd, the cdc has also created a clinical immunization safety assessment network that reviews patterns of clinical syndromes that may follow vaccination. while the safety profile of a vaccine can be relatively well defined through the efforts described above, confidence in vaccination programs has often been challenged by public perceptions, either real or unsubstantiated, about vaccine safety. in some instances, specific vaccines have been associated with increased incidence of a specific adverse experience, such as the association between the first-generation rotavirus vaccine and an increased risk of intussusception following vaccination. however, a number of other safety concerns that have emerged are not supported by scientific evidence. an example of this can be found in the case of concerns about the association of whole-cell pertussis vaccines with permanent brain damage -concerns that were later shown to be unfounded. nevertheless, public concerns about the safety of the whole-cell pertussis vaccine resulted in decreased levels of pertussis vaccination coverage that were soon followed by epidemics of whooping cough in the uk and japan. another example is the allegation that certain vaccines, such as the combination measles, mumps, rubella (mmr) vaccine, are associated with autism. highlighting how perceptions of temporal association can give rise to public concerns, mmr vaccines are generally given around year of age, and autism is generally diagnosed in the second year of life. although the alleged causal association between mmr and autism has been refuted by thorough scientific analyses, reports in the popular media in the uk resulted in a dramatic drop in vaccination rates, followed by an increased rate of new infections. , n vaccines not yet available n although an impressive armamentarium of vaccines is now available, safe and effective vaccines have yet to be developed for a number of very important infectious diseases. the reasons underlying the lack of effective vaccines for an array of important pathogens include biological considerations, safety concerns, and practical constraints. of these, the biological considerations are often the most important barrier. as discussed above, vaccines have been successfully developed for pathogens whose natural infections give rise to natural immunity wherein the infected host (at least those who survive initial infection) is no longer susceptible to re-infection (such as measles, yellow fever virus, or smallpox) or who experiences significantly less severe clinical sequelae upon re-infection (such as rotavirus). in instances where natural immunity follows natural infection, not only is a precedent for immune protection established, but the nature of protective host responses can be studied, providing a correlate of protection to guide vaccine development efforts. however, for many of the pathogens for which vaccines remain elusive, natural immunity does not follow natural infection. in the absence of natural immunity, not only is a precedent for successful immune containment lacking, but no potential correlates of protection are available to inform vaccine development. in some instances where natural immunity does not follow natural infection, persistent infections are established and maintained by active virus replication that cannot be controlled or cleared by host immune responses (such as hiv and hepatitis c). alternatively, other pathogens are able to persist in the host through establishment, via diverse mechanisms, of latent infections that are resistant to host immune clearance (such as tuberculosis or herpes viruses (such as herpes simplex virus (hsv) or epstein-barr virus (ebv))). in other instances, even when the host is cleared of an infection via drug vaccines not yet available treatment, the host remains susceptible to re-infection and disease in the future (such as malaria). although different pathogens have evolved diverse strategies for evasion of host immune responses -ranging from manifestation of tremendous genetic diversity and propensity for immune escape; to sequestration of critical structural domains that might be susceptible to antibody neutralization; to the utilization of specific mechanisms to evade host innate and adaptive immune effectors -the common end result is frustration of vaccine development. while failure of host clearance of an infection is a common theme underlying the lack of vaccines, additional obstacles to vaccine development include other immunologically related considerations as well as both practical and safety considerations. examples of immunologically related obstacles include instances where prior exposure to a given pathogen predisposes the host to more severe disease manifestations upon re-infection (as has been proposed in the case of dengue virus) or where earlier vaccine development efforts inadvertently lead to severe adverse events following infection with the targeted pathogen (such as respiratory syncytial virus (rsv )). in each of these cases, the adverse events that follow a secondary immune exposure are believed to be the result of immunopathologic responses that result from the nature of the immune response elicited by the initial exposure to pathogen-derived antigens (by either infection or vaccination). given that the mechanisms underlying these immunopathologic processes are incompletely understood, the development of vaccines that are highly immunogenic but not similarly inclined to elicit immunemediated adverse consequences represents a substantial challenge (especially given the very high expectations for vaccine safety). an additional immunologically related challenge relates to the observation that certain organisms encode antigens that resemble constituents of the human host. for example, in the case of neisseria meningitidis group b, the bacterial polysaccharide resembles those found on certain human cell lineages, thus raising concerns about whether polysaccharide-based vaccines successfully developed for group b n. meningitidis might yield undesirable autoimmune responses. an additional distinct, but important, practical barrier to new vaccine development relates to the prevention of diseases that are threats to pregnant women or their offspring (where immunization of the pregnant woman might be able to protect the neonate). although a number of inactivated vaccines are either routinely recommended for use in pregnant women (e.g., inactivated influenza vaccine) or can be used in pregnant women for pre-or postexposure prophylaxis for those at risk of infection (e.g., inactivated hepatitis a vaccine and recombinant hbsag vaccine), the development of new vaccines specifically for use in pregnant women or the study of new vaccines in pregnant women has been impeded by concerns arising from potential litigation that might follow the appearance of a congenital abnormality in a child born to a mother who was vaccinated while pregnant. given the - % prevalence of congenital abnormalities, the practical difficulties in proving the safety of a new vaccine specifically administered to pregnant women, and the current litigious environment surrounding vaccines, the development of new vaccines to address important infections of pregnant women and their neonates (e.g., group b streptococcus: gbs) faces significant challenges. there remain a number of important infectious diseases for which no effective preventive vaccines exist. below, we list the major 'missing' vaccines, comment on why they are not yet available, and highlight the major approaches currently being explored to develop them. at the end of , an estimated million people were living with hiv infection, and in the preceding year approximately . million people became newly infected, and approximately million individuals died of aids. as the most promising biomedical intervention to contain the aids pandemic, the development of an hiv vaccine is a top global health priority. yet, hiv infection represents a vexing challenge to vaccine development. , hiv infection does not result in clearance of the virus due to a host immune response. following infection of target cells, the genome of hiv -a retrovirus -is transcribed into a dna copy via the action of reverse transcriptase. the newly formed dna copy of the hiv genome then integrates into the host cell chromosomes (referred to as a provirus) as a requisite step in the viral lifecycle. once integrated into the chromosome of an infected cell, the hiv provirus can alternatively be actively transcribed, leading to the synthesis of viral mrnas and subsequently to production of new virus particles, or it can remain in a transcriptionally silent, functionally latent state in a small percentage of infected cells. as infected cells harboring latent hiv proviruses do not produce hiv protein antigens, they cannot be recognized by host antiviral immune responses and can thereby persist undetected. upon subsequent activation of latently infected cells at some later time, viral rna transcription can be coincidently activated leading to production of progeny virions. as hiv targets activated cd t cells for infection and consequent depletion, the host's ability to mount both hiv-specific and non-hivspecific immune responses is progressively impaired. the ability of the host to clear hiv infection is further complicated by the extensive genetic diversity of virus populations that emerge, and progressively diverge, within infected individuals as a function of a replicative cycle that is accomplished by the inherently error-prone reverse transcriptase and the numerous cycles of replication that occur in infected individuals. as a result of these influences, genetically diverse populations of hiv variants are established in infected persons that facilitate the outgrowth of genetic variants that can escape from selective pressures -be they effective host cellular or humoral immune responses, or the inhibitory effects of antiretroviral drugs. an extraordinary degree of genetic diversity is also manifest in the hiv variants seen in different individuals and in different geographic regions. as successful vaccines for other infectious agents have historically had to protect against pathogens exhibiting only limited genetic diversity, hiv represents an unprecedented challenge. as many successful vaccines protecting against viral infections are predicated on the induction of neutralizing antibody responses against the viral surface proteins that mediate attachment to and entry into target cells, significant efforts have focused on the potential of the hiv surface envelope (env) glycoprotein, gp , to elicit infectionneutralizing antibodies. unfortunately, hiv gp is highly resistant to the action of antibodies by virtue of its heavy glycosylation and its native conformation that shields functionally critical structural domains from antibody binding. as a result, candidate gp -based vaccines have failed to elicit meaningful levels of neutralizing antibodies in immunized human volunteers and have not protected from hiv infection in two large phase iii studies. given the inability, to date, of candidate hiv env-based vaccines to elicit appreciable levels of neutralizing antibodies, current vaccine strategies are largely focused on the induction of cd cytotoxic t-cell responses against the more constrained and conserved antigens, such as vaccines not yet available gag, pol, and nef. it has been hypothesized that induction of high levels of hiv-specific cd t-cell responses prior to infection may not prevent infection, but may enable infected individuals to control virus replication better. should this hypothesis be valid, individuals immunized with such vaccines may exhibit lower levels of ongoing hiv replication, progress to aids more slowly, and potentially be less likely to transmit hiv infection to others. much of this work involves vectored gene delivery systems (such as adenoviral vectors, described below). however, the recently announced results of a phase iib 'test of concept study' failed to demonstrate a beneficial effect on either prevention of infection or reduction of viral load among volunteers who received the vaccine despite the induction of appreciable levels of hiv-specific ctl responses by the recombinant adenovirus-based vaccine employed. while this study result does not, in and of itself, refute the 'ctl hypothesis', it represents a significant disappointment for the aids vaccine research effort, and raises important questions about the ability of vaccine-elicited cell mediated immune responses to favorably alter the outcome of hiv infection. a there are also efforts under way to utilize the recently solved three-dimensional structure of the hiv env glycoprotein to guide the derivation of nonnative structures that might serve as better immunogens to elicit broadly cross-reactive neutralizing antibodies. in addition, relatively conserved and functionally essential sequences of the extracellular domain of the hiv transmembrane env protein, gp , are being explored as immunogens to elicit broadly neutralizing antibodies. malaria is the world's most common vector-borne disease -estimated to cause approximately million clinical cases and million deaths annually. , the disease hits hardest in africa, and is especially severe in children under years of age. in addition to direct morbidity and mortality, malaria is responsible for debilitating illness with enormous social and economic consequences. of the four malaria-associated protozoal species, plasmodium flaciparum and p. vivax represent the two major agents. these parasites have a three-stage lifecycle taking place both within the mosquito, and in the liver and blood of the infected host, and each cycle is largely distinct from the others from an immunological perspective. as a result of the multiple strategies for evasion of host immune response that the parasite has evolved, parasite replication proceeds at high levels despite active host immune responses. , either as a result of these specific immune evasion strategies or the inability of the infected human host to mount immune responses that clear the parasite, prior infection does not protect an individual from repeated subsequent infections. although the severity of disease is often attenuated following repeated infection, the mechanism of disease modulation is incompletely understood, and the limited relative immunity engendered by prior infection is easily lost if an individual leaves a malaria-endemic region. as such, the limited impact and duration of host immune responses to malaria parasites suggest that any successful vaccine strategy will need to do far better than natural immune responses -a high bar for efforts to develop an effective vaccine. roughly two dozen antigens have been cloned and tested as potential vaccine immunogens, and with a few exceptions the results have been disappointing. one antigen, the circumsporozoite antigen, presented as a fusion with hbsag (rts,s), has shown modest promise in human studies. this vaccine is now undergoing larger-scale clinical efficacy testing to determine if the magnitude of protection would justify largescale implementation efforts. should 'proof of concept' be supported in these studies, but the absolute magnitude of efficacy be insufficient, future efforts will likely focus on the identification of an appropriate adjuvant to improve the magnitude and duration of immune responses. alternative approaches include immunization with irradiated or genetically attenuated sporozoites. definition of novel antigens expressed at specific stages of the parasite lifecycle, and evaluation of combinations of multiple parasite antigens. mycobacterium tuberculosis is an intracellular mycobacterial pathogen that represents one of the world's most common and most serious infectious diseases. over billion people are believed to harbor latent m. tuberculosis infections, and approximately million active cases of tuberculosis and over million deaths occur each year. furthermore, the interface of hiv infection and its attendant immune system damage both increases the severity of m. tuberculosis infection and increases the infectiousness of infected individuals. the emergence and dissemination of m. tuberculosis isolates that are resistant to multiple antimicrobial drugs represent a growing public health threat. however, while the need for a vaccine to prevent tuberculosis is clear, a significant number of challenges face vaccine development efforts. most individuals infected with m. tuberculosis can control the acute phase of mycobacterial replication, and mount vigorous innate and adaptive immune responses to the infection. however, the infection is often not cleared by the host's immune response, and the mycobacteria are able to persist and multiply within vacuoles inside macrophages. longterm latency is established in fibrotic cysts in the lung. the recrudescence and dissemination of m. tuberculosis occur at a later time in a number of infected individuals, likely as a result of waning host immune control. although the ability of m. tuberculosis to persist despite active innate and adaptive immune responses represents a major challenge to vaccine development, the fact that most individuals can contain (if not clear) m. tuberculosis infection suggests that a vaccine that can alter the course of the natural infection by limiting early dissemination and decreasing the risk of later recrudescence could provide major public health benefits. efforts to develop a vaccine against tuberculosis date back many decades. bacille calmette-guérin (better known as bcg), based on mycobacterium bovis, was first introduced in . currently, bcg is provided as a component of the routine expanded programme for immunization (epi) schedule and is administered to a significant majority of the world's children. although some protective efficacy ( - %) has been reported against miliary infection and m. tuberculosis meningitis in children, conflicting results have been obtained in different studies regarding the ability of bcg to protect against pulmonary tuberculosis in adults. one explanation for the overall limited efficacy of bcg emerges from formal genome sequencing studies that have disclosed significant differences between m. tuberculosis and of the vaccine strain of bcg. the variability in the results of bcg efficacy studies in different populations and geographies may derive from variations in the geographic prevalence of cross-reactive mycobacterial species (that may themselves confer partial protection), or the fact that bcg vaccines used throughout the world do not represent a homogenous preparation -with the root strain of bcg having been widely distributed and passaged extensively under diverse conditions. vaccine efforts against tuberculosis have primarily focused on the evaluation of specific mycobacterial antigens (e.g., esat , ag , and hsp ) that have been tested as vaccines in animal models with variable success. , some of these strategies are now being advanced into human clinical trials. an alternative strategy is based on improving the performance of the bcg vaccine by insertion of genes encoding specific potential protective antigens that it normally lacks. in addition, the development of auxotrophic mutants of m. tuberculosis is being explored as a potential immunogenic and specifically attenuated live vaccine. the determination of the sequence of the m. tuberculosis genome nearly a decade ago helped identify numerous previously unknown gene products, and increased the repertoire of antigens to be evaluated for their ability to induce protective immune responses. the pathogen sequence is also being used to elucidate virulence determinants and thereby help guide efforts to attenuate m. tuberculosis rationally. together with influenza virus, rsv and piv account for a substantial majority of pediatric upper respiratory illness and consequent acute otitis media. a variety of influenza vaccines are licensed for pediatric use, but vaccines to prevent infection with the paromyxoviruses rsv and piv remain elusive. a significant impediment to vaccine development for rsv and piv traces back to unanticipated untoward results obtained in clinical studies of inactivated rsv vaccines in the early s. these early-generation rsv vaccines -based on cultured virus that had been inactivated with formalin -raised a potent antibody response in immunized children. however, on subsequent natural exposure to rsv, vaccine recipients exhibited more frequent and significantly more severe lower respiratory tract rsv infections than did unimmunized children. as a similar phenomenon was also seen with a formalin-inactivated measles vaccine in the same era, a common immunopathologic mechanism may be operative. while the mechanism of exacerbation of rsv disease by the early inactivated vaccines is incompletely understood, it has been suggested that chemical inactivation of rsv and measles resulted in modification of a critical neutralizing structure on the surfaces of these viruses, thereby limiting the induction of the most potent neutralizing antibodies and favoring nonneutralizing and potentially immunopathologic antibody responses. (passive protection against rsv is available for premature infants in the form of monoclonal antibodies that target the rsv f protein (one of the viral envelope glycoproteins); certain anti-rsv antibody responses can clearly mediate protective as opposed to deleterious effects. ) alternatively, or in addition, it has been proposed that inactivated rsv vaccines may have preferentially induced a th -type immune response when a th -type response may be needed to effect protection of the lower respiratory tract from rsv infection and damage. while excellent live attenuated measles vaccines have been developed, rsv and piv have so far resisted the approach used for measles and mumps (these are all members of the paramyxoviridae family of viruses). based on the successful precedent provided by the live attenuated measles vaccine, an attenuated or reverse genetics-engineered rsv is considered the most promising approach. however, stable attenuation of rsv has been difficult to achieve and vaccine safety concerns result in their cautious advancement through clinical evaluation. effective vaccines for meningococcus types a, c, y, and w are available as straight capsular polysaccharides and as conjugated polysaccharides. the group b polysaccharide shares chemical similarity with a shorter sugar found on the surface of neuronal tissue. while it is possible to make highly immunogenic conjugates with the group b polysaccharide, theoretical concerns about cross-reactivity with self antigens has impeded the development of this type of vaccine. current work centers on a handful of relatively well-conserved surface proteins of meningococcus. gbs is a common component of the flora of the female genital tract, and transfer to the neonate is the cause of severe infections that are fatal or have serious sequelae. short-course intrapartum antibiotics are recommended for culture-positive women, and this approach has cut the incidence of neonatal infections by about two-thirds, thus reducing somewhat the urgency of vaccine development. however, short-course antibiotics could ultimately drive the emergence of antibiotic-resistant gbs. candidate vaccines have been shown to elicit a protective response. however, aside from a reduced market, the main impediment to development of a gbs vaccine is concern over vaccination of pregnant women or women of childbearing age. any birth defect might be attributed to the vaccine, and in a litiginous society, this would be problematic for a vaccine producer. prior to the advent of effective polymerase chain reaction methods for screening blood donations, hcv was a significant cause of transfusionrelated hepatitis. currently, transmission of hcv among the normal population is quite low; transmission among injection drug users remains high. hcv is another pathogen where infection does not typically result in an immune response that clears the infection. however, a minority of hcv patients do spontaneously clear their infection, suggesting that an appropriate immune response could do the job. current vaccine work is concentrated on vectored gene delivery vaccines, primarily adenoviruses, intended to raise antiviral cytotoxic t-cell responses. with the exception of the live attenuated varicella-zoster virus (vzv) vaccine used for the primary prevention of chickenpox and reactivation of latent vzv infections (the cause of shingles and postherpetic neuralgia in older individuals), there are no other vaccines available for use in humans to prevent infection with members of the herpes virus family. hsv types and cause recurrent vesicular eruptions "above or below the belt," respectively. like other herpes viruses, hsv infections are not cleared by the immune system and the virus can persist, remaining in a latent state that is functionally inaccessible to immune recognition and clearance. in addition, like other herpes viruses, hsv encodes a number of gene products that promote evasion of host immune responses. recent attempts to make hsv vaccines have used virus glycoproteins produced by recombinant dna methods. a recent clinical efficacy trial of this vaccine approach showed partial protection of women, but not men, who were seronegative for hsv . the reasons for this curious result are not clear, but efforts to develop this type of vaccine continue. in addition, a number of preclinical studies are exploring the ability of cell-mediated immune responses to hsv antigens induced by recombinant vaccine vectors (e.g., adenoviruses: see novel vaccine vectors, below) to prevent or ameliorate hsv infections. genetically engineered attenuated hsv variants have also been studied in experimental animal models. it is not clear when these new strategies may advance to clinical evaluation in humans. another herpes virus, cmv is a very common infection in humans, with - % of individuals being infected by adulthood. cmv is a cause of severe infections in neonates, causing debilitating neurological sequelae. following initial infection, cmv persists in infected humans, despite the fact that anti-cmv antibodies are present and that a very sizeable proportion of the overall host cd and cd immune responses are specific for cmv antigens. ongoing virus persistence and replication in the face of active host immune responses are likely explained by cmv's sophisticated repertoire of host immune evasion functions (including those that inhibit antigen presentation mechanisms and immune effector responses). for these reasons, to be successful, vaccine development efforts will need to elicit immune responses that are significantly more effective than the quantitatively impressive, but functionally limited, immune responses that are generated in the course of natural cmv infections. live attenuated vaccines have been investigated sporadically since the s. an attenuated strain, the towne strain, showed some effect, but was judged to be insufficiently immunogenic. hybrids of the attenuated towne strain and the virulent toledo strain remain in development. recent work has included recombinant dna (rdna)-derived proteins (via either dna vaccine approaches or recombinant viral vectors, such as attenuated poxviral vectors). , ebv is a herpes virus that represents the causative agent of infectious mononucleosis and is widespread among the human population. in concert with incompletely understood environmental (and perhaps additional host) factors, ebv is also etiologically associated with burkitt's lymphoma. the ability of ebv to establish persistent infections in humans (along with latent infections at the cellular level) despite readily detectable antiviral immune responses suggests that, like other herpes viruses, the development of effective ebv vaccine will likely be challenging. ebv vaccines have been in development since the s with the coat protein, gp / , as the most common vaccine antigen studied. dengue fever virus is a mosquito-borne flavivirus (the virus family that includes japanese encephalitis virus and yellow fever virus -for which successful vaccines exist). dengue virus is endemic in a substantial portion of tropical and subtropical areas and causes febrile disease as well as hemorrhagic fever. there are four distinct serotypes of dengue fever virus. prior infection with one serotype has been implicated in predisposing for more severe disease following infection with a second dengue fever virus serotype, although the evidence supporting this concept has been questioned and the underlying pathogenic mechanisms are incompletely understood. the processes of vaccine development have changed significantly in recent years -a process facilitated by substantial improvements in understanding of human immune system function, as well as the advent of powerful new technologies for vaccine development. as a result of these advances, vaccine development is now commonly pursued in a hypothesis-driven manner and is a far less empiric pursuit than in the past. however, at the same time, the infectious diseases for which no effective vaccines currently exist represent more challenging targets than those diseases that have yielded to vaccine development efforts in the past. furthermore, global changes that influence the emergence and rate of spread of infectious diseases place unprecedented challenges on the productivity and pace of new vaccine development efforts. vaccines not yet available >> the challenge of responding rapidly and effectively, with powerful new technologies, to newly emerging infectionsincluding those that haven't been seen in humans before (e.g., severe acute respiratory syndrome (sars)) or for which novel antigenic variants are anticipated but cannot be predicted (e.g., pandemic influenza) >> maximizing the value of innovative new approaches while ensuring the safety of new vaccines so derived one hypothesis proposes that antibodies against the initial infecting serotype bind to the surface of virus particles of the novel infecting serotype, but do not neutralize the infection. in a process referred to as "immune enhancement" of infection, still infectious complexes of antibody virus particles are then envisioned to be preferentially taken up by cells of the reticuloendothelial system that represent primary target cells for virus replication. although the veracity of this hypothesis in not established, it does present certain theoretical concerns about what type of antibody responses will need to be induced by vaccines to exert beneficial rather than detrimental effects. the general belief is that a vaccine providing equivalent immunity against all four serotypes will be required. vaccines based on inactivated virus, engineered chimeric viruses based on the yellow fever virus vaccine platform, engineered deletion mutant viruses, and rdna-derived proteins are in various stages of development. n new antigen discovery methods n historically, vaccine antigens were not discovered in the literal sense. rather, whole organisms were inactivated by either heat or chemistry or organisms were attenuated by forcing growth in nonphysiological conditions. the entire antigenic repertoire of the organism was delivered to the immune system. the isolation of tetanus, diphtheria, and pertussis toxins, along with chemical detoxification schemes, allowed the production of more refined vaccines. the isolation and purification of polysaccharide capsules from a range of important bacterial pathogens enabled the development of additional vaccines. with the advent of molecular biology in the late s, a new set of tools allowed a more directed approach for the discovery of pathogen virulence factors, vaccine antigen discovery, and vaccine development. the tools of molecular biology enabled for the first time the development of vaccines against pathogens that could not be propagated in culture, including the successful development of recombinant hbv and hpv vaccines. development of these vaccines was enabled by clinical and animal model studies showing that antibodies directed against a specific viral target antigen (e.g., the hbv surface antigen or the hpv l protein) were implicated in protection. in addition, molecular biologic approaches enabled the derivation of fully recombinant vaccine antigens (such as those developed using a limited set of defined antigens of bordetella pertussis), including genetically modified versions of bacterial toxins that maintain their proper antigenic structures but are no longer toxic. however, for many of the pathogens for which vaccines do not currently exist, application of these recombinant dna technology-enabled strategies are insufficient due to incomplete understanding of the pathogen antigens that would elicit a protective host immune response. as such, the development of additional techniques to discover protective antigens was needed. fortunately, several important technological advances that facilitate discovery of previously unknown protective antigens from even very complex microorganisms have opened a new era in vaccine development. the earliest rdna technology-enabled methods of antigen discovery involved the expression of individual pathogen-derived gene products (or fragments thereof ) in bacterial hosts (typically escherichia coli) using rdna expression vectors. here, the genome of a pathogen is broken up, and the fragments are inserted into a plasmid or a viral vector, typically a lambda bacteriophage. colonies, or plaques, are spread on a membrane, allowed to grow, and hopefully express the cloned gene fragments. the ability of these recombinant gene products (now isolated in individual colonies) to react with antibodies present in the serum of individuals who had recovered from infection with that pathogen could then be directly assessed. antibodies present in the immune sera are assessed for their ability to identify antigens by immunochemical reactivity. in this way, the entire genome of a given pathogen could be scanned for potential immunoreactivity. such reactivity would both indicate the in vivo expression of that gene product, as well as document its antigenicity. however, additional studies are needed to demonstrate whether antibody responses against a newly defined antigen have any protective potential. to document the ability of an antigen to elicit protective immune responses, it is necessary to immunize an experimental animal (most commonly, mice) and then, following experimental pathogen challenge, evaluate infection outcomes in immunized versus nonimmunized animals. as this approach has most often been used to identify antigens recognized by host humoral responses, sera from animals immunized with a candidate antigen can then be transferred to a naïve host to provide evidence that the antibody response to the antigen represents the relevant agent of immune protection. more recently, as dna sequencing became more efficient and scaleable, determining the entire sequence of the genomes of viruses, bacteria, and parasites has become routine, , allowing identification of previously unknown genes (and predicted gene products) that can be evaluated as vaccine immunogens. scanning the entire pathogen genome via specific computer analysis programs, genes that exhibit specific characteristics can be identified (e.g., predicted expression on the cell surface by virtue of possession of a leader sequence for secretion or membrane anchor sequences). in addition, the relative conservation of the gene within the pathogen population can be determined by assessment of gene sequences from multiple distinct isolates. once potential vaccine antigens are identified, each candidate gene is expressed in an appropriate rdna system, and the protein product is tested in an animal model. the first bacterial genome sequenced in its entirety was that of haemophilus influenzae, marking the beginning of a new approach to vaccine antigen discovery. since this initial bacterial genome sequence determination, genomic sequencing of pathogens has advanced exponentially. over bacterial genomes have now been sequenced, and hundreds more are currently in process. genome-based antigen discovery is being applied to a wide range of bacteria, including streptococci, pneumococci, staphylococci and chlamydia, as well as nonbacterial pathogens such as plasmodium falciparum. an alternate, promising approach to novel antigen discovery has been built on technological advances in proteomics. , these advances include development of high-resolution two-dimensional gel electrophoresis techniques and mass spectrometry methods that enable separation, identification, and purification of individual proteins from the complex mixture of proteins expressed by a pathogen. in proteomic analyses, a small culture of bacteria, preferably taken directly from an infected person, or otherwise grown in physiologically similar conditions, is subjected to physical or enzymatic treatment with specific proteases to generate peptide fragments that are then fractionated by a micro-high-performance liquid chromatography method and sequenced by molecular mass-by-mass spectrometry. an overlapping set of peptides of approximately - amino acids is sufficient to identify an antigen and provides the means to find the gene. although proteomic analysis is, in some ways, more involved than genomic analysis, it provides an important new approach to antigen identification, and offers a direct way to document that the specific protein identified is actually expressed by the pathogen (including, for example, demonstration that a protein of interest is expressed on the external surface of the pathogen). , a combination of proteomic and serologic methods to select potential novel vaccine immunogens, called serological proteome analysis, or serpa, , can be used to screen the pathogen proteome for expressed proteins that are recognized by antibodies present in sera obtained from individuals who have recovered from an infection with the pathogen. the proteomic approach to antigen discovery has been applied to identify novel vaccine candidates for a number of human pathogens, including helicobacter pylori, chlamydia pneumoniae, staphylococcus aureus, bacillus anthracis, haemophilus influenzae, and plasmodium falciparum. as in new genomic methods for antigen discovery, having identified a gene encoding a candidate antigen by proteomic methods, it is then necessary to show that an immune response of the desired type can be raised against the protein. in addition, it is necessary to show that immune responses elicited following immunization with the candidate antigen engender some degree of protection. often, pure protein antigens produced by recombinant methods are not very immunogenic. as such, many of the emerging recombinant vaccines so produced will likely require enhancement of their immunogenicity by means of an adjuvant. the term 'adjuvant' (derived from the latin adjuvare, to help) refers to any substance added as a component of a vaccine preparation -in addition to the vaccine antigens themselves -that improves the immunological response to the antigen. as such, 'adjuvant' is a catch-all term including a broad range of molecular entities that act via diverse -and, in a number of instances, yet to be elucidated -pathways. until recently, most adjuvants were derived empirically and the mechanisms by which they augmented immune responses were unknown . as a result, there were few, if any, principles available to guide the improvement of known adjuvants or the development of new ones. however, recent advances in understanding of the mechanisms by which dendritic cells sense the presence of pathogens and their constituents, and translate this information to shape the quantity, quality, and durability of host cellular and humoral adaptive immune responses, have transformed adjuvant discovery and optimization. what was once a process of trial and error now represents an area of hypothesis-driven research and mechanism-based discovery. a particularly promising advance emerged from the discovery that pathogen sensing by the innate immune system is mediated by recognition of specific pathogen-associated molecular patterns (pamps) by pathogen recognition receptors (prrs) such as the toll-like receptors (tlrs) that are expressed on dendritic cells (dcs) and other hematolymphoid and some epithelial cells (chapter ). the pathogen-derived pamps recognized by tlrs consist of structures that are found only in or on pathogens (including bacteria, viruses, and parasites) and are not part of normal vertebrate biology. following binding of a specific pamp to a specific prr, a specific cellular activation and response cascade is triggered that can directly confront an intruding pathogen and/or lead to the activation of specific host adaptive immune response mechanisms. these breakthroughs in basic immunology have been readily translated into what can now be considered the science of adjuvant biology. , such progress has occurred at an especially opportune time as new vaccine development strategies have transitioned from traditional approaches using attenuated or killed pathogens to highly defined and purified recombinant proteins (so-called "subunit" vaccines) or nonreplicating vectored antigens. although these newer approaches are promis-ing from the perspective of vaccine safety and the opportunity they afford to design the structures of vaccine immunogens, recombinant or synthetic vaccines are often inherently less immunogenic than traditional vaccines based on attenuated live viruses or intact killed organisms. in the context of current vaccine development and regulatory approval processes, an adjuvant is developed as part of a vaccine, not as an independent product. consequently, there are currently no adjuvants licensed by regulatory authorities as stand-alone products. most contemporary efforts to develop novel adjuvants are focused on the targeted activation of tlrs that are expressed on specific cells critical for the generation of innate and adaptive host responses to specific pathogens. a family consisting of distinct tlrs has been identified to date in humans (chapter ). tlrs are expressed in a number of innate immune cells, including dcs, macrophages, neutrophils, endothelial cells, and fibroblasts. given the importance of dcs as critical antigen-presenting cells, most studies of the biology of tlr signaling have focused on these cells. different tlrs are expressed on distinct subpopulations of dcs, and, depending on the tlr, in distinct cellular compartments. tlrs expressed on the surface of human myeloid dcs include tlr (which is heterodimerized with tlr or ), as well as tlrs , , , and ( fig. . ), while these same cells express tlrs and within endoplasmic reticulum (er) and phagolysosomes. plasmacytoid dcs (pdcs) express tlr and within er/phagolysosomes. the tlrs expressed on the cell surface are primarily activated by pamps encountered in the extracellular environment, while tlrs expressed in the er/phagolysosomes are activated by pamps (including viral pathogen-derived rna or dna) that tend to be routed through these endosomal compartments. activation of tlrs on innate immune cells leads to their production of specific cytokines, as well as their expression of co-stimulatory molecules, leading to induction of adaptive immune responses. given that different dcs express different tlrs, and that signaling via different tlrs results in the expression of a distinct pattern of cytokines, it is believed that activation of specific tlrs can variously favor the induction of th -or th -biased immune responses, or can differentially augment either direct or cross-presentation pathways for antigen presentation ( fig. . ). although most data on induction of specific types of immune responses by engagement of specific tlrs have emerged from murine studies (and have not yet been validated in humans), the ability to tailor an adjuvant preparation to achieve a desired type of immune response with a specific vaccine immunogen is a promising notion. naturally occurring ligands for tlrs include lipopolysaccharide (lps) from bacterial cell walls (recognized by tlr ), triacyl lipopeptides (recognized by tlrs + ), diacyl lipopeptides (recognized by tlrs + ), peptidoglycan (recognized by tlr ), flagellin (the monomer that makes up flagella, recognized by tlr ), singlestranded rna (recognized by tlr ), double-stranded rna (recognized by tlr ), and unmethylated dna containing the dinucleotide pair cpg (recognized by tlr ). based on these insights, a variety of approaches to develop adjuvants predicated to activation of specific tlr pathways are being actively pursued. one interesting aspect of adjuvant development is how it is revealing the mechanisms of action of adjuvants that were originally identified via a process of trial and error, as well as delineating important aspects by which certain empirically derived vaccines are able to induce high-level, long-lasting immune responses. one illustrative example can be found in the case of complete freund's adjuvant (cfa), which has long served as the benchmark for laboratory studies of adjuvants. cfa is a mixed emulsion of mineral oil, mannide monooleate, and killed mycobacteria. however, it is far too reactogenic for use in humans, causing significant pain and abcesses at the site of injection -reactions that would be exacerbated if cfa were to be used repeatedly. an alternative preparation termed incomplete freund's adjuvant (ifa) lacks the mycobacterial component, but it too is associated with injection site reactions that are severe enough to limit its use to experimental therapeutic cancer vaccines. although cfa's toxicity precludes its use as a vaccine adjuvant in humans, many of its constituents (including liposaccharides, dna, and specific bacterial cell wall components) are now understood to exert their adjuvant effects on vaccine-induced immune responses via engagement of specific tlrs. similarly, the live attenuated mycobacterium bovis strain, bcg, long widely employed as a vaccine for the prevention of tuberculosis, includes cell wall, peptidoglycan, and dna components that activate specific tlrs. interestingly, the highly effective yellow fever vaccine d has been shown to activate multiple tlrs as part of its induction of antiviral immune responses. it is quite likely that other live attenuated viruses that transiently replicate in immunized hosts also activate innate immune responses via engagement of tlrs. in yet another example involving a nonreplicating vaccine immunogen, one version of the hib polysaccharide conjugate vaccines now licensed for use in children for the prevention of invasive hib disease includes the meningococcal outer-membrane protein complex (ompc) as its protein carrier. ompc conjugates have favorable immunogenic properties that correlate with the ability of ompc to activate dcs via tlr . hundreds of different adjuvant formulations have been tested in animal models, and a few have been advanced into human studies. with a few α α ( ) toll-receptor agonists. alum, the classical adjuvant most often used in vaccines in humans, includes a range of salts of aluminum precipitated under basic conditions, usually aluminum sulfate mixed with sodium or potassium hydroxide plus a variable amount of phosphate. the relative proportions will determine the size, charge, and solubility of alum. the composition of alum used as an adjuvant varies in currently available vaccines and may influence vaccine immunogenicity. alum is utilized as an adjuvant in many of the currently available vaccines composed of inactivated toxins or recombinant proteins (live attenuated vaccines do not include alum or other adjuvants). alum serves two main purposes as an adjuvant. first, it acts as an antigen depot. vaccine antigens adsorb to alum and elute from it following injection into the host. second, alum acts a mild irritant, causing the recruitment of leukocytes necessary for generation of an immune response to the site of injection. adsorption of antigens on to alum routinely improves immunogenicity, particularly the antibody response. alum does not typically enhance cd t-cell responses. alum has been a component of many vaccines for decades and has an excellent safety record. as new adjuvants are developed, alum may remain as a component of combination adjuvant mixtures (as is the case with some newer adjuvants now approaching clinical use), or it may eventually be supplanted by other agents that more effectively provide favorable depot and local inflammatory responses to accentuate host immune responses. using lipids with polar head groups (e.g., triglycerides) and differing types of hydrophobic tails, one can form either micelles (spheres) or multilamellar sheets in aqueous environments. under the right conditions, antigens can be incorporated into the spheres or between layers of the sheets, providing a potential slow-release depot system. immunopotentiators such as qs or detoxified lps derivatives (such as monophosphoryl lipid a (mpl)) may be added to the lipid mix. iscoms are a proprietary form of liposomes made of cholesterol, saponins from quillaia bark (various members of the qs-x family of triterpene glycosides), and phospholipids that form cage-like structures into which antigens can be entrapped or intercalated. iscom complexes may provide a depot function, as well as facilitate the delivery, uptake, and processing of vaccine immunogens by apcs. purified influenza virus hemagglutinin (ha) and neuraminidase mixed with phosphatidyl choline and phosphatidyl ethanolamine (polar lipids) will form empty particles that have the surface properties of influenza virus. adding an antigen in solution before mixing the lipids results in the incorporation of the antigen inside the particle. this provides a vehicle for delivering antigens to the interior of a cell, via the influenza ha membrane fusion process, thereby enabling antigen processing and presentation via both major histocompatibility complex (mhc) class and pathways. emulsions numerous oil-in-water and water-in-oil emulsions have been tested as adjuvants. one such emulsion, mf , is used in a licensed influenza vaccine. mf consists of squalane, a metabolizable shark oil and two surfactants, polyoxyethylene sorbitan monooleate and sorbitan trioleate, in an oil-in-water emulsion. cytokines are host-produced immunomodulators that regulate immune cell action (chapter ). several cytokines are being tested as potential vaccine adjuvants, including granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin- (il- ), and il- . of the defined tlr agonists being explored as vaccine adjuvants, lps and its partially detoxified form, mpl, which activate tlr , have been most thoroughly explored in clinical trials. with evidence of enhanced ability to increase the percentage of individuals responding with protective antibody levels to hepatitis b as compared to a standard hepatitis b vaccine, one hepatitis b vaccine that employs an adjuvant formulation (termed as ) consisting of a combination of alum and mpl has been licensed for use in high-risk individuals. in addition, a vaccine against hpv that uses the same adjuvant formulation may be licensed soon, and candidate hsv- and malaria vaccines currently being studied in latestage clinical trials also include this alum-mpl combination adjuvant. a wide variety of tlr -specific agonists consisting of oligodeoxynucleotides containing unmethylated cpg motifs (cpg-odn) are being evaluated in preclinical studies. these cpg-odns resemble bacterial dna, modified to include a phosphorothioate backbone to increase their stability. two cpg-odn adjuvants have been evaluated in recent phase i and ii trials and shown to increase the timing and magnitude of induction of protective antibody levels, as well as the proportion of responding individuals, to recombinant hbsag vaccine as compared with the current commercially available version of the vaccine. one of these cpg-odn adjuvants also elicits protective antibody responses in immunized hiv-infected individuals who had previously failed to respond to the hepatitis b vaccine. this approach is now being studied as a way of inducing protective immune responses to hepatitis b earlier after initiation of the vaccination regimen or with fewer doses of the vaccine. in addition to the cpg-odn-based tlr adjuvants described above, small chemical compounds with structures that resemble nucleic acid bases have been identified that activate tlr (e.g., imiquimod) or both tlr and (e.g., resiquimod). these compounds are being evaluated as vaccine adjuvants in preclinical studies. flagellin, a tlr agonist, is also being explored as an adjuvant. recently, attention has also been focused on coupling, rather than mixing, tlr agonists to antigens. cpg oligonucleotides conjugated to antigens have been tested in preclinical studies of hepatitis b vaccines and in human clinical trials for treatment of allergy. ligands for tlr / have been coupled to hiv antigens, and the ligand for tlr (flagellin) has been fused to a variety of antigens. , in some instances, coupling a tlr ligand to an antigen resulted in a substantial improvement of the immune response compared to mixtures -potentially the result of enabling the antigen and the tlr ligand to co-locate in the same dc compartments. numerous preclinical studies have confirmed that many natural and synthetic tlr agonists possess adjuvant activity. importantly, early human clinical trials of tlr-predicated adjuvants have supported the promise of this approach to mechanism-based strategies to augment vaccine immunogenicity. an important challenge is to define the most potent and best-tolerated variants, and to define rules by which activation of specific tlr pathways might translate into predictable augmentation of desired types of immune responses. it is hoped that general rules will emerge to suggest which of an increasing number of novel adjuvants in development performs best with which type of vaccine immunogen, and if results obtained with a specific type of immunogen-adjuvant combination can be extrapolated to predict the likelihood of enhanced immunogenicity with other vaccines. although beyond the reach of available experimental results, the ability to tailor, titrate, and otherwise optimize immune responses to vaccines by manipulation of specific tlr pathways appears a realistic future possibility. however, important challenges remain. in particular, a primary challenge for nextgeneration adjuvant development is finding a combination that retains immunopotentiating action while minimizing vaccine-associated adverse experiences. short-term adverse experiences, such as local injection site reactions, represent undesirable side effects that may disqualify candidate adjuvants early in clinical development. however, given that vaccines are administered to healthy people to prevent potential future infectious diseases, the potential for rarer adverse experiences (such as autoimmunity) that may only be manifest with much longer latency from the time of vaccine adjuvant administration will undoubtedly be important considerations for use in prophylactic vaccines. as induction of cell-mediated immune responses is considered an important component of vaccine strategies for many diseases for which no vaccines are currently available (many of which are caused by intracellular pathogens), there is a need to develop safe and readily scalable approaches to elicit durable cd t-cell responses in immunized humans. further, given the critical role that cd t cells play in induction, differentiation, and maintenance of cd t-cell responses, any such novel vaccine strategy will likely also require appropriate cd tcell responses. as elicitation of cd t-cell responses against a foreign antigen usually depends on the de novo expression of the antigen within a host cell and its subsequent processing and presentation via class i mhc pathways (chapter ), most novel vaccine strategies are predicated on the need to achieve synthesis of pathogen-derived antigens within apcs of immunized human hosts. with an increasing appreciation of the role that cross-presentation pathways can play in elicitation of class i-restricted cd t-cell responses, such de novo antigen synthesis may not need to occur within apcs themselves (which may be an advantage for potential vaccine delivery strategies that do not directly target apcs). one of the many attractive attributes of effective live attenuated vaccines is their ability to recapitulate (to various degrees) many of the processes that lead to the generation of potent immune responses following natural infection. these processes include the fact that replication of all viruses depends on gaining access to host cells for genome replication and for the synthesis of essential components of virus particles that permit further propagation of the infection within and between hosts. one immunologic benefit of this requirement is that de novo synthesis of viral gene products within infected cells provides a key opportunity for viral antigen presentation (via mhc class i pathways) and elicitation of antiviral cellular immune responses. along with the processing and presentation of intact virus proteins via mhc class ii pathways leading to production of antiviral antibody responses, live attenuated viral vaccines have a strong track record for induction of broad cellular and humoral immune responses that likely both contribute to conferring protective immunity. however, despite their track record of success, it is likely that few, if any, new live attenuated viral vaccines will be derived in a manner that resembles previous successful efforts (e.g., the empiric derivation of live attenuated polio, yellow fever, or varicella-zoster vaccines). important reasons for this change include the desire for safe and well-characterized vaccines whose mechanisms of attenuation are defined and that can be monitored in the course of vaccine production and use. indeed, most of the recently developed live attenuated vaccines were derived using new approaches for genetic reassortment (fig. . ) wherein genome segments encoding pathogen-derived antigens of interest are recombined with a common set of genome segments that carry attenuating mutations (derived either by use of attenuating viral passage under specific conditions in cell culture (e.g., the cold-adapted influenza vaccine or use of a virus obtained from a nonhuman host that is itself inherently unable to replicate to high levels in humans, e.g., the reassortant rotavirus vaccine prepared via genetic reassortment between human and bovine rotavirus strains (fig. . ) ). although such approaches have proven successful, they are limited in that they can only be applied to homologous viruses (e.g., those derived from the same virus type) whose genomes are segmented and capable of ready genetic reassortment in culture, or to viruses that can be manipulated by reverse genetics. in response to the desire to produce vaccines that can safely and reliably elicit desired immune responses, especially t-cell responses, several approaches are being explored to develop novel vector systems that permit the expression of pathogen-derived antigens. as many of these approaches are based on viruses distinct from the viral pathogen targeted for induction of host immune responses, the inserted pathogen-derived gene products are expressed via recombinant methods as heterologous antigens. alternatively, in nonviral expression systems, such as dna vaccines, the pathogen-derived antigen is expressed in isolation and does not depend on virus-mediated antigen delivery to apcs following host inoculation. collectively, such recombinant heterologous expression systems are commonly referred to as 'vaccine vectors.' in some instances, such recombinant vectors express only a specific antigen (in the case of dna vaccines or certain viral vectors, e.g., adenovirus), while in others both the inserted pathogen-derived antigens and antigens encoded by the viral vector 'backbone' are expressed (e.g., poxvirus vectors). most new approaches employ expression systems that are inherently nonreplicating (e.g., dna vaccines) or that employ viral vectors that can replicate at high levels in tissue culture but not in vivo (e.g., complemented adenovirus deletion variants or host range-restricted poxviruses). while numerous approaches are being pursued to develop novel vaccine vectors, they will all need to meet certain common criteria to emerge as vaccine approaches applicable for widespread use. in particular, any successful approach must be safe in healthy and immunodeficient humans (given their increased representation in the population as a result of hiv infection and therapeutic immunosuppression), desirably immunogenic (including in individuals who may have been previously exposed to the virus from which the vector was derived, e.g., vaccinia or adenovirus), and able to be produced in large quantities and in a stable manner. a limited number of vaccine vector approaches now being pursued are likely to meet these criteria. should successful approaches emerge, there will likely be interest in applying them for use in vaccines targeting diverse pathogens. thus, while definition of promising, broadly applicable, vaccine vector approaches may help simplify certain aspects of vaccine regulatory review and manufacture, they may also present challenges to prioritize use for specific applications should administration of a given vaccine vector on one occasion compromise or preclude successful administration at a later time. nevertheless, the development of novel vaccine vectors is laying essential groundwork for the development of next-generation vaccines. several novel vaccine vectors currently being studied in preclinical studies and human clinical trials are described below, all of which depend on the delivery and expression of a candidate pathogen-derived gene sequence. in a number of ways, dna vaccines represent the simplest approach to deliver pathogen-derived genes. viral vectors similarly serve to deliver pathogen gene sequences to host apcs, either directly or indirectly, but do so in a manner that depends on and takes advantage of the lifecycle and tropism of the virus that is being adapted to express the exogenous pathogen gene products. the ability of purified plasmid dna containing heterologous antigens expressed under the control of eukaryotic transcriptional regulatory and rna processing signals to elicit immune responses when injected into experimental animals was discovered serendipitously. however, since the initial, quite surprising, description, the development of so-called dna vaccines has become an active area of preclinical and clinical vaccine development. reasons for this enthusiasm include the attractive simplicity and facile preparation of vectors that encode only the defined antigen of interest (which can itself be manipulated via recombinant methods to assume a desired configuration), a reasonably straightforward method for vaccine production, and the inherent stability to temperature (which is much greater than most currently live or subunit vaccines). although the dna vector is most commonly injected intramuscularly, the generation of specific immune responses depends on the uptake of the vector dna by apcs followed by the expression, processing, and presentation of vector-encoded antigens. as tissue and tissue fluids present a hostile environment for purified dna, and the process of dna uptake by apcs appears to be relatively inefficient, much of the dose of injected dna is degraded before it can be reached by an apc that can initiate the desired immune response. most dna vaccine research has been pursued in mice, although studies have now been performed in numerous animal species. studies have usually utilized intramuscular injection of vaccine vector dna, but various intradermal and transdermal approaches have also been explored. murine studies have shown that administration of antigen-encoding plasmid dna can elicit appreciable cellular and humoral immune responses that may confer protection against experimental challenge. however, translation of these promising results in animal models to humans has proven frustrating. while dna vaccines have been generally well tolerated in immunized volunteers, in most human studies of dna vaccines, administration of even substantial quantities of dna vaccine vectors has elicited relatively low-level immune responses. it is not yet known whether these disappointing results reflects fundamental differences in the immunogenic behavior of dna vaccines in humans and mice, or the fact that the dna doses administered to humans do not match those administered to mice (dna per weight of the immunized host). given the substantial size differences between humans and mice, it would likely be impractical (for reasons of both vaccine supply and the actual process of administration of sufficiently high doses of dna) to administer the relative murine dose to humans. as such, a variety of approaches are being explored to prolong dna survival in tissue, promote more efficient targeting of dna to apcs, or to develop novel adjuvants that might specifically amplify immune responses to dna vaccines. , dna vaccines are currently being used as candidate preventive vaccines for a wide variety of infectious diseases, including hiv, tuberculosis, malaria, and cmv. poxviruses represent the family of viruses that are physically the largest viruses and that possess the largest genomes. much of the poxvirus genome encodes gene products that serve to evade host immune responses, and that are not required for virus replication in tissue culture. further, facile techniques for the insertion and deletion of specific viral genes have been developed. the ability to accommodate sizeable foreign gene inserts is, in part, a function of the large size of the poxvirus genome (and the large packaging capacity of poxvirus virions). as a result of these favorable attributes, poxviruses have been utilized extensively in laboratory studies of virus biology, recombinant protein production, and host immune responses. although poxviruses encode multiple gene products that help the virus evade host immune responses, they are, nevertheless, potent immunogens. studies of individuals immunized decades ago with vaccinia virus (in the course of smallpox eradication efforts) have shown that this virus induces long-lasting memory t-and b-cell immune responses. in contrast to most of the other viral vectors currently being developed, poxviruses can replicate readily in culture and do not require an engineered host cell to support propagation ex vivo. one important limitation of all poxvirus vectors developed to date is that, given the large size of the poxvirus genomes and the multitude of gene products they naturally express, even large inserts derived from foreign pathogens of interest will present only a minority of the vaccine vector antigens delivered to and recognized by the host immune system. to be effective, approaches to focus immune responses on the antigen of interest will need to be developed. toward this end, a variety of so-called 'prime-boost' approaches are being explored where the host immune response is primed with one type of recombinant vaccine vector (such as a dna vaccine or adenovirus vector) and then boosted with subsequent delivery of poxvirus vectors encoding the same antigen. in this manner, immune responses to antigens of interest have been significantly augmented in a number of preclinical studies. vaccinia virus represents the prototypic vaccine vector. this virus is the same one that was employed in the successful smallpox eradication campaign, and has been used as a laboratory tool for decades. however, given current high expectations for vaccine safety, and the increased number of immunodeficient individuals present in the population (as a result of the new antigen discovery methods emergence of the hiv pandemic and the increased use of immunosuppressive therapies in clinical medicine) at high risk of serious adverse events, and potentially fatal consequences, from vaccinia immunization, the original vaccinia strains used in smallpox eradication efforts are not considered safe for general use. however, studies of vaccinia-based vaccine vectors have provided a strong basic foundation for research on other more highly attenuated poxvirus variants. modified vaccinia ankara (mva) is an attenuated vaccinia virus that was originally derived by prolonged passage of a vaccinia virus isolate on chicken embryo fibroblasts in culture. in the course of extensive passage in culture, a viral variant emerged that had fortuitously deleted large sections of the viral genome, including those that encode important poxvirus immune evasion genes and those that determine the ability of the virus to replicate on cells obtained from different animal species. specifically, while mva grows well on chicken cells, it cannot replicate in human cells in culture or in vivo, conferring an inherent safety feature. mva was safely administered to over individuals at high risk of adverse consequence for vaccinia immunization toward the end of the smallpox eradication effort. more recently, it has garnered renewed interest as a potential safer smallpox vaccine in the wake of concerns about bioterrorism threats. even though mva cannot replicate in mammalian cells, the virus demonstrates favorable immunogenic properties. mva has been used as a vector expressing genes for a wide variety of genes, including hiv and malaria antigens either alone or, as described above, in 'prime-boost' regimens, where mva has been administered following initial priming immunizations with other vaccine vectors. a concerted effort is under way to improve further the performance of mva by manipulating a series of poxvirus genes that dampen the human immune response to the virus (and to any antigens inserted in it). avipox is a family of poxviruses that infect birds and cause respiratory diseases in poultry. canarypox, a member of the avipox group, has been adapted as a vaccine vector. canarypox replicates well on avian cells in culture but cannot replicate on human cells in culture or in humans in vivo. as a result, canarypox, like mva, provides an interesting vector system with inherent safety features. canarypox vectors carrying hiv genes have been tested in several clinical studies, either alone, or in 'prime-boost' regimens following priming with adenovirus vectors and recombinant protein antigens. in a large ongoing phase iii hiv vaccine trial, a recombinant canarypox vector is being used as a priming vector, followed by boosting with a recombinant version of the hiv gp surface env protein. to date, the results from human clinical trials of canarypox vectors have been disappointing, with only low-level specific immune responses generated in human volunteers. adenoviruses, one of the common causes of upper respiratory and gastrointestinal infections, have seen extensive use in clinical trials and were one of the first gene therapy vectors. most adenovirus vectors currently being studied in preclinical and clinical settings are disabled by deletion of the early e genes that are necessary for replication in an immunized host. most adenovirus vaccine vectors developed have used the well-characterized and readily produced adenovirus serotype (ad ) as the vector 'backbone.' disabled adenovirus vectors are grown in cells that express the e genes artificially inserted into the cell's genome. once these disabled vectors, encoding a heterologous pathogen-derived antigen of interest, enter a cell, the pathogen gene product is expressed, processed, and presented by host apcs. as adenoviruses can directly infect dendritic cells, they promise to provide efficient vaccine vectors. robust antibody and cd t-cell responses to heterologous antigen genes expressed by adenovirus vectors have been observed in preclinical animal models. furthermore, in early-phase human clinical trials, adenovirus vectors have been generally well tolerated, and proven to be the most effective of any recombinant vector system studied to date in eliciting high-level cd t-cell responses. the main potential drawback to widespread use of adenovirus vectors in humans is that, depending on the adenovirus type and the geographic location, variable levels of pre-existing immunity are found in humans as a result of prior naturally acquired adenovirus infections. high levels of antibody against the adenovirus vector might blunt the immunogenicity and efficacy of an adenovirus vector-based vaccine, but it remains to be seen if this will be a significant limitation. should pre-existing immunity to adenovirus vectors derived from epidemiologically prevalent serotypes (e.g., ad ) limit vaccine immunogenicity, current efforts to develop vaccine vectors based on serotypes that are rare in human populations or novel adenovirus vectors specifically designed to avoid preexisting antibody responses may yield effective alternative approaches. adenovirus vectors are currently used in clinical trials for vaccines against hiv, malaria, influenza, and a range of other pathogens. alphaviruses are rna viruses that cause zoonotic diseases, such as venezuelan equine encephalitis. these viruses do not normally circulate in humans, so immunity to these viruses is quite rare in humans. alphaviruses have a strategy for overexpressing the proteins that make up the virion by making a separate subgenomic rna specifically encoding these gene products. current recombinant alphavirus vaccine vector strategies take advantage of this subgenomic transcript, replacing the viral genes with selected genes for other antigens, but maintaining the signals for translation and protein production. in addition, through use of genetic complementation, it is possible to generate virus particles that only contain this heterologous antigen-encoding expression cassette. such virus particles can efficiently mediate infection of host cells, but because they lack other alphavirus genes needed for virus replication cannot spread beyond the initial target cell infected. , alphavirus vectors rival the adenoviruses in efficiency of protein production in tissue culture and have induced robust antibody and t-cell responses in preclinical studies. one current limitation of the alphavirus vector system is the difficulty of scaling the production system; however, this is a technical matter that should be addressable. in addition, ample safety data will be needed before widespread use of alphavirus vaccines achieves endorsement by regulatory authorities for use in healthy populations. adeno-associated viruses (aav ) belong to a family of single-stranded dna viruses (parvoviruses) that include the b parvovirus that causes a rash in children known as 'fifth disease' (measles, mumps, rubella, and varicella make up the first four). aav is transmitted in conjunction with adenovirus infection, and is not known to cause any significant disease. it is poorly immunogenic in the course of natural infections. aav can integrate into the genome of the infected cell, usually in a particular place on chromosome , although integration does not appear to be efficient or site-specific when replicationdefective adenoviruses of the type being developed as vaccine vectors are used. the propensity for chromosomal integration and poor immune response to the virus made aav a good candidate for gene therapy; cells with an integrated viral genome could deliver a gene product for a long time without the immune system killing the infected cell. recently, efforts have been made to adapt replicationdefective aav as a vaccine vector. although encouraging results have been reported in preclinical studies, phase i studies in humans have demonstrated disappointing immunogenicity. n summary n the challenges to optimizing the full public health potential of existing vaccines largely relate to programmatic considerations. in contrast, the terrible impact of infectious diseases that cannot now be prevented by vaccines (such as the 'big three' killers of hiv, tuberculosis, and malaria) pose direct challenges to the scientific community to develop new generations of vaccines that overcome the largely biological obstacles to control and elimination of these diseases. the nature of the challenges posed by such pathogens necessitates that future vaccine efforts will not simply recapitulate the immune responses engendered by natural infection (as has been the premise of traditional vaccine development efforts), but rather, substantially improve upon them. as the development of vaccines to prevent infections with the so-far refractory pathogens is pursued, improved understanding of the immune response to natural infection, as well as delineation of the reasons why host immune responses fail either to clear incipient infections or prevent future new ones, will be essential. fortunately, early empiric approaches have now been replaced with hypothesis-driven strategies enabled by improved insight into the functioning of the human immune system, as well as new technologies, including higher-resolution tools to describe and quantitate pathogen-specific immune responses; novel methods for antigen discovery and targeted optimization of immunogenicity; the development of new, mechanism-based adjuvants; and the advent of innovative methods for vaccine vector-mediated antigen delivery. thus, although the challenges may be vexing, the scientific and technical foundations on which vaccine development efforts rest have never been stronger. n references n for many important infectious diseases for which no vaccines are currently available, successful derivation of effective vaccines will depend on improving upon natural immunity, especially in those instances where natural immunity does not follow natural infection (such as human immunodeficiency virus (hiv) and malaria) or where safety concerns limit the development of specific protective antigens (neisseria meningitidis group b). in addition, for other pathogens that typically manifest significant genetic (and antigenic) diversity (such as influenza, hiv, and bacteria such as streptococcus pneumoniae), a need exists to develop novel vaccines that can protect against a wide range of variants with a limited number of vaccine immunogens. towards these ends, a number of new approaches, enabled by new vaccine technologies, are being pursued, including: >> targeted alteration of protective antigens to increase their ability to elicit protective immune responses (e.g., efforts to alter the structure of the hiv env glycoproteins gp and gp so that they elicit higher-level, more potent, neutralizing antibody responses than their native counterparts) >> the development of synthetic consensus antigens able to elicit broader immune responses than would sequences obtained from individual pathogen isolates (e.g., efforts to develop consensus immunogens able to elicit cytotoxic t-lymphocyte (ctl) responses against genetically diverse hiv- variants) >> techniques for new antigen discovery to identify novel conserved antigens within otherwise genetically diverse pathogens (e.g., streptococcus pneumoniae) or those for which currently known protective antigens cannot be developed as vaccines (e.g., neisseria meningitidis group b) >> the use of novel adjuvants or vaccine vectors to enable generation of higher-level and/or more functional immune responses to pathogen antigens by vaccination than are seen following natural infection, and to enable high-level, fully functional memory immune responses to be activated at the time of initial infection (e.g., efforts to elicit high-level hivspecific or hepatitis c virus-specific ctls by recombinant viral vectors) >> use of novel methods to shift relative immunodominance of specific pathogen gene products to increase the immunogenicity of conserved antigens from otherwise diverse pathogen genomes that are typically poorly immunogenic in the course of natural infections (e.g., efforts to augment the antibody response to the influenza a virus m protein via the use of potent adjuvants or conjugation to immunogenic carrier proteins) ten great public health achievements -united states principles and lessons from the smallpox eradication programme advance market commitments for vaccines against neglected diseases: estimating costs 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computational analysis of a deadly pathogen: combating mycobacterium tuberculosis from multiple fronts identification of in vivo-expressed immunogenic proteins by serological proteome analysis of the bacillus anthracis secretome identification of tumor antigens in renal cell carcinoma by serological proteome analysis innate immune recognition on regulation of phagosome maturation and antigen presentation translating innate immunity into immunological memory: implications for vaccine development innate immune induction of the adaptive immune response liposomes and iscoms understanding the role of innate immunity in the mechanism of action of the live attenuated yellow fever vaccine d haemophilus influenzae type bouter membrane protein complex glycoconjugate vaccine induces cytokine production by engaging human toll-like receptor (tlr ) and requires the presence of tlr for optimal immunogenicity structure and properties of aluminum-containing adjuvants immunoactivating potential of 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adaptive immunity the development of multivalent bovine rotavirus (strain wc ) reassortant vaccine for infants intramuscular injection of plasmid dna heterologous protection against influenza by injection of dna encoding a viral protein dna vaccines: recent developments and future possibilities from plasmids to protection: a review of dna vaccines against infectious diseases poxvirus-based vaccine candidates for hiv: two decades of experience with special emphasis on canarypox vectors drug evaluation: dna/mva primeboost hiv vaccine improving recombinant mva immune responses: potentiation of the immune responses to hiv- with mva and dna vectors expressing env and the cytokines il- and ifn-gamma applications of canarypox (alvac) vectors in human and veterinary vaccination high-dose recombinant canarypox vaccine expressing hiv- protein, in seronegative human subjects biology of adenovirus and its use as a vector for gene therapy adenovirus-based expression vectors and recombinant vaccines established immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. potential for immunosuppression and vector engineering to overcome barriers of immunity attenuation of simian immunodeficiency virus sivmac infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing gag venezuelan equine encephalitis virus vectors expressing hiv- proteins: vector design strategies for improved vaccine efficacy an alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector recombinant alphaviruses as vectors for anti-tumour and anti-microbial immunotherapy immune responses to adeno-associated virus and its recombinant vectors key: cord- -kocwsyi authors: shannon, m. frances title: genomic approaches to the host response to pathogens date: - - journal: genomic and personalized medicine doi: . /b - - - - . - sha: doc_id: cord_uid: kocwsyi nan following a period of relative disinterest in infectious disease research due to the enormous impact of vaccines and antibiotics on the spread of and mortality from these diseases, there is now renewed and growing interest in this area of research. this has been driven by several recent worldwide developments including: (i) the rising incidence of diseases such as acquired immune defi ciency syndrome (aids) and antibiotic-resistant tuberculosis; (ii) antibiotic-resistant bacterial strains presenting a severe health threat in hospitals; (iii) the rapid spread of new pathogens such as severe acute respiratory syndrome (sars); and (iv) the threat of bioterrorism. indeed, nearly % of annual deaths worldwide are due to infectious disease ( morens et al., ) . thus, the need to develop new diagnostic methods, more effective vaccines and better therapeutic strategies is urgent. in order to effectively deal with infectious disease threats, it is important to understand both the pathogen and the response of the host, since the outcome of infection is determined by complex host-pathogen interactions. pathogens are initially detected by the surveillance cells of the innate immune system using cell surface receptors known as toll-like receptors (tlrs) (reviewed in ). these tlrs recognize specifi c components of the pathogen, for example, bacterial lipopolysaccharide (lps) or double-stranded (ds) rna from viruses. while many cell types express tlrs, cells of the innate immune system such as dendritic cells (dcs) and macrophages play particularly important roles in detecting and responding to pathogens. the response of these cells to a pathogen is determined by the specifi c pathogen component that interacts with the tlr and the specifi c tlr family member that is activated. widespread changes in gene expression are detected following tlr activation and the activated cells produce a plethora of cytokines and chemokines that then activate the adaptive arm of the immune system. the specifi c cytokines and chemokines produced by the tlr-activated cells tailor the response of the adaptive immune system to deal with the specifi c pathogen . thus, the initial host response to a pathogen through the tlrs determines the outcome of the infection. host response to infection can be a double-edged sword in that sometimes the response itself can create an adverse outcome for the host. in addition, the aberrant response of the host to self instead of foreign pathogens can create severe pathologies involving chronic infl ammatory and autoimmune diseases. the urgent need to better understand host-pathogen interactions has come at a time when genomics and related technologies are expanding rapidly. the availability of complete genomic sequences of an expanding number of pathogens, the human and mouse genome sequences and the advent of genome-wide genotyping and gene expression profi ling has opened up new avenues of investigation in the fi eld. the genotype of the pathogen plays a major role in the response of the host to infection with more virulent pathogenic strains often possessing the capability to interfere with the host immune response ( fitzgerald and musser, ; kato-maeda et al., ; schoolnik, ) . in addition, different individuals in a population can have very different responses to a genetically identical pathogen. while there are many complex reasons for this, it is clear that part of the differential response is governed by underlying genetic differences between individuals ( clementi and di gianantonio, ; zhang and zhang, ) . studies in mouse models of infection have clearly demonstrated that these genetic differences are complex and may involve more than one genetic locus for a given susceptibility or resistance trait (e.g., [delahaye et al., ] ). while there are some classic examples of genetic mutations affecting the response of the host to a pathogen (e.g., malaria and sickle cell mutations) there is much to be learned before the genetics of host susceptibility is fully understood. the advent of genome-wide genotyping using single nucleotide polymorphisms (snps) or microsatellite markers, leading to major advances in molecular epidemiology, will revolutionize our ability to determine the complexities of the genetic component of pathogen-host interactions ( weiss and terwilliger, ) . it is well known that the cells of the host immune system are activated upon detection of a pathogen by tlrs as described above. this activation process includes widespread changes in the gene expression profi le of the cells with hundreds of genes being either switched on or off in response to signals generated from the pathogen-detecting tlrs. the response of individual genes has been studied in minute detail for a handful of genes and while this has produced an understanding of some aspects of host response to infection it by no means gives us the total picture. understanding the molecular response of the host to infection has been greatly improved by using microarray-based technologies and these technologies are opening up new diagnostic possibilities as well as presenting new therapeutic options ( aderem and smith, ; bryant et al., ; feezor et al., ; hedeler et al., ; korth and katze, ; ng et al., ; ricciardi-castagnoli, ; ricciardi-castagnoli and granucci, ; smith and bolouri, ) . this chapter will focus on two aspects of the host response to pathogens where major advances are being made using genomics approaches and will describe the future impact of these approaches on the development of diagnostics and therapeutics for infectious disease. these are (i) defi ning the basis of genetic susceptibility to infection and (ii) the defi nition of the system-wide molecular response to a pathogen. it is now relatively easy to map genes associated with genetic diseases that show a mendelian pattern of inheritance. however, these diseases account for only a very small proportion of the human disease burden and many of the more common and fatal diseases have a complex etiology with many genetic and environmental contributions. while it is clear that most complex disease has a genetic component, defi ning that genetic component has been diffi cult to date since many complex diseases such as coronary heart disease, diabetes and others are polygenic with different genetic loci contributing in major or minor ways to disease susceptibility. in addition, in different populations or under different environmental conditions, distinct but overlapping sets of genetic loci are likely to contribute. the sequencing of the human genome and the genome-wide genetic and functional mapping that has followed have raised hopes of mapping the genetic component of complex disease and there are many large efforts around the world with this aim. studies in both animal models and human populations have shown that infectious disease and the response of the host to a specifi c infection also has a complex genetic component ( clementi and di gianantonio, ; lipoldova and demant, ; marquet et al., ; mira et al., ) . thus, inbred mouse models have been developed that clearly show a genetic component to susceptibility for specifi c pathogens and in some cases at least part of the underlying genetic reason has been defi ned ( beck et al., ; mak et al., ; rogner and avner, ) . mapping the genetic components of susceptibility to infection in human populations has been much more difficult due to the large natural variation in humans, the polygenic nature of this trait and the low penetrance of many of the susceptibility alleles. for infectious disease, this is complicated even more by the complex nature of the environmental infl uences particularly the fact that these diseases, unlike other complex diseases, are transmissible in populations. however, a combination of animal and human population studies, combined with the latest genomic technologies, is beginning to unravel the issues of genetic susceptibility to infection. the use of inbred and congenic strains of mice are well established systems for identifying susceptibility loci ( beck et al., ; rogner and avner, ) . in recent years genetic manipulation of specifi c loci by deletion or mutation has provided many mouse models for screening ( mak et al., ) . the use of ethylnitrosourea (enu) mutagenesis to randomly create point mutations in the mouse genome has opened up a new forward genetics approach to identifying susceptibility loci (papathanasiou and goodnow, ) . this chemical mutagen, when used at appropriate doses and at the correct stage of development, can introduce single point mutations into the mouse genome. by screening libraries of mutant mice for susceptibility to specifi c pathogens, it should be possible to identify genetic loci that dictate susceptibility or resistance to a range of pathogens on a large scale. it is relatively straightforward to identify a chromosomal region involved in susceptibility in these mouse strains by genotyping with microsatellite markers, but identifying the specifi c gene that is mutated is still very time-consuming. the speed with which this can be achieved depends on the presence of candidate genes within the chromosomal interval or the ability to resequence large amounts of dna. the latter is becoming achievable with the advent of new rapid sequencing technologies and is set to revolutionize forward genetic approaches to disease understanding ( bennett et al., ; serre and hudson, ) . the recent explosion in genetic information for the human genome including the complete genome sequence and detailed genetic and physical maps has increased our ability to fi nd variation in the human genome and correlate it with disease. family studies, especially twin studies, and population studies have clearly shown a genetic component to susceptibility to infectious disease ( frodsham and hill, ; lipoldova and demant, ; strunk and burgner, ) . susceptibility generally follows a complex pattern of inheritance and there are two main methods of mapping and identifying genetic loci involved in such complex heredity. these are either association studies or linkage studies. association studies involve screening populations for specifi c mutations in a candidate gene(s) in case-control studies or in family studies. this type of approach identifi ed the link between human immunodefi ciency virus (hiv) resistance and the chemokine receptor, ccr described below ( dean et al., ; samson et al., ) . genome-wide association studies although still quite expensive are now becoming feasible and being used to defi ne linkage between specifi c markers and susceptibility. these linkage studies depend on the availability of markers and the density of these markers is rapidly increasing t a b l e . list of well-described susceptibility loci for resistance or susceptibility to infectious disease. genes references with the large scale identifi cation of new snps across the human genome. the selection of which snps to use and the large numbers of samples needed to generate statistically significant associations for low penetrance alleles are still challenges. the haplotype map (hapmap) project is starting to identify haplotypes within different population groups and together with improvements in large scale genotyping technology and bioinformatics should be useful in studies of complex disease inheritance. table . summarizes the best studied genetic susceptibility loci for response to different infectious agents in both mouse models and human studies. one of the classical examples of genetic susceptibility to infection is the role of the hemaglobinopathies in the outcome of malaria infection ( patrinos et al., ) . there are also certain chromosomal regions and families of genes that have attracted attention in terms of searching for susceptibility alleles or polymorphisms. because the tlr family of receptors plays a major role in recognizing pathogens, it was speculated that genetic variation in these receptors or their signaling pathways might be responsible for some susceptibility phenotypes (reviewed in [schroder and schumann, ] ). one of the best examples to date is the occurrence of a single polymorphism in the region of the human tlr gene encoding the extracellular domain of the receptor which confers reduced sensitivity to inhaled escherichia coli ( e. coli) lps ( arbour et al., ) . interestingly, when septic shock patients were compared with a control group, these lower-responding alleles were found only in the septic shock group and these individuals had a higher incidence of gramnegative bacterial infection ( feterowski et al., ) . such studies need further confi rmation since there are also a number of studies that failed to fi nd any linkage between tlr mutations and response to various infections ( schroder and schumann, ) . there is also enormous variation in the response of individuals to lps even in the absence of tlr mutations implying that variation may occur in other components of the tlr signaling system. an example of this is the link between irak mutations and increased susceptibility to infection with pyogenic bacteria ( picard et al., ) . variation in other tlr genes has also been associated with disease susceptibility. for example, a mutation in the extracellular domain of tlr is linked to susceptibility to leprosy ( alcais et al., ) and a mutation in tlr increases susceptibility to legionella . taken together these data support the idea that variation in the innate immune recognition of pathogens play an important part in governing susceptibility to an array of infectious diseases. however, caution needs to be exercised until larger population groups have been studied. the extensive polymorphism at the chromosomal regions encoding major histocompatibility complex (mhc) proteins is thought to have arisen through natural selection in response to selective pressure from infectious disease. although human leukocyte antigen (hla) association with resistance or susceptibility to infectious disease has been diffi cult to identify because of the complex array of antigenic epitopes involved, a number of studies have implicated this locus in genetic susceptibility to infectious disease ( ghodke et al., ; little and parham, ) . mhc molecules fall into two classes, class i that present foreign antigens to cd ϩ cytotoxic t cells and class ii that play a similar role for cd ϩ helper t cells. variation in specifi c class i genes has been shown to confer susceptibility to pulmonary tuberculosis and to hiv whereas mutations in other class i genes confer resistance to hiv and to severe malaria. class ii mutations that confer resistance to hepatitis b or hepatitis c have been identifi ed and susceptibility to typhoid fever and leprosy are also associated with specifi c class ii mutations. further molecular analysis of these and other associations may in the future have an impact on the development of new vaccines and immunotherapeutics. to date the most successful manner of identifying susceptibility genes in human populations has been the candidate gene approach. candidate genes have emerged from many sources including mouse genetic studies as well as biochemical and function dissection of the immune system. once a candidate gene is identifi ed, the chromosomal region spanning this gene in the human genome is then scanned for the occurrence of specifi c mutations or for functional polymorphisms in casecontrol studies across populations or in linkage studies in family groups. such studies have identifi ed a number of well described susceptibility loci for infection with various pathogens. one of the most heralded example was the identifi cation of a deletion in the chemokine receptor, ccr , which was shown to confer resistance to hiv infection ( dean et al., ; samson et al., ) . biochemically, this can be explained by the fact that ccr is a co-receptor for hiv on the surface of t cells (dragic et al., ) . a mutation in another chemokine receptor, ccr , has also been shown to confer hiv resistance in certain caucasian populations ( o'brien and moore, ) . some genes have been associated with susceptibility or resistance to multiple pathogens. for example, variation in the nramp /slc a gene is associated with susceptibility to leishmania and to specifi c intracellular bacteria such as tuberculosis ( barton et al., ; govoni et al., ; lipoldova and demant, ; sebastiani et al., ) . mutations in the tumor necrosis factor (tnf) locus, mainly gene promoter mutations, have been linked with malaria and leprosy susceptibility ( lipoldova and demant, ) . gene promoter or control region mutations have an impact on the level of protein produced from the gene rather than the function of the protein. this is an area of great interest but more diffi cult to study for several reasons, including the inability to identify control regions simply from sequence information and the complexity and fl exibility of transcriptional control. a recent review detailing the genes associated with leishmania susceptibility describes a number of genes that can affect disease outcome including the interferon-gamma receptor type ( ifngr ), the interleukin- (il- ) gene and the nramp- /slc a gene ( lipoldova and demant, ) . these genes and others such as interleukin- ( il- ) and its receptor are also linked with salmonella and certain mycobacterial infections ( lipoldova and demant, ) . thus, it is likely that variation in many genes can contribute to disturbing the fi nely balanced tuning of the immune system and lead to an altered response to a pathogen. it is clear from such studies that the same genes may be involved in susceptibility to an array of pathogens indicating a core immune response critical for any pathogen. the identifi cation of susceptibility loci for infection with various pathogens will aid in developing new diagnostic screens based on the detection of genetic variants in these loci. it could be envisaged that a person's susceptibility or resistance to a pathogen could be defi ned by a simple genotyping screen either prior to exposure to any pathogen or upon presentation with an infection. it may also be possible to determine the likely outcome of the infection through a genotyping screen. the defi nition of susceptibility loci will also contribute to our ability to develop new vaccines and therapeutics. the use of microarray technology to generate expression profi ling data is becoming common place in biomedical research (reviewed in [ quackenbush, ; sherlock, ] ). this technology allows the documentation of mrna levels for thousands of genes from total rna prepared from cells or tissue samples. the data obtained can be compared from sample to sample allowing the changes between samples to be documented and quantifi ed. the "expression profi le " for any cell or tissue is simply the list of genes whose expression can be detected using microarrays. the differences in the expression profi le from one cell or tissue to the next or in cells treated in a specifi c manner is a surrogate measure of the cell/tissue phenotype and shows how that phenotype responds to its environment. expression profi ling is most useful when large datasets become available and when the data is combined with other data types and detailed bioinformatics studies. for example, using functional clustering of expression profi ling data can help identify pathways that are important for a particular process and co-expression clustering combined with other technologies can help defi ne regulatory networks within the cell. over the last - years, this technology has been applied to identifying the changes in gene expression that occur in response to infection by various pathogens ( aderem and smith, ; boyce et al., ; bryant et al., ; feezor et al., ; foti et al., ; jenner and young, ; korth and katze, ; korth et al., ; ricciardi-castagnoli and granucci, ) . to date there are more than papers in the literature that describe gene expression changes that occur in response to infection with a plethora of pathogens and in many cell types (reviewed in [ jenner and young, ] ). many of these are in vitro studies, taking specifi c cell types and infecting them with specifi c agents including bacteria, viruses, parasites and yeasts. in addition, cellular responses to bacterial components have also been documented, helping to identify pathogen-specifi c responses as well as determining the pathogenic component responsible for the major gene expression effects. virulent or non-virulent strains of specifi c pathogens as well as mutant organisms have been used to determine the gene expression profi le associated with a negative or positive clinical outcome. few in vivo studies have been carried out and have shed light on the more complex responses seen in whole animals and helped to validate the in vitro data. several pioneering studies demonstrated that microarrays could be used to determine changes in the gene expression profi le of cells in response to virus or bacterial infection ( boldrick et al., ; gao et al., ; huang et al., ; nau et al., ) . these studies paved the way for the analysis of the host response to a wide variety of infectious agents. the most signifi cant of these studies compared the response of macrophages or dcs to a variety of infectious agents in a single study. in these studies a strong shared response to all infections, be they bacterial, viral or parasitic in nature, was identifi ed. not only was there commonality from one infectious agent to another but there was also some commonality across cell types. this expression signature has been interpreted as a general "alarm signal " for infection (reviewed in [ jenner and young, ] ). studies of infection with gram-positive and gram-negative bacteria also revealed a common expression signature in peripheral blood mononuclear cells. recently, the young lab has interrogated all of the publicly available expression profi ling data related to the host response to infection ( jenner and young, ) . the dataset includes experiments in cells ranging from macrophages and dcs to cells of the adaptive immune response, endothelial and epithelial cells and spanning a wide range of infecting agents. this meta-analysis revealed that a "common host response " can be detected across all of these cell types and infectious agents and show that although cells such as macrophages and dcs specialize in detecting infection, other cells of the body can mount the same " alarm response " as described above ( figure . ) . not surprisingly this expression signature contains many genes associated with the immune system particularly those encoding infl ammatory cytokines, chemokines and their receptors. however, some more surprising patterns of expression were also detected. it has been long known that interferon-stimulated genes (isgs) are regulated by virus infection, but it has only recently been recognized that bacteria and other infecting agents can also illicit the interferon response. this was borne out in these meta-analyses where upregulation of an isg set is observed across a broad range of infecting agents and cell types. not only do these cells change the expression of secreted factors and their receptors during infection but the intracellular milieu is also modifi ed. once again, there is a common pattern of change observed in all of these studies. the upregulation of signaling and transcription pathways that both augment and attenuate the immune response are observed leading to the interpretation that both positive and negative feedback loops operate within the cell to heighten or dampen the immune response. temporal profi ling can reveal extra layers of complexity and in one study a pro-infl ammatory profi le followed by an anti-infl ammatory profi le was identifi ed in macrophages activated with lps ( wells et al., ) . meta-analysis of temporal studies of activation through different tlrs also revealed that the infl ammatory chemokines/cytokine signature was an early response while the isg response was later, presumably refl ecting the need for an indirect activation of the isgs through interferon production ( jenner and young, ) . changes in the expression level of genes involved in both activation and repression of apoptosis also fall under the "common signature " banner and this is interpreted as sending the cells into a state of high alert where apoptosis can be either initiated to eliminate infected cells or terminated if the infection resolves ( jenner and young, ) . although these in vitro studies have provided an overview of the response of isolated cell types to pathogenic infection, in vivo studies are needed to validate any of these results before application to clinical medicine. a number of animal models have been used to profi le the host response to infection with a variety of agents. these studies are complicated especially if whole tissue samples are used in that changes in gene expression can result not only from genuine changes within the cells of the tissue but also from the recruitment especially of immune cells into the infected or infl amed tissue. nevertheless, in vivo studies have, in some cases, shown good correlation with the expression profi les found from in vitro studies. for example, profi ling the brains of mice infected with virulent sindbis virus revealed that isgs as well as infl ammatory chemokines were upregulated ( johnston et al., ) and in terms of diagnosis or treatment the exact reason behind these changes in expression signature may be irrelevant. in vivo studies also have some advantages in that the gene expression profi les detected in infected tissues will often make more sense when combined with other physiological or cell biology data from studies of the infected host. thus an "infection signature " would not only describe the altered gene expression of the immune cells that are recruited to the sites of infection but also would include changes in the gene expression of the resident cells of the tissue and may provide a more robust profi le of the infection process for use in diagnostic applications. what can be applied to clinical medicine from these studies? the ability to detect an "infection signature " using focused microarrays could potentially be used as a diagnostic tool. there would be an immediate need to identify a core set of genes with suffi ciently robust changes in gene expression to form the basis of a diagnostic array. arraying technology would need to be priced for diagnostic use and the technology would have to be deemed suffi ciently robust to pass all the quality control requirements of a diagnostic laboratory. no doubt progress will be made toward these goals in the near future. in addition to the "common host response " described above, microarray studies have revealed that pathogen-specifi c responses also exist. this is not surprising since it has long been known that different pathogens induce distinct arms of the adaptive immune response. for example, distinct types of helper t cells are activated in response to bacterial and viral infection compared to parasite infection and dc products such as cytokines and chemokines control the differentiation of t helper cell into the correct response type ( mosmann et al., ) . thus, the fact that cells of the innate immune system display a pathogenspecifi c transcriptional response as well as a general alarm signal helps to dictate the subsequent immune response. different tlrs are involved in recognizing and responding to different pathogens. for example, tlr is responsible for activation in response to gram-positive bacteria while tlr responds to lps, a component of gram-negative bacteria (beutler and rietschel, ; . tlr responds to dsrna and thus dictates the viral immune response for many dsrna viruses ( beutler and rietschel, ; . studies using bacterial components such as lps and fl agellin as well as dsrna have revealed that each tlr induces a specifi c as well as general transcriptional response. the transcriptional response of macrophages and peripheral blood mononuclear cells is more robust in response to gram-negative (tlr ) compared with gram-positive (tlr ) bacteria and the isg response is considerably reduced for the gram-positive expression signature ( boldrick et al., ; nau et al., ) . these differences are further observed when bacterial components are used to activate cells. for example, lps from gram-negative bacteria, a specifi c ligand for tlr , can activate the isg profi le but tlr ligands such as lta and mdp cannot ( jenner and young, ) . not only the activation of specifi c gene sets but also the strength of the specifi c response signature may be important for the immune detection of the type of pathogen involved. e. coli infection of dcs strongly upregulates the chemokines/cytokine infl ammatory cluster whereas infection with infl uenza or other single stranded (ss) rna viruses (through tlr ) has a weaker ability to regulate this cluster but a stronger ability to regulate the isg signature ( huang et al., ; lund et al., ) . these types of results raise the possibility that the diagnosis of the type of pathogen involved in an infection would be helped by the development of customized microarrays that could distinguish the gene expression profi les elicited by particular pathogens. additionally, arrays that also detect rnas produced by the pathogen may be even more signifi cant as a diagnostic tool. an infecting agent can either be cleared from the body by the immune system mounting an appropriate response or cause severe or terminal pathology. the outcome depends on a multiplicity of events ranging from the genotype of the host, that is, whether the host displays a resistant or susceptible phenotype, the genotype of the infectious agent, that is, virulent or non-virulent strains and many other less defi ned environmental factors. can genomic approaches be used to determine the outcome of infection? clearly, as discussed above, the technology is developing to defi ne susceptible and resistant host genotypes especially in animal models of infection but the ability to do this routinely in human populations is some way into the future. given the smaller genomes of pathogenic organisms, defi ning virulence genotypes is progressing at a faster rate ( chan, ; dorrell et al., ; fitzgerald and musser, ; kato-maeda et al., ; macfarlane et al., ; schoolnik, ; zhang and zhang, ) . expression profi ling studies have been used to investigate the differences in the host response to pathogenic and nonpathogenic strains of specifi c infectious agents. in one example, mice infected with a pathogenic strain of pneumonia virus upregulated the expected infl ammatory chemokines/cytokine profi le as well as the isg profi le but an attenuated strain of the same virus could not, although the virus replicated in the lungs of these mice to the same degree ( domachowske et al., ) . temporal profi ling of the infection process in animals will also help to defi ne the expression signatures associated with the ability of the host to clear specifi c pathogens. while considerable information and progress is being made with the approaches described above, there is now much interest in combining both a genetic screening approach with an expression profi ling approach in a new area of investigation being dubbed "genetical genomics " (reviewed in de koning et al., ; schadt et al., ] ). the rationale behind this combination is twofold: fi rst, expression profi ling data can be used to identify those genes in a chromosomal region previously identifi ed from genetic approaches, whose expression is altered between two different genotypes. these genes then immediately become strong candidates for a susceptibility gene at that genetic locus. in this way, mutations that affect the expression but not the function of a protein can also be identifi ed. second, expression profi ling combined with genotyping can identify functional pathways that can be affected by a single mutation. for example, if a mutation affects a transcription factor that controls expression of a group of functionally related genes then not only the transcription factor but the entire pathway will be identifi ed opening up better opportunities to design new therapeutics. in a prelude to these types of approaches, hume and colleagues ( wells et al., ) carried out expression profi ling studies on lps-stimulated macrophages from various mouse strains that show differential response to infection. these studies showed that while there was a common core response to tlr activation, each strain of mice showed a unique gene expression program implicating many different genetic loci in this variable response. mapping the genetic loci responsible for these different transcriptional responses will help to shed light on genetic loci that may play a role in human susceptibility to infection. these type of approaches move into the realms of " systems biology " which is the study of an organism, viewed as an integrated and interacting network of genes, proteins and biochemical reactions, focusing on all the components and the interactions among them, as part of one system. given the complexity of the immune response to infection, ultimately this type of approach may provide more answers. although genomic approaches to understanding the host response to infectious disease are still very much at the developmental stage, it is likely that such approaches will be applied to diagnosis in the near future. in fact there are already some examples of the application of these approaches. a number of studies have been described using oligonucleotide-based arrays for the detection of multiple pathogens in a single experiment (campbell and ghazal, ; dietel and sers, ; hervas, ; kostrzynska and bachand, ; pompe et al., ) . recent studies have investigated the use of expression profi ling of whole blood to determine human subjects at risk of recurrent tuberculosis ( mistry et al., ) and to identify high and low responders to lipopolysaccharide ( wurfel et al., ) . a study in macaques has used whole blood expression profi ling to examine molecular signatures of infl uenza infection ( baas et al., ) . there are many efforts currently to optimize expression profi ling for biomarker discovery and for clinical trials ( debey et al., ; shou et al., ; zheng et al., ) . there are still many hurdles to be over come in terms of application to the clinical setting including standardization of sample preparation, robustness of the microarray platform and data analysis, cost quality assurance and so on ( abdullah-sayani et al., ) . the introduction of genetic or genomic screening into the clinical setting for infectious 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( ) . insights into host responses against pathogens from transcriptional profi ling. nat rev microbiol ( ), - . this review describes a meta-analysis of expression profi ling data from the literature of cells infected with different pathogens or treated with pathogenic components. lipoldova, m., demant, p. ( ) . genetic susceptibility to infectious disease: lessons from mouse models of leishmaniasis. nat rev genet ( ), - . this paper reviews the literature on the genetic susceptibility to infection with leishmania and compares susceptibility loci to those identifi ed for other infections. cook, d.n., pisetsky, d.s., schwartz, d.a. ( ) . toll-like receptors in the pathogenesis of human disease. nat immunol ( ), - . this review describes the role of toll-like receptors in detection of pathogens and summarizes their involvement in infectious disease susceptibility. http://www.genmapp.org/ gene map annotator and pathway profi ler is a computer application designed to visualize gene expression data on maps representing biological pathways and groupings of genes. http://www.ensembl.org/index.html ensembl is a joint project between embl-ebi and the sanger institute to develop a software system which produces and maintains automatic annotation on selected eukaryotic genomes. http://www.informatics.jax.org/ mgd includes information on mouse genetic markers, molecular clones (probes, primers and yacs), phenotypes, sequences, comparative mapping data, graphical displays of linkage, cytogenetic and physical maps, experimental mapping data, as well as strain distribution patterns for recombinant inbred strains (ris) and cross haplotypes. http://www.geneontology.org/ the gene ontology project provides a controlled vocabulary to describe gene and gene product attributes in any organism. http://www.ncbi.nlm.nih.gov/geo/ gene expression omnibus is a gene expression/molecular abundance repository supporting miame compliant microarray data submissions, and a curated, online resource for gene expression data browsing, query and retrieval. http://www.genome-www .stanford.edu/ the stanford microarray database stores raw and normalized data from microarray experiments, and provides data retrieval, analysis and visualization. http://www.expression.microslu.washington.edu/expression/index. html public microarray data download site powered by expres-sion array manager. http://www.ncbi.nlm.nih.gov/projects/snp/ the national center for biotechnology information has established the single nucleotide polymorphism (dbsnp) database to serve as a central repository for both single base nucleotide substitutions and short deletion and insertion polymorphisms.recommended resources ■ key: cord- - bma authors: xu, yingying; yuen, pak-wai; lam, jenny ka-wing title: intranasal dna vaccine for protection against respiratory infectious diseases: the delivery perspectives date: - - journal: pharmaceutics doi: . /pharmaceutics sha: doc_id: cord_uid: bma intranasal delivery of dna vaccines has become a popular research area recently. it offers some distinguished advantages over parenteral and other routes of vaccine administration. nasal mucosa as site of vaccine administration can stimulate respiratory mucosal immunity by interacting with the nasopharyngeal-associated lymphoid tissues (nalt). different kinds of dna vaccines are investigated to provide protection against respiratory infectious diseases including tuberculosis, coronavirus, influenza and respiratory syncytial virus (rsv) etc. dna vaccines have several attractive development potential, such as producing cross-protection towards different virus subtypes, enabling the possibility of mass manufacture in a relatively short time and a better safety profile. the biggest obstacle to dna vaccines is low immunogenicity. one of the approaches to enhance the efficacy of dna vaccine is to improve dna delivery efficiency. this review provides insight on the development of intranasal dna vaccine for respiratory infections, with special attention paid to the strategies to improve the delivery of dna vaccines using non-viral delivery agents. majority of the current licensed vaccines for the prevention of infectious diseases are live-attenuated vaccines, inactivated vaccines, or subunit vaccines. each of them has its pros and cons. the live-attenuated vaccines can stimulate both cellular and humoral immune responses, and induce prolonged immunity that closely resembles natural infection. however, there are safety concerns associated with the use of the live attenuated virus or bacterial vaccines as they may revert to disease causing forms. it is also difficult to target multiple viral subtypes or pathogens using live-attenuated vaccines. inactivated and subunit vaccines are safer options as they cannot replicate and do not cause disease. they confer protection mainly through humoral immune responses with little or no cellular immunity. the induced immunity lasts for a shorter period of time; therefore, supplemental doses are always required. in recent years, dna vaccines have attracted considerable attention as an alternative vaccination method against infectious diseases, with the potential to provide broad immune responses similar to the live-attenuated vaccines without the risk associated with the replicating micro-organisms. dna vaccine approach relies on the in situ production of target antigens. plasmid dna encoding antigenic proteins is delivered to the appropriate tissues in the body, leading to the expression of the desired antigens, eliciting specific immunogenic responses and thereby inducing immune protection against the pathogens. since the host cells are responsible for antigen production, the natural glycosylation and folding of the protein are warranted. plasmid dna encoding different bacterial and viral antigens have been tested for their immunogenicity and protective efficacy in vivo, confirming their clinical potential [ ] [ ] [ ] [ ] . in addition, dna vaccines offer several distinct advantages over conventional vaccines. the double helical structure of dna is simple and stable at high temperature, allowing easy storage and transportation. large-scale manufacture of dna vaccines is convenient and relatively cheap. it only requires standard cloning of antigen coding sequence into plasmid vectors, avoiding the complex procedures of repeated culture and inactivation of infectious pathogens, or the purification of recombinant proteins. apart from their advantageous physicochemical properties, dna vaccines have the ability to generate the cellular immunity in addition to the humoral immunity. they are also highly flexible, encoding several types of genes including viral and bacterial antigens, as well as immunological proteins. the advantages of dna vaccines compared to conventional vaccines are summarized in table . the field of dna vaccination is developing rapidly. dna vaccines are currently approved for veterinary use against various infectious diseases [ ] [ ] [ ] [ ] . however, the results in clinical trials have been less encouraging. dna vaccines are generally safe and well tolerated in human, but the immune response is often too low to offer sufficient protection [ , [ ] [ ] [ ] . in early studies, dna vaccines alone were not able to generate t cell responses at a magnitude that was enough to protect against difficult diseases in humans [ , ] . recent attempts still failed to overcome this problem. a plasmid pthr dna hiv- vaccine candidate evaluation in phase i clinical trials on healthy volunteers showed that it had weak immunogenicity in human. no significant hiv- -specific cell-mediated immune response difference was found between vaccine recipients and placebo recipients, in addition to no hiv specific antibody production [ ] . another phase i trial of hiv vaccine using dna prime-virus vector vaccine boost strategy on healthy volunteers was proved effective in eliciting t-cell responses but incapable of inducing neutralizing antibody activities [ ] . in , a human hiv- gag dna vaccine with or without interleukin (il)- and/or il- plasmid cytokine adjuvant was reported to produce poor cellular immunogenicity with no vaccine-induced anti-gag humoral immune responses on healthy volunteers, which contrasted with the previous findings in macaques [ ] . several strategies have been introduced to optimize dna vaccines [ ] . one of them is to enhance the dna delivery efficiency, which is the focus of this review. dna delivery efficiency is dependent on the administration route and the delivery system used. mucosal surfaces are attractive sites of vaccine administration against infectious diseases as they are the portals of entry for many pathogens. vaccination at the mucosal sites where pathogens initiate infections can be more efficacious than parenteral administration as invading pathogens may be neutralized at the front lines before generating any systemic effect. in particular, intranasal vaccine has been extensively investigated in recent years. vaccination at nasal mucosa can stimulate respiratory mucosal immunity by interacting with the nasopharyngeal-associated lymphoid tissue (nalt) where large amounts of local lymphocytes are present. furthermore, intranasal delivery is a needle-free, non-invasive route of administration with the possibility of self-administration. intranasal dna vaccination has become a promising approach in offering immune protection against various pathogens that affect the respiratory system including tuberculosis, coronavirus infection, influenza and respiratory syncytial virus (rsv). in this article, the current developments of dna vaccine delivery systems that are specifically designed for intranasal administration against respiratory infectious diseases are discussed in detail. typically, dna vaccines are administered by intramuscular injection although other administration routes including intradermal, subcutaneous, oral and intranasal routes are also investigated. upon administration, somatic cells (e.g., myocytes or keratinocytes) and professional antigen presenting cells (apcs) are transfected. as the antigens are expressed intracellularly, both humoral and cell-mediated immunity can be activated to offer broad immune protection. the host-synthesized antigens become the subject of immune surveillance in the context of both major histocompatibility complexes (mhc) class i and class ii molecules of apcs. antigen expressed apcs then travel to the draining lymph nodes where they present the antigenic peptide-mhc complexes and stimulate t cells. alternatively, b cells are activated, initiating the antibody production cascades. the major advantage of dna vaccines is their ability to activate cd + t-cells, the cytotoxic t lymphocytes, which are important in controlling infections [ ] . this action is lacking in inactivated or subunit vaccines. the induction of cd + t-cells by dna vaccines can occur in two major ways: (i) direct dna transfection of the apcs such as dendritic cells (dcs); (ii) cross-presentation which occurs when somatic cells such as myocytes are transfected with dna and the expressed antigens are taken up by the apcs, or when the transfected apoptotic cells are phagocytosed by the apcs. the mechanisms of dna vaccines are illustrated in figure . it is interesting to note that immunization may occur rapidly before a dna vaccine expresses the antigens, and antigens expressed in somatic cells may not qualitatively be the major player in eliciting immune response. comparing to the secondary role of somatic cells, bone marrow derived antigen presenting cell (apc) activation is an important indicator for successful induction of dna vaccine [ ] . in this study, dna vaccine was delivered into the skin of mouse ears by gene gun. immune response was produced after the inoculation site (i.e., the ear) was rapidly removed after immunization before antigens were expressed, indicating that mobile cells are important in elaborating immunity. in another similar study, surgical removal of the injected muscle within min of dna vaccines administration did not affect the magnitude or longevity of antibody responses to the encoded antigen [ ] . again, the results confirmed the importance of apcs over somatic cells such as myocytes and keratinocytes for eliciting immune responses. early studies showed that dna vaccines are poorly immunogenic with low levels of antigen expression. to improve the immunogenicity of dna vaccines, cpg motifs are commonly employed in the construct of the plasmid backbone [ ] . bacterial or viral dna contains unmethylated cpg motifs, whereas in mammalian cells, cpg dinucleotide motifs are rare and are usually methylated [ ] . it has been demonstrated that the unmethylated cpg motifs have immunostimulatory effect and are considered by mammalian cells as pathogen-associated molecular patterns (pamps). unmethylated cpg activates innate immune cells through binding to toll-like receptor (tlr- ), which is constitutively expressed in the endosomal compartments of apcs and b cells. once bound to the dna that is rich in cpg motifs, tlr- activates the immune system by initiating pro-inflammatory response that result in the production of cytokines such as interferon (ifn)-γ and interleukin (il)- . however, it was found that tlr- deficient mice also responded to dna vaccines, suggesting that tlr- may not be the sole mediator of the adjuvant effect [ , ] . dna vaccines also interact with cytoplasmic dna sensors including tank-binding kinase (tbk ) [ ] and stimulator of ifn genes (sting) [ ] , activating tlr-independent pathways and inducing ifn-γ. these pathways are crucial in contributing to the immunogenicity of dna vaccines. although persistent antigen expression of dna vaccine is usually expected to provide long-term immune protection against infectious diseases, the effect of sustained expression of antigen must be carefully examined and controlled. it has been reported that prolonged expression of antigen may lead to the switch of type- ifn from an antiviral cytokine to an immunosuppressive cytokine [ ] , or may deplete the pool of memory t cells [ ] . to evaluate the efficacy of dna vaccine in humans, serum antibody titer or the enzyme-linked immunospot (elispot) assays are the commonly employed methods to measure the immunogenic response, although the induction of antigen-specific immune effectors by an immunization process does not imply that these antibodies or cytokines represent surrogates or correlates of vaccine efficacy. in early stage of vaccine development, in vitro serum antibodies and elispot assays are the direct detectable indicators of the clinical potential of a vaccine formulation. at later stage of development, morbidity and mortality (especially the improvement of survival rate after vaccination) in animals upon target pathogen challenge is a more certain way to confirm the protective efficacy of vaccines [ , ] , as the ultimate goal of vaccine is to prevent the targeted disease. the efficacy of vaccine such as influenza vaccine could be monitored in human during subsequent influenza epidemic season [ , ] or challenged with a controlled influenza virus [ ] . however, some lethal virus challenge studies are difficult to conduct directly on human. hence, the measurement of antibodies production and immune responses in humans remain the most direct way to assess vaccine efficacy. longer study is required to investigate if the vaccine is indeed able to prevent disease. safety is always a primary concern with any vaccine products. dna vaccines are generally considered to be safer than conventional vaccine approaches as they lack the risk of reversion to a disease causing state or secondary infection. similar to other gene therapy, the major safety issue related to dna vaccines is the risk of integration of the plasmid dna into the host genome, causing insertional mutagenesis, which may lead to the inactivation of tumour suppressor genes or activation of oncogenes, resulting in devastating adverse effects. according to preclinical and clinical studies, there is little evidence of genomic integration following dna vaccines administration, and the risk of integration is found to be significantly lower than the spontaneous mutation rate [ ] [ ] [ ] [ ] . another safety issue of dna vaccines is related to the development of anti-dna immune responses. animal studies showed that there is no increase in anti-nuclear or anti-dna antibodies after dna vaccination. in clinical trial studies, signs and symptoms of autoimmunity, and the markers of autoimmunity are sometimes monitored in the human subjects. there has been no evidence that autoimmunity is associated with dna vaccines [ , [ ] [ ] [ ] . it has been suggested that proper purification of e. coli can effectively prevent pathogenic anti-dna antibody production [ , ] . antibiotic resistance is another issue related to dna vaccines. typically, large-scale manufacture of plasmid dna involves the use of antibiotic-resistant marker. there is a safety concern that resistance to the same antibiotic might be introduced. in response to this issue, antibiotic-resistance genes in dna vaccine should be restricted to those that are not used to treat human infections. alternatively, the use of antibiotic-resistance genes should be avoided completely [ ] . another concern of dna vaccines is the development of tolerance to the encoded antigen, which appears to be age-related. newborns have immature immune system and are more likely to develop tolerance rather than protection when exposed to foreign antigens. in contrast, immunity instead of tolerance occurred when dna vaccines are administered to young animals [ ] [ ] [ ] . in recent years, there has been an increasing concern that vaccination in general may induce harmful systemic inflammation, which may lead to increase of cardiovascular risk [ ] [ ] [ ] [ ] [ ] . dna vaccine is still considered as a relatively new approach to vaccination but its potential to induce systemic inflammation must not be overlooked. it was reported that little or no local inflammatory infiltration was observed at the dna vaccine injection site, especially after the acute effects of the vaccination have disappeared [ ] . the first clinical trial of a dna-based vaccine for hiv- infection was published in in asymptomatic hiv-infected patients who were not using antiviral drugs. the immunization was well tolerated with neither local, systemic reaction nor laboratory abnormalities were detected after three doses of vaccines [ ] . in addition, no patient developed anti-dna antibody or muscle enzyme elevations. no consistent change of cd + or cd + lymphocyte counts occurred. another early experiment conducted on pigs showed that electroporation of dna vaccines was more efficient in enhancing immune response, but also stimulated inflammatory response and accompanying cellular infiltration, whereas the conventional intramuscular injection of dna vaccines only showed low gene expression and low inflammatory cell infiltration [ ] . it was suggested that improved antigen presentation was one of the possible mechanisms by which increased inflammatory cell infiltration may enhance immune responses to dna vaccines delivered with electroporation. however, the long-term safety effect was not investigated. overall, many recent preclinical studies and clinical trials have indicated that dna vaccines are generally well tolerated with good safety profile, and no systemic inflammation was reported [ , , [ ] [ ] [ ] [ ] . nonetheless, dna vaccines are relatively new vaccination approaches and yet to be approved in human use, the long term safety of their uses must be thoroughly evaluated for routine prophylactic and therapeutic use in human, especially when new delivery systems or adjuvants are introduced into the formulation. conventional vaccines are usually administered by parenteral injections which mainly target the systemic immune system, eliciting only weak mucosal immune response. when the vaccines are delivered directly to the mucosal site, mucosal immune response can be more efficiently potentiated. in particular, nasal mucosa has attracted considerable attention as the site of vaccination in recent years, including dna vaccines, due to several distinct advantages. however, there are also some formidable barriers that need to be overcome to allow successful development of intranasal dna vaccines. the intranasal route of drug administration has been frequently used to treat local conditions such as nasal congestion and allergy. intranasal administration is characterized by easy administration, rapid onset of action and avoidance of first-pass metabolism. the needle-free administration route is non-invasive and can avoid the risk of spreading blood-borne infections, which is a particular problem in developing countries. these desirable features lead to the exploration of the systemic delivery of polar drugs or biomolecules including vaccines that are not feasible in other administration routes. intranasal vaccination has been investigated for over a decade. the majority of currently available vaccines are administered by intramuscular, subcutaneous or intradermal injection. although these parenteral routes of administration are effective in inducing systemic immune responses, they are ineffective in inducing local immunity at mucosal sites. as many as % of pathogens infect human through the mucosal surfaces [ ] . mucosal vaccination could provide better protection than injectable vaccines against infectious diseases by inducing both systemic and mucosal immunity [ , ] . since the strongest immune response is usually induced at the vaccination site and the adjacent mucosal sites, intranasal immunization is able to elicit protective immune response effectively in the lungs and the upper respiratory tract [ ] . nasal mucosa appears to be an appropriate site of vaccine administration against respiratory infectious diseases, not only because the nasal cavity is the first site of contact with inhaled macromolecules and a common site of infection by respiratory pathogens, it can also stimulate respiratory mucosal immunity by interacting with the nalt. current, licensed intranasal vaccines include flumist ® , a live-attenuated vaccine that targets influenza types a and b [ ] ; and nasovac ® , a live-attenuated vaccine that targets h n influenza virus [ ] . apart from live-attenuated vaccine, intranasal route of administration is also favorable to protein-based vaccination, as evidenced by many studies including the intranasal pneumococcal protein immunization against pneumonia [ ] , and a recent study on the intranasal respiratory syncytial virus (rsv) vaccine based on a recombinant fusion protein [ ] . with the success of intranasal live-attenuated virus vaccine and the promising effect of protein-based vaccine, it is highly plausible that dna vaccines can adopt the same delivery route to achieve efficient immunization. it is well established that mucosal vaccination can induce humoral and cell-mediated immune response systemically as well as at mucosal sites [ , ] . immune response induced by mucosal vaccination is mainly initiated at specific mucosa-associated lymphoid tissue (malt). the malt lining the nasal cavity is known as the nasopharyngeal-associated lymphoid tissue (nalt), which include the waldeyer's ring of tonsils, adenoids and a collection of isolated subepithelial lymphoid follicles [ ] . the nalt is rich in immunocompetent cells, including b cells, t cells and phagocytic apcs such as macrophages and dcs [ ] . in addition, the overlying epithelium of mucosal follicles forms a specialized cell layer. these cells have microfolds on their apical surface and are known as microfold cells (m cells). m cells play a crucial role in the initial phase of induction of mucosal immune responses. therefore, m cell targeting is an important strategy to achieve mucosal immunity [ , ] . m cells are efficient in taking up particles from the epithelial surface, transporting them across the cells and releasing them to the underlying extracellular space. this process is known as transcytosis. at their basal surface, the cell membrane of m cells is extensively folded around the underlying immune cells including b cells, t cells and apcs, which take up the particles released from m cells and process them for antigen presentation. the initiation of mucosal immune response is summarized in figure . upon b cells activation following nasal vaccination, the production of antigen-specific secretory immunoglobulin a (siga) is triggered. siga is a critical component in the mucosal immune system. it is protease resistant, and can effectively bind and neutralize pathogens and their toxic products on the nasal mucosa surface despite the protease rich environment, thereby preventing the pathogens from breaching the mucosal barrier [ ] . local immunoglobulin g (igg) production is also detected after mucosal vaccination [ ] and partly contributes to the neutralization of pathogens. however, igg concentration is around - -fold lower than that of the siga due to its susceptibility to protease degradation [ ] . indeed, siga provides the first barrier to pathogens invasion, so induction of potent siga response is an important goal of mucosal vaccination. in addition, nasal immunization can also result in the production of serum iga and serum igg, which can potentially neutralize pathogens that enter the mucosa and prevent systemic spread. when the dcs at the mucosa are presented with antigens, the activated cells may migrate to the proximal draining lymph node and disseminate immune responses to other sites of the body. apart from the humoral immune response, cell-mediated immune response is also induced after mucosal vaccination. although the cytotoxic t cells in the mucosal tissues may not prevent pathogen entry, they are crucial for the clearance of pathogens [ ] . overall, cells of nalt are involved in the regulation of both humoral and cell-mediated immune responses locally and also systemically, offering a broad immune response. since the nasal mucosa is an important portal of entry for respiratory pathogens, the nasal route has become attractive for the administration of vaccines by reinforcing the nasal mucosal immune response. the defense mechanism of the nasal cavity presents a significant barrier to the entry of pathogens and potentially harmful substances; however, it has also become an important barrier to intranasal dna vaccines. the nasal mucosa, which constitutes the outmost layer of the nasal passage, consists mainly of ciliated columnar cells, non-ciliated columnar cells, goblet cells and basal cells. the proportions of these cells vary in different regions of the nasal cavity. nasal mucus, which is produced by goblet cells and submucosal glands, provides a protective physical barrier to foreign materials. it is a highly viscous, gel-like heterogeneous mixture that contains glycoproteins, enzymes, immunoglobulins, salts, proteins and lipidic components [ ] . dna vaccines that are administered to the nasal cavity have a propensity to be trapped by the nasal mucus, leading to enzymatic degradation. the effect of mucus depends on its viscosity and pore size, which affect the diffusivity of agents delivered to mucosal surfaces. in addition, the entrapped dna vaccines may also be removed by the mucociliary action of the ciliated cells that drives the overlying mucus layer continuously towards the nasopharynx, clearing the mucus from the nasal passage, resulting in short residence time at the mucosal surface. another challenge of intranasal vaccine is that the vaccine formulation may be diluted in mucosal fluids, and bulk fluid may limit effective deposition onto the epithelium of the mucosal system. to overcome these barriers, a safe and efficient dna delivery system must be developed. ideally such delivery system should target the mucosal apcs for antigens processing that lead to specific b and t cell activation. the ultimate goals of dna delivery systems are to facilitate the uptake of dna to the target tissues and cells, protect dna from enzymatic degradation, increase the residence time of the formulation in the nasal cavity, enhance the expression of the antigens and to increase the immune response without compromising safety. the different dna vaccine delivery systems currently being investigated for intranasal administration are discussed in detail in section . despite the numerous merits of intranasal immunization, the potential hazard of nasal vaccines must not be overlooked. the concern of the safety of intranasal vaccination was raised after an intranasal inactivated influenza vaccine called nasalflu, developed by berna biotech, was found to be associated with bell's palsy, a temporary neurological paralysis of one side of the face [ ] . nasalflu consists of influenza virosomes which are formulated to contain hemagglutinin (ha) and neuraminidase (na) antigens, as well as heat-labile enterotoxin (lt) from e. coli as mucosal adjuvant. since parenteral administration of inactivated influenza vaccine did not confer an increased risk of bell's palsy, nor the natural influenza virus infection, it was soon concluded that adjuvant lt from e. coli. was the culprit of these cases. for this reason, flumist ® and nasovac ® -both are intranasal live-attenuated influenza vaccines without enterotoxin as adjuvants-do not appear to confer an increased risk for this condition. understanding the pathogenesis of the bell's palsy that was connected to nasalflu has become an important research focus for vaccine development. following intranasal administration in mice, enterotoxins were found in the olfactory nerve and the olfactory bulb for an extended period. since the olfactory epithelium is the only part of the central nervous system (cns) that is exposed to the external environment, drugs and nanoparticles, including intranasal vaccines, may bypass the blood brain barrier and enter the cns through olfactory transmission. there is a reason to concern the neurotoxic effects of vaccine containing enterotoxin adjuvant for intranasal administration. while the nasal delivery of neuronal-binding lt-derived adjuvants is inadvisable [ ] , other toxin-derived adjuvants such as cholera toxin-derived cta-dd and double mutant cholera enterotoxin (ct) are claimed to be safe and effective adjuvant candidates without causing any inflammation or cns toxicity [ , ] . nevertheless, thorough evaluation must be performed with the use of toxin derivatives as intranasal vaccine adjuvants. tremendous efforts are now focusing on the development of alternative adjuvants with better safety profile. mankind has been haunted by respiratory infectious diseases for aeons. they have created public health concerns since ancient times. with the emergence of new or drug-resistant strains, it is becoming a challenge to protect the public from infections using conventional vaccine methods. dna vaccines have huge potential for the prevention of respiratory infections due to their ability to offer broad immunity, the relatively rapid process of designing new dna vaccine construct and the possibility of large-scale production in a short period of time. in this section, four pathogens that cause severe diseases in the airways are highlighted, including tuberculosis, coronavirus, influenza and respiratory syncytial virus, with a brief discussion of current dna vaccine development against these infections. tuberculosis (tb) is a bacterial infectious disease caused by mycobacterium tuberculosis which is transmitted by respiratory aerosols. tb has become a major public health problem that threatens the progress made in tb care and control in the world. the only tb vaccine currently available is an attenuated strain of mycobacterium bovis, bacillus calmette-guérin (bcg), developed in the s. however, its efficacy against adult pulmonary tb remains controversial [ ] . with the emergence of drug-resistant tb and increasing rates of hiv/aids and tb co-infection, a new effective tb vaccine is urgently in need. effective protective immunity to mycobacterium tuberculosis requires cell-mediated immune responses, including both cd + and cd + t cells [ ] [ ] [ ] . since dna vaccines have the ability to induce strong cellular immunity, it has become an attractive vaccine approach against tb. the first two studies that reported promising protective effect with dna vaccine against tuberculosis were conducted in mice using plasmid dna encoding antigen a (ag a) of mycobacterium tuberculosis [ ] and the kda heat-shock protein of mycobacterium leprae (hsp ) [ ] . other different antigens such as ag b esat- , il- , mpt , psts- and other fusion proteins have also been explored as dna vaccines against tuberculosis [ ] [ ] [ ] [ ] [ ] [ ] . most of these dna vaccines encode mycobacterial proteins that are either secreted in mycobacterial culture filtrate or exposed on the mycobacterial cell-wall surface. coronaviruses (cov) are potentially lethal pathogens, characterized by the presence of spike proteins on the viral surface. two new strains, severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov), have been identified. both of them could cause acute respiratory distress syndrome (ards) and are associated with high mortality rates [ ] . cov vaccines have historically exhibited poor capacity for cross-protection [ ] , the development of safe, broad spectrum and effective vaccines that can be rapidly made available during an emerging epidemic is required. currently, there are no approved vaccines for human cov infections, and most of the studies have focused on the sars-cov. spike and nucleocapsid proteins, which are the immunodominant cov proteins, are the antigens of interest for vaccine development [ , ] . dna vaccines that encode nucleocapsid protein induced strong cell-mediated immunity but are not protective after high titer of viral challenge [ , ] . in addition, nucleocapsid dna vaccine could induce delayed-type hypersensitivity even in the absence of an antibody response. this effect was not observed with spike protein dna vaccines. influenza is caused by orthomyxoviruses which are rna viruses that affect mainly the upper respiratory tract. in recent years, zoonotic or variant influenza has become a serious threat to human health, including the avian influenza virus h n and h n , and the swine influenza virus h n and h n . although these animal viruses are distinct from human influenza viruses and do not usually transmit between humans, they may still occasionally infect humans and cause severe pneumonia and even death. furthermore, if such a virus acquired the capacity to spread easily among people, it could start an epidemic or even a pandemic [ , ] . to protect the populations from influenza infection, highly effective, broad-spectrum influenza vaccines that could be prepared rapidly are in demand. current influenza vaccines mainly target the induction of antibodies against the viral glycoproteins, particularly surface glycoproteins hemagglutinin (ha) and neuraminidase (na). antibodies to ha neutralize the infectivity of the virus while antibodies to na prevent the release of the virus from the infected cells. apart from surface glycoproteins, internal proteins such as nucleoprotein (np) and matrix protein (m ), as well as the ion channel protein (m ), which are highly conserved between and within different subtypes, have also become very attractive target antigens for vaccines to provide broad, cross-strain protection [ , ] . dna vaccines can potentially solve the mismatch problem by shortening the lag time [ ] , which is particularly useful when facing influenza pandemic. in addition, the strategy of combined immunization with dna vaccines encoding surface protein (e.g., ha) and internal protein (np and m ) could offer better protection against influenza virus than single dna vaccine alone in mice and ferrets [ ] [ ] [ ] [ ] . with the success of dna vaccines in various animal models, several phase i & ii clinical trials on dna vaccine against influenza have been being carried out. results so far have been encouraging, demonstrating both safety and immunogenic response in human [ , , ] . respiratory syncytial virus (rsv) is a single stranded rna pneumovirus which belongs to paramyxoviridae family. it accounts for one of the leading pathogeneses of lower respiratory tract infections and hospitalization in infants and young children [ ] , as well as the elderly and high-risk population [ ] . immunity against rsv is dependent on the induction of antibody responses. in addition, cd + t cells responses have been shown to reduce disease severity [ ] . although maternal antibodies appear to protect infants against infection, their amount gradually decreases within the first few months of life. human rsv lacks an approved vaccine or an antiviral therapy. to prevent infant and childhood infection, vaccine should be able to induce immune responses rapidly after birth. this could be a challenging task because the immune system at the first few months of life is immature, and the persistence of maternal antibodies may limit the induction of infant antibodies responses. three rsv proteins, namely the fusion (f) protein, attachment glycoprotein (g) and matrix protein (m ), are the leading candidates for rsv vaccine development [ ] [ ] [ ] [ ] [ ] [ ] [ ] . delivery is one of the major barriers to dna vaccine. administration of naked dna is usually inefficient with only a small fraction of dna being taken up by the cells and subsequently expressed [ ] . this is because dna is a negatively charged, hydrophilic macromolecule; it is incapable of crossing the biological membrane unassisted. therefore, a safe and efficient dna delivery system is sometimes employed as adjuvant to facilitate efficient cellular uptake of dna vaccines, promote dna release inside the cells, induce high level of antigen expression and hence immune responses. physical method such as gene gun, also known as the particle-mediated epidermal delivery, has been studied to deliver dna to the skin [ , [ ] [ ] [ ] . gold beads coated with dna vaccines are discharged directly into the cytoplasm and nuclei of skin cells. this method of delivery has enjoyed some success, but is not applicable for intranasal administration. considerable efforts have been made to improve the efficacy by developing effective dna delivery systems for intranasal vaccines. formulation of dna vaccines in synthetic non-viral vectors such as polymeric nano-/micro-particles and liposomes has been reported to increase the uptake of plasmid dna by cells, increasing immunogenicity in animal models and humans. additional adjuvants may also be used to further improve the immunogenicity of these delivery systems. the elicitation of immune responses of dna vaccines mainly relies on professional apcs that present antigens to both b cells and t cells. to ensure good immune response, the dna vaccine delivery systems should be able to target apcs. in addition, m cells in nalt, which is a major site of pathogen entry, are also a target of dna vaccine. apcs are a heterogeneous group of immunocompetent cells that mediate immune response by processing and presenting antigens to the t cells. t cells recognize only antigenic peptide fragments on the surface of apcs through the t cell receptors. helper t cells recognize antigen in association with class ii mhc proteins, whereas cytotoxic t cells recognize antigen in association with class i mhc proteins. an additional co-stimulatory signal is then produced by the apcs, leading to the activation of t cells. non-professional apcs lack the co-stimulatory signaling, and therefore do not simulate t cells sufficiently. there are three types of professional apcs, namely dcs, macrophages and b cells. among them, dcs have the broadest range of antigen presentation and are considered as the most efficient cells for induction and regulation of immune responses. they play a central role in bridging the innate immune system with the adaptive immune system [ ] [ ] [ ] . to achieve efficient dna vaccination, it is logical to target the plasmid dna to dcs where the encoded antigen could be expressed endogenously. dcs express a large number of surface receptors such as c-type lectin receptors (clrs) and tlrs, which are engaged in the recognition of pathogens. it has been reported that targeting antigens to receptors on dcs can significantly enhance immune responses [ ] . these receptors could be exploited for dna vaccine targeting with the aid of antibodies or natural ligands. in particular, clrs are endocytic receptors which recognize carbohydrate structures that resemble pathogen cell-wall components. they are responsible for internalizing pathogens. one of the most commonly studied receptors for vaccine targeting is the dc c-type lectin receptor (dec- ) which is specifically expressed on dcs. however, ligands for dec- have yet to be identified. several studies have demonstrated the employment of anti-dec- antibodies to achieve dc targeting for dna vaccines, including intranasal immunization [ ] [ ] [ ] . another identified dc-specific target is c-type lectin domain family member a (clec a). activation of clec a leads to the stimulation of antibody production [ ] . alternatively, dcs could be targeted by using the natural ligands to the mannose receptors [ ] . however, the effectiveness of these targeting components in mucosal vaccines remains to be investigated. there are also several molecules being investigated to target apcs in general. one of the most widely studied molecules is the flt ligand. flt ligand is a growth factor that stimulates the proliferation of hematopoietic cells. it binds to the fms-like tyrosine kinase receptor flt . flt expression is, in hematopoietic tissue, restricted to cd + progenitors, including dc progenitors. in vivo treatment of flt ligand is found to up-regulate the number of dcs, but not their activation [ , ] . furthermore, flt ligand treatment could also enhance immune response when delivered via the mucosal route [ ] . it has been reported that when plasmid dna encoding flt ligand was co-administered with plasmids encoding protein antigens, effective immune responses were induced [ ] . in addition to its apcs targeting ability, flt ligand is an efficient and safe mucosal adjuvant that facilitated expansion of dcs following nasal administration [ , ] . as discussed in section . , improving m cells uptake is another strategy to enhance vaccine immunity. effective mucosal immunity often correlates with the uptake of antigen by mucosal inductive tissues, such as nalt in the upper respiratory tract following intranasal immunization. since m cells are responsible for antigen sampling on the mucosal surface for eventual antigen presentation to mucosal b and t cells, targeting of vaccines to m cells can be an effective method to achieve strong immune response. particle size is an important parameter for m cell uptake. a number of studies have been conducted to identify the optimal particle size for cellular uptake of the mucosal system. some studies suggest that particle size of less than μm is optimal for oral vaccine delivery for peyer's patch m cell uptake [ ] [ ] [ ] . another nalt nanoparticle uptake study also suggested that particles with sub-micron size are optimal for mucosal m cells uptake [ ] . it is generally accepted that nalt m cells can uptake nanosized particles rapidly with no definite size range being established [ ] . apart from cellular uptake, particle size also affects the kinetics of lymphatic drainage. it appears that nanoparticles less than nm are more readily transported by the draining lymph compared to larger particles [ ] . apart from controlling particle size to achieve specific targeting passively, inclusion of targeting ligand could also increase uptake by m cells [ , ] . a number of pathogens including reovirus [ ] , salmonella typhimurium [ ] and mycobacterium tuberculosis [ ] target m cells as a mode of entry into the host. by identifying the key molecules expressed by these pathogens that are crucial for their invasion, it would be extremely helpful to design an effective delivery system for m cells targeting [ ] . one example is related to reoviruses which target m cells using their surface protein sigma- (σ ). in this regard, wu et al. reported an m cell targeting dna vaccine delivery system consisting plasmid dna and the recombinant protein σ as targeting ligand which was covalently attached to poly-l-lysine (pll) for intranasal vaccination in mice. the results showed significant mucosal siga production as well as enhanced cell-mediated immunity [ ] . other ligands such as co- peptide [ ] , claudin targeting peptide [ , ] and m cell specific monoclonal antibody (nkm - - ) [ ] have been investigated for m cell targeting in mucosal protein vaccine, with the potential to be explored for mucosal dna vaccine delivery. however, mucosally induced tolerance may develop with m cell targeting delivery system. following nasal administration of protein σ genetically conjugated with ovalbumin, systemic unresponsiveness was induced instead of mucosal iga immunity [ ] . therefore, special attention must be paid with the development of m cell targeting delivery system. high versatility is one of the attractive features of polymer-based dna delivery systems. cationic polymers can form complexes (polyplexes) with nucleic acids through electrostatic interaction. polymer synthesis is relatively cheap and is easy to scale-up. particle size and surface properties of polymeric particles can be controlled by using different polymers and fabrication methods in order to optimize their cellular uptake and transfection efficiency. the polymeric particles can also be modified to include specific function groups or ligands to enhance immune responses. polyethylenimine (pei) (figure ) is one of the early generation polymers being investigated for gene delivery. it has high transfection efficiency and is frequently regarded as the gold-standard of non-viral gene delivery vectors. pei has high ph buffering capacity, which allows its cargo to escape from endosomal entrapment via a mechanism known as "proton sponge hypothesis" [ ] . transfection efficiency of pei depends on its molecular weight and the level of branching. shim et al. described the use of a simple method to prepare pei ( kda)-dna complexes for vaccine delivery [ ] . plasmid dna encoding sars-cov spike protein without transmembrane domain was employed in the study. mice that were immunized intranasally with the pei-dna vaccines produced significantly higher systemic spike protein specific igg and mucosal secretory iga in the lung compared to those immunized with naked dna. furthermore, cellular immune responses were detected with an improvement of specific t cell responses. in another study, torrieri-dramard et al. demonstrated the utilization of pei (in vivo-jetpei ® ) as dna vaccine carrier for intranasal administration [ ] . plasmid dna encoding ha from influenza a viruses was used. the intranasal administration of the pei/dna vaccines induced cellular and humoral immune response capable of providing protective immunity against a divergent virus of h n subtype in mice. the protection could be further improved by including the plasmid dna encoding na. although pei appeared as a promising vector for gene delivery including dna vaccines, the cationic pei is highly charged and non-biodegradable, and it often encounters toxicity problems which cannot be ignored. in this regard, many groups are developing low toxic or biodegradable pei derivatives for gene delivery application. mann et al. has developed a pei derivative, deacylated pei (dpei), as dna vaccine delivery agent [ ] . dpei is a nearly fully hydrolysed linear pei with % additional free protonatable nitrogen atoms, enabling more efficient binding with dna, reduced toxicity and high transfection efficiency. it is an effective dna vaccine carrier for pulmonary delivery to elicit both systemic and mucosal immune responses, and offers protection against influenza challenge in vaccinated mice. this system has a potential to be exploited for intranasal administration. (pei) (a,b) , chitosan (c) and poly(lactic-co-glycolic acid) (plga) (d). chitosan (figure ) is a natural polysaccharide that has been frequently studied for drug delivery. it is derived from chitin which is found abundant in crustacean. it is biodegradable and biocompatible with low toxicity [ , ] . the properties of chitosan can be tuned by changing its molecular weight and degree of deacetylation. because of its cationic nature, chitosan has strong binding affinity with nucleic acids, making it a suitable candidate for dna delivery agent. in addition, chitosan and its derivatives are found to display strong mucoadhesive property, making them particularly suitable to facilitate intranasal delivery [ ] [ ] [ ] . moreover, chitosan was also reported to have an immunostimulating effect, such as increasing the accumulation and activation of macrophages, promoting resistance to infections by cytokines, and enhancing cytotoxic t cell response [ , ] . a number of studies have described the use of chitosan nanoparticles to deliver dna vaccine formulation for intranasal administration. kumar et al. reported the exploitation of chitosan nanoparticles to deliver dna vaccine against acute respiratory syncytial virus (rsv) infection [ ] . a cocktail of plasmid dna encoding a number of rsv antigens was used to complex with chitosan to form nanoparticles. following the nasal vaccination in mice, high levels of serum igg and mucosal iga antibodies, as well as cytotoxic t cells responses were induced. there was also an elevated lung-specific production of ifn-γ with antiviral action. a single dose of dna vaccine was able to decrease the rsv titers by -fold on primary infection. similar study was performed by another group who described the use of chitosan nanoparticles to deliver plasmid dna encoding a t cell epitope from the m protein of rsv [ ] . it was found that intranasal administration of the formulation in mice induced specific cytotoxic t cell response that was comparable to those induced via intradermal immunization. following rsv challenge of the nasal immunized mice, the virus load in lungs was significantly reduced. in a recent study, raghwanshi et al. investigated a sophisticated dc targeted chitosan nanoparticle system for nasal dna immunization against sars-cov [ ] . the chitosan nanoparticles were surface functionalized with ligands to achieve dc selective targeted delivery. dec- receptor is c-type lectin receptor found in dcs for recognition and uptake of pathogens. the authors developed a bifunctional fusion protein (bffp) vector which consists of truncated core-streptavidin fused with anti-dec- single chain antibody. the core-streptavidin arm of the fusion protein binds with biotinylated chitosan nanoparticles while anti-dec- scfv imparts targeting specificity to dc dec receptor. plasmid dna encoding nucleocapsid protein of sars-cov was loaded into the chitosan nanoparticles. following intranasal administration of the dc targeted nanoparticles in mice, the levels of mucosal iga and systemic igg against nucleocapsid proteins were significantly enhanced, whereas no mucosal and systemic immune responses were detected when the naked plasmid dna was intranasally administered. the results showed that such dc targeting delivery system could be a promising strategy for low-dose nasal dna vaccines. to enhance the transfection efficiency of chitosan for intranasal administration, thiolated chitosan derivative has been introduced. thiolated chitosan derivative has strong mucoadhesive properties due to the formation of disulphide bonds between thiol groups of the modified polymer and cysteine-rich subdomains of glycoproteins in the mucus layer, leading to an improvement in mucoadhesion of up to -fold when compared to unmodified chitosan [ ] . improved and sustained gene expression could be achieved both in vitro and in vivo with thiolated chitosan derivative [ ] . this technology has a huge potential to be adopted for intranasal dna vaccine delivery. poly(lactic-co-glycolic acid) (plga) (figure ) is a synthetic biodegradable copolymer that has been extensively investigated for the delivery of different therapeutic agents including proteins and nucleic acids [ ] . due to its biocompatibility and excellent safety profile, plga is approved by the fda in various drug delivery systems for human use. since the degradation rate of plga can be adjusted by modifying the molecular weight of the polymer and the lactic acid to glycolic acid ratio, the rate of drug release can also be controlled accordingly. however, the negative charge and hydrophobic nature of plga limit its interaction with the negatively charged dna. cationic surface modification of plga micro/nanoparticles using polycations such as pei and chitosan can overcome this problem and allow efficient nucleic acids delivery [ ] [ ] [ ] [ ] . this strategy has also been applied to the delivery of dna vaccines. oster et al. first employed the use of micro-particles consisting plga and pei as dna vaccine carrier for injection [ ] . later on, wang et al. reported the use of chitosan coated plga nanoparticles to deliver plasmid dna encoding fmdv (foot-and-mouth disease virus) capsid protein together with il- as mucosal adjuvant for intranasal vaccination [ ] . chitosan coated plga nanoparticles were first prepared by emulsion-diffusion-evaporation technique [ ] , followed by the incorporation of plasmid dna to the nanoparticles by simple complexing. the samples were then freeze-dried with mannitol before use. after intranasal administration in guinea pigs and rats, both cellular and humoral immune responses were detected. il- was also found to be an effective mucosal adjuvant which significantly enhanced mucosal and systemic immune responses. more importantly, the vaccines could offer some immune protection to animals against fmdv challenge. liposomes are vesicles comprised of phospholipid bilayers. they have been extensively investigated for delivering dna into mammalian cells as well as vaccine adjuvants. the duo functions make them an excellent carrier system for dna vaccine [ ] [ ] [ ] [ ] . for dna delivery, the negatively charged plasmid dna can be absorbed to the surface of cationic liposomes through electrostatic interaction to form complexes. alternatively, dna can be encapsulated in the aqueous core of the cationic, non-ionic or anionic liposomes. in general, transfection efficiency of cationic liposomes is superior to their non-ionic or anionic counterparts [ ] , whereas anionic liposomes provided enhanced antibody responses [ ] . surfaces of the liposomes can be decorated with targeting ligands or antigenic components to improve the immune response in vaccine formulation [ ] . currently, there are at least two approved liposomal vaccine formulations on the market for antigen delivery, including inflexal ® v (influenza vaccine) and epaxal ® (hepatitis a vaccine). both formulations employed the virosomes technology (figure ) , in which the viral proteins are bound to the surface of a liposome in an array, similar to what is seen on viral particles [ ] . the idea is to create a safe viral-like particle that can induce strong protective immune response. similar technology could be applied to dna vaccines to improve immunogenicity. in fact, liposomes on their own could elicit immune response even in the absence of other antigens. it has been demonstrated by lay et al. that dotim (octadecenoyloxy-ethyl-heptadecenyl- -hydroxyethyl imidazolinium chloride)/cholesterol cationic liposome-dna complexes (jvrs- ), in which an empty plasmid dna vector was incorporated, were able to induce high levels of antibody and t cell immunity in mice and non-human primates [ ] . since lipid composition, particle size, surface charge and dna entrapment efficiency of the liposomes can affect their immunogenicity and potency, these parameters must be carefully characterized and controlled. the major limitation of liposome as dna vaccine carrier is long-term stability, as lyophilization of liposomes may not be always possible [ ] . [ ] . plasmid dna encoding ag a was complexed with the lipids. following intranasal immunization in mice, naked dna was completely ineffective, probably due to the degradation of the dna by mucosal nuclease. the lipid-dna formulation was capable of inducing a weak cell-mediated immune response, and no humoral immune response was detected. the co-lipid intranasal formulation was compared with another cationic lipid formulation, vaxfectin, which was used for intramuscular administration. it was found that the intramuscular formulation was able to induce a better immune response. however, combining intranasal and intramuscular administrations resulted in stronger immune responses in the lungs. considerable improvement is needed to further develop the formulation for intranasal use. rosada et al. developed another liposome-based formulation of dna vaccines against tb [ ] . the non-toxic, cationic liposome, epc/dope/dotap (egg phosphatidylcholine/ , -dioleoyl-snglycero- -phosphoethanolamine/ , -dioleoyl- -trimethylammonium-propane) was used. plasmid dna encoding the kda mycobacterial hsp was either entrapped inside or complexed with the cationic liposomes, and the intramuscular and intranasal routes of administration were compared in mice. when administered intramuscularly, both liposomal formulations were ineffective in preventing tuberculosis infection in mice even after two doses. on the contrary, the complexed liposomal formulation of dna vaccine was able to offer protection against infection even after a single dose, with a significant reduction of colony forming unit (cfu) in lungs after the immunized mice were challenged with mycobacterium tuberculosis. however, four doses of intranasal administration of naked dna vaccines failed to offer any protection. the authors reasoned that the intranasal vaccination enhance the immune response by stimulating the mucosal immunity, but the naked dna failed to cross the mucosal barriers in the nasal cavity, demonstrating the importance with the use of delivery carrier for intranasal dna vaccination. apart from tb vaccine, there are studies that reported the use of liposome to deliver influenza dna vaccine [ , ] . cationic liposomes dodac/dope/peg ( , -dioleoyl-sn-glycero- - phosphoethanolmine/dioleylphosphatidylethanolamine/polyethylene glycol) were used to encapsulate plasmid dna encoding influenza virus ha. after intranasal immunization in mice, the liposome system was effective at eliciting both igg and iga humoral responses systemically. local iga level was enhanced. cell-mediated immune response was also successfully induced. in addition, the immunized mice were able to withstand a lethal challenge of influenza virus. on the other hand, intramuscular immunization of the same system enhanced igg level only with no effect on iga level either locally or systemically. intranasal administration of naked dna failed to induce antibody response. the promising results demonstrated the potential of the intranasal liposomal dna vaccine system. to improve the dna vaccine delivery efficiency of liposomes for intranasal administration, khatri et al. modified the surface of liposomes by coating with glycol chitosan [ ] . the major function of glycol chitosan is to provide mucoadhesive and immune stimulating property [ ] . in the study, cationic liposomes, pc/dope/chol (phosphatidylcholine/dioleylphosphatidylethanolamine/cholesterol) were used to entrap plasmid encoding the s region of hepatitis b antigen, and the glycol chitosan was adsorbed on the liposome surfaces through electrostatic interaction and hydrogen bonding. following intranasal administration in mice, the surface modified liposomes could elicit both humoral mucosal and cell-mediated immune responses which were better than the uncoated liposomes. such system has the potential to be exploited for intranasal dna vaccine of respiratory infectious diseases. a number of studies have already demonstrated the potential of liposomal dna vaccine system for intranasal administration, and some could offer considerable immune protection against respiratory infections in animals. however, the lipid composition of different liposomal systems varied greatly, and currently there is a lack of knowledge of how the composition may affect the immune response. to enable the utilization of liposomal dna vaccine for clinical application and approval, a better understanding of how these factors govern the efficacy and immunity of the liposomal delivery system must first be sought. to further enhance the immune responses of the dna vaccines, adjuvants are included in the formulation in many studies. adjuvants are generally defined as agents that could enhance the immune response of the vaccinated subjects to an antigen. in dna vaccine, since the delivery of dna is a major hurdle, dna carrier system using bacterial, viral or non-viral vectors, as well as the cell specific targeting ligands, which are discussed above, are also considered as dna vaccine adjuvants. the summary of dna vaccine adjuvants being investigated are shown in table . in this section, proteins and other macromolecules with immunopotentiating properties but are not directly involved in the delivery of dna are discussed, especially those that are commonly employed for intranasal vaccination. enterotoxins are protein exotoxin released by pathogens that infect the gut. enterotoxin-based mucosal adjuvants are the most potent and well-established strategy for the induction of both mucosal and systemic immunity to co-administered protein antigens [ ] . heat-labile enterotoxin (lt) from e. coli and cholera enterotoxin (ct) are very potent adjuvants but they are too toxic to be used in human. therefore, detoxified mutants of enterotoxins have been produced by site-directed mutagenesis and they are extensively investigated as adjuvants for mucosal vaccine including intranasal vaccine. intranasal antigen immunization with lt mutant adjuvants can provide effective protection against infectious diseases in animals [ ] [ ] [ ] [ ] [ ] . it is suggested that the lt mutant adjuvants could induce potent cytotoxic t lymphocyte responses. the mechanism of action is believed to arise from enhanced permeation of antigens across epithelial barriers and a marked increase in antigen presentation by apcs [ ] . mutants of ct have also showed strong adjuvant activity [ , , ] , and could retain good adjuvant function when administered intranasally [ ] . it is expected that lt mutants and ct mutants have similar mechanisms of adjuvant activities [ ] . the major concern with the intranasal administration of mutant lt or ct is that these toxin derivatives may gain access into the central nervous system through the olfactory nerve. it has been reported that both native and mutant lt used as adjuvants were associated with the development of bell's palsy following intranasal delivery in humans [ , , ] . the risk of enterotoxin as mucosal adjuvant for intranasal administration is already discussed in section . . lipopolysaccharides (lps) are the major component of the outer membrane of gram-negative bacteria, and could elicit strong immune response. however, they are also highly toxic. in order to make them safe and suitable as vaccine adjuvants, lps derivatives are produced to reduce the endotoxic effect but to retain their immunostimulatory function. monophosphoryl lipid a (mpl) is one example of lps derivative that is investigated as vaccine adjuvant. mpl is prepared by removal of a phosphate and fatty acid group from the lipid a of salmonella minnesola. mpl is thought to interact with toll-like receptor (tlr- ) on apcs. it has been demonstrated that mpl could activate macrophages, increase their cytokine secretion and hence immune response [ ] [ ] [ ] [ ] [ ] . regarding safety, mpl appears to retain the immunogenic activity of lps but with significantly reduced toxicity [ ] . mpl has been extensively evaluated in the clinic as adjuvants for various diseases including infectious diseases with an acceptable profile of adverse effects [ ] . mpl has been used successfully as a mucosal adjuvant when formulated with liposomes or virosomes for intranasal administration in animals [ ] [ ] [ ] . cytokines are small proteins that are important in regulating immunological response by recruiting and stimulating t cells, or by directly acting on infected cells. they have potential to be natural adjuvants for dna vaccines [ ] . cytokines that have been evaluated as possible dna or intranasal vaccine adjuvants include il- , il- , il- , il- , il- and gm-csf [ , , , ] . in particular, il- is found to be an effective mucosal adjuvant [ , ] . lynch et al. demonstrated that intranasal administration of pneumococcal polysaccharide conjugate vaccine in the presence of il- was able to enhance systemic and mucosal immune responses to pneumococci in mice [ ] . however, cytokines have short half-life in vivo and poor stability. they are also expensive and are associated with dose related toxicity. therefore, these molecules may not be suitable for use as adjuvants in vaccines designed to protect against infectious diseases [ ] . nevertheless, intranasal administration of il- induced less toxicity than parenteral administration [ ] . alternatively, cytokines can be expressed from plasmid dna to allow long-lasting expression in vivo and to reduce the cost of production [ , ] . with the emergence of new or antimicrobial-resistant bacteria and viruses, and the ease of transmission, especially the respiratory pathogens, respiratory infections are becoming serious threats to human health. safe and effective vaccines are important to safeguard public health. intranasal dna vaccination appears to be a promising non-invasive approach to provide protection against various infectious diseases. evidence shows that intranasal dna vaccine could elicit strong and long-lasting humoral as well as cell-mediated immune responses in many animal models. dna vaccines are already successfully used in veterinary products for protection against infections, but their immunogenicity needs to be further enhanced to make them suitable for human use. improving dna delivery and formulation is one of the several strategies to enhance the immune response. various studies have demonstrated that significant improvement of immune response that could be achieved by the employment of dna carrier system, or to target the dna vaccines to apcs. dna vaccines generally have good safety profile, but the potential toxicity associated with dna delivery systems, especially when they are used at high concentration, must not be neglected. dna vaccines may circumvent many problems associated with conventional vaccines such as high costs of protein vaccine purification and bacterial/viral inactivated or attenuated process, the incorrect folding of antigen and viral mutation risk, thereby offering a 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against intranasal challenge infections efficacy and safety of an intranasal virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid a in mice and cotton rats chemical adjuvants for plasmid dna vaccines cytokines as immunological adjuvants cytokines: the future of intranasal vaccine adjuvants increased protection against pneumococcal disease by mucosal administration of conjugate vaccine plus interleukin- cytokine requirements for induction of systemic and mucosal ctl after nasal immunization delivery of il- intranasally leads to reduced il- -mediated toxicity the authors would like to thank aoe on control of pandemic and inter-pandemic influenza & centre of influenza research (aoe/m- / ) and health and medical research fund (hmrf ) for financial support. y.x. is supported by hong kong phd fellowship scheme, research general council (pf - ). the authors declare no conflict of interest. key: cord- -g qe ml authors: di sotto, antonella; vitalone, annabella; di giacomo, silvia title: plant-derived nutraceuticals and immune system modulation: an evidence-based overview date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: g qe ml immunomodulators are agents able to affect the immune system, by boosting the immune defences to improve the body reaction against infectious or exogenous injuries, or suppressing the abnormal immune response occurring in immune disorders. moreover, immunoadjuvants can support immune system acting on nonimmune targets, thus improving the immune response. the modulation of inflammatory pathways and microbiome can also contribute to control the immune function. some plant-based nutraceuticals have been studied as possible immunomodulating agents due to their multiple and pleiotropic effects. being usually more tolerable than pharmacological treatments, their adjuvant contribution is approached as a desirable nutraceutical strategy. in the present review, the up to date knowledge about the immunomodulating properties of polysaccharides, fatty acids and labdane diterpenes have been analyzed, in order to give scientific basic and clinical evidence to support their practical use. since promising evidence in preclinical studies, limited and sometimes confusing results have been highlighted in clinical trials, likely due to low methodological quality and lacking standardization. more investigations of high quality and specificity are required to describe in depth the usefulness of these plant-derived nutraceuticals in the immune system modulation, for health promoting and disease preventing purposes. immunomodulators are defined as agents able to affect the immune response, which represents the set of reactions activated to protect the organism against infective agents, environmental injuries and illness; moreover, immune response can counteract the invasion of harmful native cells, such as precancerous and cancerous ones [ ] . immune response is mediated by a first line of defence, namely innate immunity (figure ), which is characterized by physical and biochemical barriers, alongside a non-specific cell-mediated immune response, including granulocytes, macrophages, natural killer cells and humoral elements, which cooperate to counteract pathogen infection and malignant transformation [ ] . moreover, an adaptive immunity is activated as a second defense line after a macrophage-mediated presentation of antigens to b lymphocytes, with the help of t lymphocytes; then, b cells can mediate the humoral immunity through the production of high-affinity antibodies and establish immunological memory [ ] . moreover, t lymphocytes can mediate cellular immunity after activation by cytokines released from helper t cells [ ] . responses involved in innate and adaptive immunity. fast and nonspecific responses, occurring against all factor identified as nonself, are involved in innate immunity; conversely, adaptive immunity is a highly specific, complex and slow response mediated by t and b lymphocytes, which release antigen-specific antibodies and cytokines. immunomodulators can directly affect innate and adaptive response or the factors involved, thus leading to immunostimulant or immunosuppressive effects. a further group of immunomodulators is represented by immunoadjuvants, able to enhance immune response to vaccines without producing specific antigenic effects, and more recently approached as adjuvant pharmacological treatments, especially for viral infections and cancers [ ] [ ] [ ] [ ] [ ] . immune-associated disorders, including autoimmune diseases, viral or bacterial infections, and chronic diseases, are usually associated with acute inflammation, which represents a key component for the activation of immune response [ ] . on the other hand, the chronicity of inflammatory response can negatively influence the immune function, affecting both innate immune cells and t and b lymphocytes, thus suggesting a possible usefulness of anti-inflammatory immunomodulators [ ] . accordingly, immunomodulatory agents, with antioxidant and anti-inflammatory activity, have attracted great attention as possible chemopreventive agents, due to their ability to counteract chronic inflammation, which provides favorable conditions for the transition from normal to cancer cell [ ] . responses involved in innate and adaptive immunity. fast and nonspecific responses, occurring against all factor identified as nonself, are involved in innate immunity; conversely, adaptive immunity is a highly specific, complex and slow response mediated by t and b lymphocytes, which release antigen-specific antibodies and cytokines. immunomodulators can directly affect innate and adaptive response or the factors involved, thus leading to immunostimulant or immunosuppressive effects. immunomodulating agents can affect immunity in a negative or positive manner, thus being categorized as suppressing or stimulant [ ] . particularly, immunosuppressors inhibit the activation of immune response or decrease the activity of its components, thus restoring normalcy. they are of interest in organ transplantations and in autoimmune disorders, wherein the immune system mistakenly activates an immune response against the own body tissues, leading to their destruction [ ] . for instance, vitamin d has been shown to counteract the aberrant immune responses of systemic lupus erythematosus, without compromising the physiological defences and to produce benefits in atopic dermatitis too [ , ] . conversely, immunostimulants boost the endogenous immune defences, thus allowing one to restore or maintain the body homeostasis [ ] . they can be usefully exploited as immunotherapeutic agents by individuals with immunocompromised conditions; however, they can represent suitable prophylactic strategies for healthy individuals or more susceptible subjects against viral infections [ ] . in support, during the current sars-cov- pandemic, the trained immunity by vaccines, which induce heterologous protection, have been proposed as a rational strategy to boost antiviral defences and reduce susceptibility to infection [ ] . a further group of immunomodulators is represented by immunoadjuvants, able to enhance immune response to vaccines without producing specific antigenic effects, and more recently approached as adjuvant pharmacological treatments, especially for viral infections and cancers [ ] [ ] [ ] [ ] [ ] . immune-associated disorders, including autoimmune diseases, viral or bacterial infections, and chronic diseases, are usually associated with acute inflammation, which represents a key component for the activation of immune response [ ] . on the other hand, the chronicity of inflammatory response can negatively influence the immune function, affecting both innate immune cells and t and b lymphocytes, thus suggesting a possible usefulness of anti-inflammatory immunomodulators [ ] . accordingly, immunomodulatory agents, with antioxidant and anti-inflammatory activity, have attracted great attention as possible chemopreventive agents, due to their ability to counteract chronic inflammation, which provides favorable conditions for the transition from normal to cancer cell [ ] . furthermore, immunostimulants can act as adjuvant anticancer treatments, to counteract their immunosuppressive side-effects [ ] . particularly, aristolochic acid, an alkaloid from aristolochia clematitis l., showed immunostimulatory properties, by enhancing the phagocytic activity of peritoneal macrophages and leukocytes; however, its potential cannot be exploited, because of its carcinogenic risk [ ] . likewise, vincristine and staurosporine act as immunostimulants at low doses, while as immunosuppressors particularly, aristolochic acid, an alkaloid from aristolochia clematitis l., showed immunostimulatory properties, by enhancing the phagocytic activity of peritoneal macrophages and leukocytes; however, its potential cannot be exploited, because of its carcinogenic risk [ ] . likewise, vincristine and staurosporine act as immunostimulants at low doses, while as immunosuppressors at higher doses [ ] . among polyphenols, resveratrol stimulated both cellular and humoral immunity in preclinical models, thus preventing pathogen replication and inflammation, and promoted antitumor immune response too [ ] . furthermore, cichoric acid from echinacea promoted phagocytic activity, both in vitro and in vivo [ ] . anti-inflammatory and immune-modulatory effects has been highlighted for curcumin too, although the poor bioavailability limits its clinical application [ ] . in the present review, up to date knowledge on the scientific basis for the immunomodulatory activity and clinical relevance of some emerging classes of plant-derived nutraceuticals, including polysaccharides, fatty acids and labdane diterpenes, has been reported. a comprehensive search was made using pubmed and scopus electronic databases and selecting english as the preferred language, although no language limitations nor filters were applied. for more specific requirements, google scholar and clinicaltrials.gov were considered too. the following searching keywords and their combinations through the boolean logical operators were used: "herbal immunomodulators", "phytochemicals", "immune system", "nutraceuticals", "medicinal plants", "immunomodulation", "immune system boosters", "immunosuppressors", "immunoadjuvants", "gut microbiome", "natural occurrence", "chemical features", "preclinical studies", "clinical trials", "polysaccharides", "echinacea", "astragalus", "β-glucan", "fatty acids", "pufa", "oleic acid", "punicic acid", "γ-linolenic acid", "linoleic acid", "evening primrose oil", "borage oil", "flaxseed oils", "labdane diterpenes" and "andrographolide". this overview allows one to identify novel immune system modulators to be usefully exploited for health promoting and disease preventing purposes. polysaccharides are carbohydrate macromolecules containing at least monosaccharide units, joined by glycosidic linkages to form long-chain molecules, which can be both linear and highly branched. they are called homopolysaccharides when constituted of the same monosaccharide unit, while heteropolysaccharides if different units are present. some of them are also referred to as dietary fibres, meaning that these macromolecules are neither digested nor absorbed in the human small intestine [ ] . several polysaccharides have been found to modulate both innate and adaptive immune responses, among which, glucans, mannans, pectins, fucoidans, galactans, fructans, and xylans are the most studied ( figure ) [ ] . chemical structure, molecular weight, conformation, the presence of functional groups (i.e., acetyl and sulfate groups), and branching have been identified as structural features for the immunostimulatory properties of polysaccharides. the chemical structures of the polysaccharides associated with immunomodulatory properties are displayed in figure . glucans are based on the d-glucopyranosyl unit (homoglucans); the different glycosidic bonds, namely (β → ), (β → ), and (β → ) or (α → ), (α → ), and (α → ), allow the production of linear and branched glucans. it seems that (β → )-d-glucan moiety, triple helix conformations, sulfation and carboxymethylation of (β → )-d-glucans, and chain acetylation are involved in glucan immunostimulatory activity. regarding (α → ) (α → )-d-glucans, their structure activity relationship is less characterized [ ] . while heteropolysaccharides if different units are present. some of them are also referred to as dietary fibres, meaning that these macromolecules are neither digested nor absorbed in the human small intestine [ ] . several polysaccharides have been found to modulate both innate and adaptive immune responses, among which, glucans, mannans, pectins, fucoidans, galactans, fructans, and xylans are the most studied ( figure ) [ ] . chemical structure, molecular weight, conformation, the presence of functional groups (i.e., acetyl and sulfate groups), and branching have been identified as structural features for the immunostimulatory properties of polysaccharides. the chemical structures of the polysaccharides associated with immunomodulatory properties are displayed in figure . glucans are based on the d-glucopyranosyl unit (homoglucans); the different glycosidic bonds, namely (β → ), (β → ), and (β → ) or (α → ), (α → ), and (α → ), allow the production of linear and branched glucans. it seems that (β → )-d-glucan moiety, triple helix conformations, sulfation and carboxymethylation of (β → )-d-glucans, and chain acetylation are involved in glucan immunostimulatory activity. regarding (α → ) (α → )-d-glucans, their structure activity relationship is less characterized [ ] . mannans consist of a d-mannose backbone, linked mainly by β → bonds, which can be ramified with other monosaccharide, so originating glucomannan, galactomannan, and galactoglucomannan [ ] . furthermore, (β → )-or (α → )-, (β → )-, and (β → )-or (α → )-dmannosidic bonds are reported [ ] . the (β → )-d-mannan moiety (e.g., galctoglucomannans), acetyl and sulfate group presence, and this kind of branching seems to confer a high immunostimulatory activity [ ] [ ] [ ] . pectins are complex polysaccharides which contain a common galactopyranosyluronic acid. homogalacturonans, xylogalacturonan, apiogalacturonan, rhamnogalacturonan, type i and ii arabinogalactans belong to this class. particularly, type i arabinogalactans (ag-i) possess an α- arabinofuranosyl and β-d-galactopyranosyl units linked via position at the main chain, while type ii arabinogalactans (ag-ii) comprise highly branched polysaccharides with ramified chains of (β → )-and (β → )-d-galactopyranosyl units [ , ] , to which the arabinosyl units might be attached. the degree of branching, methyl esterification, acetylation, and the type of branched chains and mannans consist of a d-mannose backbone, linked mainly by β → bonds, which can be ramified with other monosaccharide, so originating glucomannan, galactomannan, and galactoglucomannan [ ] . furthermore, (β → )-or (α → )-, (β → )-, and (β → )-or (α → )-d-mannosidic bonds are reported [ ] . the (β → )-d-mannan moiety (e.g., galctoglucomannans), acetyl and sulfate group presence, and this kind of branching seems to confer a high immunostimulatory activity [ ] [ ] [ ] . pectins are complex polysaccharides which contain a common galactopyranosyluronic acid. homogalacturonans, xylogalacturonan, apiogalacturonan, rhamnogalacturonan, type i and ii arabinogalactans belong to this class. particularly, type i arabinogalactans (ag-i) possess an α- -arabinofuranosyl and β-d-galactopyranosyl units linked via position at the main chain, while type ii arabinogalactans (ag-ii) comprise highly branched polysaccharides with ramified chains of (β → )-and (β → )-d-galactopyranosyl units [ , ] , to which the arabinosyl units might be attached. the degree of branching, methyl esterification, acetylation, and the type of branched chains and molecular weight determine the structural diversity [ ] . moreover, flexible chain conformation and branched regions are the main ones responsible for the immunomodulatory properties [ , ] . galactans are polysaccharides rich in galactose and include, beside type i and ii arabinogalactans, carrageenans, chemically characterized by repeating disaccharide units of sulfated or unsulfated d-galactose, that are linked by (β → )-and (α → )-bonds. low molecular weight (< kda) and a high degree of sulfation have been reported as features that high influence their immunomodulatory properties [ , ] . fucoidans are heteropolysaccharides rich in l-fucopyranosyl sulfated units linked by (α → ), (α → ) or (α → ) bonds. other monosaccharides can be present, such as galactopyranosyl, mannopyranosyl, xylosepyranosyl and uronic acids [ ] . the naturally higher content of sulfate groups and the presence of acetyl groups are associated with a higher stimulatory activity [ ] . fructans are polysaccharides which constitute up to fructose units with a sucrolose terminal molecule. they are classified in inulin with a (β → )-d-fructofuranosyl, levan with a (β → )-d-fructofuranosyl, and mixed type, with both (β → )-and (β → )-linked d-fructofuranosyl moieties. a helical conformation has been associated with the modulatory activity on the immune system [ , ] . at last, xylans are polysaccharides containing predominantly a backbone of (β → )-dxylosepyranosyl units. other monomers attached to their backbone include α-dglucopyranosyl a units (glucuronoxylans) and α-l-arabinofuranosyl units (arabinoxylans). a correlation between their structure and activity has not been elucidated yet [ , ] . polysaccharides are naturally occuring in animal body fluids, cell walls, bacteria, yeast and fungi, extra cellular fluids, and in plant seeds, stems and leaves, which represent the focus of the present review. the main advantage of plant polysaccharides seems to be the low toxicity with respect to immunomodulatory bacterial polysaccharides and synthetic compounds [ ] . thus, they represent an ideal alternative for immune modulation. a variety of polysaccharides with immunomodulatory properties have been discovered in different species of plants (table ). among the most studied, there are type i and ii arabinogalactans from astragalus membranaceus (fisch.) bge., fructans from allium sativum l. [ ] , fucogalactoxyloglucan and type ii acidic arabinogalactan from echinacea purpurea l. (moench), ginsan and panaxanes from panax ginseng c.a. meyer, acemannan and aloeride from aloe vera l. [ ] , and glucomannan from amorphallus konjac koch [ ] . several studies have shown that polysaccharides from plants can modulate both innate and acquired intestinal immunity, by direct and indirect mechanisms. the former include the activation of immune cells (e.g., macrophages, dendritic cells, natural killer cells, t cells, b lymphocytes), while the latter the short-chain fatty acid (scfa) formation ( figure ). of immune cells (e.g., macrophages, dendritic cells, natural killer cells, t cells, b lymphocytes), while the latter the short-chain fatty acid (scfa) formation ( figure ). the immunomodulatory effects of plant polysaccharides on macrophages are mainly achieved through the generation of reactive oxygen and nitrogen species (ros and nos), and the stimulation of cytokines secretion, cell proliferation, and macrophage phagocytic activity [ ] . for example, a. membranaceus polysaccharides have been shown to promote nitric oxide (no) synthesis in macrophages, by inducing the gene expression of inducible nitric oxide synthase (inos), through the activation of nuclear factor kappa-b (nf-κb)/rel [ , ] . moreover, they were also able to increase the macrophage phagocytic activity, by enhancing their secretion of release factor and intracellular ca + concentration [ , ] . pectic polysaccharides from citrus unshiu marc. have been shown to simultaneously regulate the expression of pro-and anti-inflammatory cytokines. particularly, they increased the production of the pro-inflammatory cytokines tumor necrosis factor (tnf)-α and interleukin (il)- and the antiinflammatory cytokine il- in macrophage raw . , so showing a regulatory mechanism to maintain an equilibrium state [ ] . arabinogalactan from e. purpurea has been reported to increase macrophages activation and il- , tnf-α and interferon (ifn)-β production [ ] . the activation of macrophages by plant polysaccharides seems to be due to specific receptors present on their surface, which initiates the immune response, and exerts an immunomodulatory effect. these receptors are called pattern recognition molecules, and include: toll-like receptor (tlr ), cd , complement receptor (cr ), scavenger receptor (sr), mannose receptor (mr), and dectin- . their activation determines a series of intracellular signaling cascades, leading to the transcriptional activation and production of inflammation-related cytokines [ ] . immunity modulation by plant polysaccharides can be achieved also by modulating the cytokine release from intestinal dendritic cells. indeed, pectin has been shown to reduce il- and il- release induced by the synthetic lipopeptide p csk [ ] . moreover, inulin, pectin, arabinoxylan and β-glucan have been found to elevate il- /il- ratio and to reduce the release of ifn-γ, il- , il- , il- , il- , monocyte chemoattractant protein (mcp)- , macrophage inflammatory proteins the immunomodulatory effects of plant polysaccharides on macrophages are mainly achieved through the generation of reactive oxygen and nitrogen species (ros and nos), and the stimulation of cytokines secretion, cell proliferation, and macrophage phagocytic activity [ ] . for example, a. membranaceus polysaccharides have been shown to promote nitric oxide (no) synthesis in macrophages, by inducing the gene expression of inducible nitric oxide synthase (inos), through the activation of nuclear factor kappa-b (nf-κb)/rel [ , ] . moreover, they were also able to increase the macrophage phagocytic activity, by enhancing their secretion of release factor and intracellular ca + concentration [ , ] . pectic polysaccharides from citrus unshiu marc. have been shown to simultaneously regulate the expression of pro-and anti-inflammatory cytokines. particularly, they increased the production of the pro-inflammatory cytokines tumor necrosis factor (tnf)-α and interleukin (il)- and the anti-inflammatory cytokine il- in macrophage raw . , so showing a regulatory mechanism to maintain an equilibrium state [ ] . arabinogalactan from e. purpurea has been reported to increase macrophages activation and il- , tnf-α and interferon (ifn)-β production [ ] . the activation of macrophages by plant polysaccharides seems to be due to specific receptors present on their surface, which initiates the immune response, and exerts an immunomodulatory effect. these receptors are called pattern recognition molecules, and include: toll-like receptor (tlr ), cd , complement receptor (cr ), scavenger receptor (sr), mannose receptor (mr), and dectin- . their activation determines a series of intracellular signaling cascades, leading to the transcriptional activation and production of inflammation-related cytokines [ ] . immunity modulation by plant polysaccharides can be achieved also by modulating the cytokine release from intestinal dendritic cells. indeed, pectin has been shown to reduce il- and il- release induced by the synthetic lipopeptide p csk [ ] . moreover, inulin, pectin, arabinoxylan and β-glucan have been found to elevate il- /il- ratio and to reduce the release of ifn-γ, il- , il- , il- , il- , monocyte chemoattractant protein (mcp)- , macrophage inflammatory proteins (mip)- α, rantes and tnf-α by dendritic cells [ ] . polysaccharide enriched extracts of e. purpurea have been found to promote the phenotypic and functional maturation of dendritic cells by modulating c-jun n-terminal kinase (jnk), p mitogen-activated protein kinase (mapk) and nf-κb pathways [ ] . the activation of natural killer (nk) cells also contributes to the immunity modulation by polysaccharides. indeed, it has been shown that a. membranaceus polysaccharides can enhance the activity and killing effects of nk cells and promote their proliferation in rats with gastric cancer [ ] . moreover, they were able to increase cd -cd -cd + nks in peripheral blood lymphocytes [ ] . nks activation is probably due to the polysaccharides interaction with the killer cell lectin-like receptor k (klrk ) of nks [ ] . arabinoxylans extracted from wheat bran have been shown to inhibit the growth of transplantable tumors, and to promote the nk cell activity in s tumor-bearing mice [ ] . moreover, the mgn- rice bran arabinoxylan showed to enhance natural killer (nk) cell activity in aged c bl/ and c h mice upon its intraperitoneal injection [ ] . adaptive immunity is also modulated by plant polysaccharides. particularly, fan et al. have shown that polysaccharides from a. membranaceus significantly up-regulated the proliferation of b lymphocytes, probably through the interaction with immunoglobulin on the surface of b cells [ ] [ ] [ ] . a. membranaceus polysaccharides were also able to increase the number of cd +cd +cd + memory t helper (th) cells and cd +cd -cd + cytotoxic t cells [ ] . moreover, they also enhance the cd +/cd + t cell ratio [ ] . furthermore, arabinoxylan were found to increase the activation of tand b-cells and humoral and cell-mediated immunity in tumor bearing mice [ ] . at last, β-glucan microparticles enhanced t-cell activation and proliferation in vitro [ ] . their ability to affect the immune system by inducing th and/or th type immune response makes polysaccharides suitable adjuvants of the vaccine. among them, inulin, chitosan, glucans and mannans have been most extensively studied. particularly, the gamma and delta forms of inulin fructan have shown adjuvant activity against infectious pathogens by stimulating both th and th responses without inducing immunoglobulin e (ige) production [ ] . moreover, advax, a polysaccharide derived from delta inulin, has demonstrated to increase the immunization derived from influenza vaccine in mice. particularly, an induction of neutralizing antibody and memory b-cell against influenza, an increase in cd and cd t-cell proliferation, and enhanced levels of il- , ifn-γ, il- , il- were highlighted [ ] . advax also enhanced the immunogenicity of hepatitis b surface antigen (hbs) in mice and guinea pigs, by increasing both anti-hbs antibody titers and anti-hbs cd and cd t-cells. th , th and th responses were increased too [ ] . astragalus polysaccharides were also used as adjuvants of hepatitis b virus dna vaccine in a mice model, showing increased hbsag-specific antibody levels, higher activity of t cells, the production of il- , il- and ifn-γ by cd + t cells, and ifn-γ expression of cd + t cells. moreover, a stimulation of cytotoxic lymphocytes and dendritic cells maturation, and a reduction in the frequency of regulatory t cells were observed [ ] . mannans and fructooligosaccharide have also been shown to possess adjuvanticity [ , ] . furthermore, indirect effects are involved in the immunomodulatory properties of polysaccharides. in particular, dietary fibers (e.g., inulin, mannan, β-glucan, pectin) are metabolized by intestinal bacteria in the anaerobic environment of the cecum and colon, so generating scfa, such as acetate, propionate and butyrate [ ] . these molecules are able to cross the gut epithelium and interact with surface receptors on the immune cells, such as the g-protein coupled receptors (gprs) and [ ] . the activation of gprs by scfa modulates inflammatory signalling pathways, such as nf-κb, erk and p mapk [ , ] . moreover, it has been highlighted that scfa can reach t lymphocyte nucleus, so modulating several functions through a histone deacetylase (hdac) inhibition. recently, scfa have been reported able to induce t cells metabolic alterations by enhancing the mtor complex activity. particularly, after absorption into t cells, scfa can stimulate the activity of mtor complex, so increasing the conversion of pyruvate into acetyl-coa. moreover, the acetyl groups from scfa can be link to coa and enter the tricarboxylic acid cycle. the increased levels of citrate are exported from mitochondria into the cytoplasm, where the enzyme atp citrate lyase converts it into acetyl-coa, then used by histone acetyltransferases (hats) for histone acetylation and the regulation of cytokine gene expression [ ] . some clinical studies have been carried out on the potential immunomodulatory properties of polysaccharides, and a. membranaceus, e. purpurea and β-glucan have been most investigated. in a clinical trial on a. membranaceus by jiang et al. [ ] , twenthy-eight stable continuous ambulatory peritoneal dialysis patients were treated with peritoneal dialysis fluid containing astragalus ( ml/ l) for one week. an increase in the macrophage phagocytic capacity, no and tnf-α contents were observed in patients compared to those before the treatment [ ] . furthermore, ji et al. [ ] investigated the effect of astragalus pre-operative treatment of colorectal cancer patients (n = ) on immune function. results showed that astragalus pre-operative treatment promoted the nk cell activity in postoperative patients. in addition, the possible immunomodulatory activity of astragalus in patients with acute exacerbations of bronchial asthma (n = ) has been investigated [ ] . particularly, it was observed that the combination of conventional therapy with astragalus injection for days improved the effects of routine treatment, by enhancing t lymphocyte and nk-cells immune function. results of clinical trials on the immune system modulation by e. purpurea are controversial. particularly, a randomized blinded trial carried out on patients revealed that there was no significant difference in the incidence and severity of colds and respiratory infection between echinacea treatment ( weeks) and placebo groups. however, a small decrease of total lymphocyte counts was observed [ ] . another randomized, placebo controlled, double-blind clinical trial investigated the effect of different echinacea preparations, namely echinaforce ® (e. purpurea preparation from % herba and % radix), e. purpurea concentrate (same preparation at times higher concentration), special e. purpurea radix preparation (totally different from that of echinaforce ® ) on the reduction of the complaint index, defined by symptoms in healthy, adult volunteers who caught a common cold. the treatment continued until the enrolled patients felt healthy again, but not longer than days. the supplementation with echinaforce ® and its concentrated preparation showed to be significantly more effective than the special echinacea extract or placebo. moreover, all treatments were well tolerated [ ] . furthermore, prevention trials have been carried out, showing that echinacea products slightly reduce the risk of getting a cold in healthy individuals [ ] . however, the heterogeneity (e.g., different species and part used) of preparations used in the trials makes the conclusions on the potential immunomodulatory properties of echinacea difficult. clinical trials concerning the β-glucan immunomodulatory properties have also been carried out, although in some cases, yeast-derived-glucan were used. particularly, three randomized, double-blind, placebo-controlled studies have evaluated the effects of short-term β-glucan supplementation on children with chronic respiratory problems. after days' treatment, significant improvements in immunoglobulin, lysozyme, exhaled nitric oxide, and calprotectin production were found [ ] [ ] [ ] . furthermore, the combination of resveratrol plus carboxymethyl-β-glucan as a solution for aerosol has been tested in clinical trials. particularly, the ability of the combination to prevent or treat recurrent respiratory infections in children was studied [ , ] . in both cases, resveratrol plus carboxymethyl-β-glucan had a positive impact on children clinical conditions. indeed, nasal obstruction, rhinorrhea, sneezing, cough, fever, medication use, medical visits, and school absence were significantly reduced. moreover, resveratrol plus carboxymethyl-β-glucan have also been shown to relief nasal symptoms in children with allergic rhinitis, due to pollen allergy [ ] . at last, mannans should be mentioned. they have been reported to possess adjuvant-vaccine properties in clinical studies, probably mediated by its interaction with mannose receptors. particularly, it has been shown that oxidized mannan-mucin can be useful as an adjuvant in the breast cancer immunotherapy. indeed, a - years follow-up has highlighted that it decreases the cancer recurrence rate and prolongs recurrence time, without inducing toxicity or adverse reactions [ ] . fatty acids (fa) are a large group of lipids, characterized by a different number of carbons, arranged in a linear carbon chain skeleton of variable length with a terminal carboxylic group [ ] . based on the number of carbons in the chain, fatty acids can be classified as shortchain fatty acids (scfa; aliphatic tails up to a maximum of six carbons), medium-chain fatty acids (mcfa; aliphatic tails of - carbons), long-chain fatty acids (lcfa; aliphatic tails of to carbons) and very long-chain fatty acids (vlcfa; aliphatic tails of and more carbons) [ ] . among them, scfa, such as acetate, propionate, and butyrate, are produced by gut microbiota enzymes (i.e., propionate-coa transferase and propionaldehyde dehydratase) during the metabolism of carbohydrates and peptides containing branched-chain amino acids [ ] . bacteroidetes are reported to be mainly responsible for the production of acetate and propionate, while firmicutes are the primary contributors of butyrate; however, other bacteria such as lactobacillus and bifidobacterium spp. are involved too [ ] . based on the presence of different double bonds in this structure, fatty acids can be distinguished in saturated fatty acids (sfa), lacking double bonds in their carbon backbone, and unsaturated fa (ufa), which may contain one or more double bonds, thus leading to monounsaturated (mufa) and polyunsaturated fa (pufa) [ ] . sfa include palmitic acid (c : ), lauric acid (c : ), myristic acid (c : ), and stearic acid (c : ), whereas n- oleic acid (c : ) is an example of mufa. furthermore, pufa class includes fatty acids such as α-linolenic acid (ala; c : ), linoleic acid (la; c : ) and further long-chain metabolites [ ] . the number of carbon atoms and unsaturated bond position are used for the systematic nomenclature of fa. moreover, the greek letters omega (ω) and delta (∆) are included, to indicate how far a double bond is from the terminal methyl carbon and the presence and position of one or more double or triple bonds in the carbon backbone, respectively [ ] . a further "ω" or "n" classification designates the position of the first double bond in the skeleton from the end opposite to the carboxy group. accordingly, oleic acid is classified as a ω- (or n- ) fatty acid, while linoleic acid and α-linolenic acid are ω- (or n- ) and ω- (or n- ) fatty acids, as they contain the double bond nine, six and three carbons from the methyl end [ ] . nomenclature of the major representative fatty acids in the different fa classes is displayed in table . unsaturated fatty acids can be characterized on the basis of the cisor transorientation of the double bonds. usually, natural fatty acids carry a cisconfiguration, although some trans-fatty acids can also occur in foods as a consequence of the hydrogenation process, which can move double bonds from their naturally occurring position to a trans-configuration [ ] . trans-fatty acids are considered undesirable compounds in foods, as their intake is associated with an increased risk of cardiovascular and metabolic diseases [ ] . pufa can be further classified depending on the relative positions of the double bonds, as conjugated (double-bonded carbon atoms alternate with single bonds) and unconjugated (double bonds separated by one or more single bonds) [ ] . unconjugated pufa, especially ω- , ω- , and ω- series, are the most occurring in nature. the most common conjugated pufa are trienes, such as octadecatrienoic acids (e.g., punic acid, calendic acid). fatty acids within the series are biosynthetically related, being synthesized through enzymatic processes of desaturation, chain elongation, and chain shortening [ ] . particularly, the biosynthesis of ω- and ω- pufa starts from α-linolenic acid (ala or linolenate; , , - : ) and linoleic acid (la or linoleate; , - : ), respectively ( figure ). these precursors cannot be synthetized by mammals, which lack the ∆ and ∆ desaturases responsible for the convertion of : ω- fa to : ω- and : ω- pufa, and must be supplied by the diet, thus being considered as essential fatty acids. the initial rate-limiting step for the biosynthesis of ω and ω fatty acids is the insertion of a further double bond at the ∆ carbon into the carbon chain of ala and la, through the help of a ∆ desaturase enzyme: stearidonic acid (sa; , , , - : ) and γ-linolenic acid (gla; , , - : ) are formed, respectively. these compounds are converted to eicosatetraenoic acid (eta; , , , - : ) and dihomo γ-linolenic acid (dgla; , , - : ) by the elongase , being further converted to eicosapentaenoic acid (epa; , , , , - : ) and arachidonic acid (aa; , , , - : ) , by the addition of a double bond at the ∆ position, through a ∆ desaturase. further elongations convert epa and aa to docosapentaenoic acid ( , , , , - : ) and adrenic acid ( , , , - : ) ; then, a desaturation by ∆ desaturase generates docosahexaenoic acid (dha; , , , , , - : ) [ ] . both series of fatty acids can be further metabolized by cyclooxygenase and lipoxygenase enzymes, to obtain eicosanoids, including prostaglandins, thromboxanes and leukotrienes, acting as central modulators of the inflammatory process [ ] . the byosynthetic pathways of ω and ω fatty acids are interconnected; indeed, it is known that long-chain derivatives from linolenic acid are accumulated in tissue only slightly when competing ω analogues exceed their amounts. therefore, suitable levels can be reasonably obtained through diet and when an optimum ratio of ω and ω series is maintained. both series of fatty acids can be further metabolized by cyclooxygenase and lipoxygenase enzymes, to obtain eicosanoids, including prostaglandins, thromboxanes and leukotrienes, acting as central modulators of the inflammatory process [ ] . the byosynthetic pathways of ω and ω fatty acids are interconnected; indeed, it is known that long-chain derivatives from linolenic acid are accumulated in tissue only slightly when competing ω analogues exceed their amounts. therefore, suitable levels can be reasonably obtained through diet and when an optimum ratio of ω and ω series is maintained. figure . biosynthetic pathways of ω- and ω- fatty acids. fatty acids occur widely in nature, being identified in both animal tissue and plants. particularly, short-chain saturated acids are components of milk fats: in bovine milk, butanoic acid along with other scfa and mcfa have been reported [ ] . likewise, the mcfa lauric acid and myristic acid are the major components of the oils obtained from some lauraceae and myristiceae species [ ] . moreover, palmitic acid is the most representative sfa in vegetable oils, such as palm oil [ ] . fatty acids with immune modulating properties mainly belong to the long-chain classes; among them, punicic acid is a peculiar conjugated triene, found to be a unique component of pomegranate seed oil [ ] , while oleic acid is one of the most widely distributed fatty acids: it represents % to % of total fa in olive oil, although it does occur in high amounts in other oils, such as those from grape seeds, canola and sufflower [ , ] . moreover, ω and ω fatty acids have been highlighted in several natural sources, wherein both series co-occur (table ) , although in different amounts [ ] . table . major natural sources of long-chain monounsaturated (mufa) and polyunsaturated fatty acids (pufa) associated with immune system modulating activities and relative amounts. some vegetable oils, including rapseed, hemp seed, and sunflower oils, contain higher levels of la (essential ω pufa), while ala (essential ω pufa) is in lower proportion; a similar trend has also been reported for soybean, corn, and for dried black walnuts and brazilnuts; conversely, higher amounts of ala respect to la are reported in flaxseed oil and in the seeds of chia and perilla [ ] . likewise, green leafy vegetables seem to be an interesting source of ala [ ] . fish oils are also sources of both epa and dha (ω pufa), with lower amounts of dpa (ω pufa) [ ] . wild marine species showed to contain higher ω pufa levels compared to farmed ones, likely due to the feed composition [ ] . some vegetables can supply both the essential pufa and some derivative fatty acids. particularly, the oils obtained from the seeds of borago spp., echium spp., ranunculus spp. and oenothera biennis l. have been reported to contain high levels of both la and γ-linolenic acid (gla) [ , ] . fatty acids play energetic, metabolic, and structural functions, being the main component of phospholipids, triglycerides, diglycerides and monoglycerides. a separate category is represented by scfa, which act as metabolites of carbohydrates, produced by gut microbiota: their role in the modulation of immune function is described in section . . long-chain fatty acids have been found involved in immune modulation, being able to affect both innate and adaptive response. although specific profiles characterize each class of fatty acids, these effects are mainly ascribed to their ability to target the cell membrane, where they can be incorporated, thus changing membrane composition and fluidity and modulating membrane-protein interaction and signal transduction. furthermore, a role in the control of inflammation has been reported. epithelial growth factor receptors (a critical crossroad of multiple receptor pathways which is potentially implicated in the regulation of proliferation and possibly involved in atherogenesis) are considered possible targets for unsaturated fatty acids [ ] . mufa, especially oleic acid, have attracted great attention in the years as possible immunomodulating nutrients. preclinical studies demonstrated the ability of oleic acid to modulate the immune system, through affecting both innate and adaptive immunity response [ ] . indeed, it diminished nk cell activity [ ] and the expression of the leucocyte adhesion molecules, which have shown to be implicated in some pathophysiological conditions, such as rheumatoid arthritis [ ] . furthermore, it enhanced neutrophil aggregation and neutrophil-endothelial cell attachment, phagocytic and candidacidal capacities [ , ] . in regard to adaptive response, it inhibited the proliferation of immune cells, such as jurkat t cells and lymphocytes, likely through the regulation of the cell cycle, although the true mechanisms remain to be clarified [ ] . similar suppressive effects were also highlighted for its synthetic analogue minerval and confirmed in animal models [ , ] . furthermore, the treatment with oleic acid and minerval induced proapoptotic effects in jurkat (t lymphocyte) and raji (b lymphocyte) cells, likely due to mitochondrial depolarization and ros production [ ] [ ] [ ] . recently, oleate has been reported to be able to protect macrophages from palmitate-induced lipotoxicity; moreover, it has been associated with an increase in the regulatory phenotype of the myeloid msc- suppressor cells and suppression of activated t cells [ , ] . in the skin, oleic acid, along with other unsaturated fa, seems to be incorporated into the lipid moiety of staphylococcus aureus lpp, inducing an immune response against the pathogen [ ] . regarding conjugated pufa, punicic acid has been shown to improve the immune system development, stimulate the cd + and cd + lymphocyte-mediated immunity and increase the immune response against viruses [ ] . these immune boosting effects are due to nuclear peroxisome proliferator-activated receptor (ppar)γ-and δ-dependent mechanisms, as punicic acid is able to act as an agonist of these receptors; in support, the loss of pparγ in immune cells impaired its effects [ , ] . moreover, punicic acid inhibited the tnf-α-induced priming of ros production by inhibiting the ser -p phox phosphorylation and upstreaming kinase p mapk; likewise, it blocked the tnf-α-induced release of myeloperoxidase from neutrophils, and decreased neutrophil-activation and ros/mpo-mediated tissue damage in vivo [ ] . antinflammatory properties were found to be related to the activation of pparγ and the suppressed expression of inflammatory genes (encoding cytokines, chemokines, cyclooxygenase, no synthase, and metalloproteinases) [ ] . immunomodulatory properties of ω- and ω- pufa have been highlighted in different preclinical models and have been associated with their ability to modulate the inflammatory process [ ] . these fatty acids share common biosynthetic enzymes which mediate the production of different series of eicosanoids, starting from typical precursors, including dihomo-γ-linoleic acid (dgla), arachidonic acid (aa) and eicosapentaenoic acid (epa). among prostanoids, three types of prostaglandins (pg), including pg , pg and pg , can be obtained. pg is associated with beneficial effects and lower inflammation, thus being considered as an antinflammatory prostanoid; conversely, pg has opposite behaviour, increasing inflammation, vasoconstriction and blood clotting. pg acts through a mixture of functions and is able to reduce the pg -mediated inflammation [ ] . starting from dgla, both anti-inflammatory pg and pro-inflammatory pg , through the conversion into arachidonic acid, can be produced (figure ) . effects and lower inflammation, thus being considered as an antinflammatory prostanoid; conversely, pg has opposite behaviour, increasing inflammation, vasoconstriction and blood clotting. pg acts through a mixture of functions and is able to reduce the pg -mediated inflammation [ ] . starting from dgla, both anti-inflammatory pg and pro-inflammatory pg , through the conversion into arachidonic acid, can be produced (figure ) . this synthesis is controlled by the activity of Δ -desaturase and Δ -desaturase enzymes, which are often compromised during inflammatory conditions and diseases. it has been found that diets enriched in ω- fatty acids are able to activate the conversion of dgla into pg , whereas low ω- intake induces the conversion in aa with the synthesis of proinflammatory prostanoids [ ] . aa can also be released by the cell membranes through the action of phospholipase a during cell injuries or changes in biomembrane composition, thus representing a physiological activator of inflammation as a defence response. aa and eicosapentaenoic acid (epa) compete for the synthesis of different series of pg, mediated by cyclooxigenase, while -lipooxygenase (lox) is involved in their conversion into thromboxanes and leukotrienes. particularly, tromboxane a and leukotriene b are produced from aa, while tromboxane a and leukotriene b from epa. epa and dha are also precursors of lipoxins, resolvins and protectins, which produced anti-inflammatory effects and regulate vascular tone and blood pressure [ ] . like punicic acid, the anti-inflammatory effects of epa and dha are mediated by the activation of the pparα/γ [ ] . despite the antinflammatory role of ω- and ω- -based diets, aa increases the plasmatic levels of proinflammatory eicosanoids, associated with an increased incidence of allergic and inflammatory disorders and with excessive cell proliferation. anyhow, it is not clear the usefulness to select ω- with respect to ω- in the diet: a balance between ω- and ω- pufa seems to be essential for mantaining ahealth status. the ability of fatty acids to be incorporated in the cell membrane seems to represent a key mechanism accounting for the immunomodulating properties of ω- and ω- pufa. indeed, immune cells (i.e., t cells and neutrophils) can incorporate exogenous fatty acids into membrane with a lateration in the function of cell surface pattern recognition receptors [ ] . dietary ω- pufa has been shown able to modulate the macrophage function, through the activation of g protein coupled receptors (gpr) and to induce a shift to an anti-inflammatory phenotype [ ] . modulating signalings through the gpr receptor activation can also affect leukocyte function. likewise, an inhibition of the pro-inflammatory phenotype of dendritic cells and of the t cell responses has been reported [ ] . they are also able to inhibit neutrophil and monocyte adhesion, depending on the activation of ppar-α [ ] . conversely, ω- pufas seem to promote inflammation, associated with incresead ros levels, in neutrophils [ ] . particularly, linoleic acid increased the marginated pool of neutrophils in tissues by the induced expression of adhesion molecules;it also complexed with the anti-inflammatory this synthesis is controlled by the activity of ∆ -desaturase and ∆ -desaturase enzymes, which are often compromised during inflammatory conditions and diseases. it has been found that diets enriched in ω- fatty acids are able to activate the conversion of dgla into pg , whereas low ω- intake induces the conversion in aa with the synthesis of proinflammatory prostanoids [ ] . aa can also be released by the cell membranes through the action of phospholipase a during cell injuries or changes in biomembrane composition, thus representing a physiological activator of inflammation as a defence response. aa and eicosapentaenoic acid (epa) compete for the synthesis of different series of pg, mediated by cyclooxigenase, while -lipooxygenase (lox) is involved in their conversion into thromboxanes and leukotrienes. particularly, tromboxane a and leukotriene b are produced from aa, while tromboxane a and leukotriene b from epa. epa and dha are also precursors of lipoxins, resolvins and protectins, which produced anti-inflammatory effects and regulate vascular tone and blood pressure [ ] . like punicic acid, the anti-inflammatory effects of epa and dha are mediated by the activation of the pparα/γ [ ] . despite the antinflammatory role of ω- and ω- -based diets, aa increases the plasmatic levels of proinflammatory eicosanoids, associated with an increased incidence of allergic and inflammatory disorders and with excessive cell proliferation. anyhow, it is not clear the usefulness to select ω- with respect to ω- in the diet: a balance between ω- and ω- pufa seems to be essential for mantaining ahealth status. the ability of fatty acids to be incorporated in the cell membrane seems to represent a key mechanism accounting for the immunomodulating properties of ω- and ω- pufa. indeed, immune cells (i.e., t cells and neutrophils) can incorporate exogenous fatty acids into membrane with a lateration in the function of cell surface pattern recognition receptors [ ] . dietary ω- pufa has been shown able to modulate the macrophage function, through the activation of g protein coupled receptors (gpr) and to induce a shift to an anti-inflammatory phenotype [ ] . modulating signalings through the gpr receptor activation can also affect leukocyte function. likewise, an inhibition of the pro-inflammatory phenotype of dendritic cells and of the t cell responses has been reported [ ] . they are also able to inhibit neutrophil and monocyte adhesion, depending on the activation of ppar-α [ ] . conversely, ω- pufas seem to promote inflammation, associated with incresead ros levels, in neutrophils [ ] . particularly, linoleic acid increased the marginated pool of neutrophils in tissues by the induced expression of adhesion molecules; it also complexed with the anti-inflammatory molecule -antitrypsin, thus reducing lps-induced il- secretion in neutrophils [ ] . on the whole, preclinical evidence highlighted that these fatty acids could increase neutrophil function, thus promoting innate immunity. regarding adaptive immunity, ω- pufa have been reported able to improve the mitogen-mediated activation of immune cells and to promote the development of a th -type immune response [ ] . moreover, an increased production of associated anti-inflammatory cytokines like il- , in spite of a reduction of pro-inflammatory tnf-α, was found [ ] . similar effects were highlighted with both fish oil-enriched diets and the purified epa and dha [ ] . the beneficial influence of ω- pufa has been highlighted also on epithelial cells during inflammation, being able to restore impaired barrier function and reduce the production of pro-inflammatory mediators [ ] . moreover, a strictly interplay between omega- fatty acids, immunity and gut microbiota has been reported and seems to be an essential factor to maintain the intestinal wall integrity. these effects have been ascribed to the ability of ω- pufa to positively affect the microbiota composition and increase the production of anti-inflammatory compounds, like short-chain fatty acids [ ] . although a major interest over the years has been focused on marine sources of ω- -enriched oils or on pure compounds, some plant species have been studied for their immunomodulating and anti-inflammatory properties, likely ascribable to ω- and/or ω- pufa, although the major evidence has been highlighted for linum usitatissimun l., oenothera biennis l. and borago officinalis l. [ , , ] . the seed oil from l. usitatissimum, also known as flaxseed oil, has been reported to induce immunomodulating effects, likely through suppressing cell mediated immunity, without the involvement of humoral immunity. being a rich source of ala, its effects are mainly ascribed to this compound, although further studies suggested a possible contribution of bioactive phenolics [ ] . flaxseed oil was found to be effective in reducing skin inflammatory responses, although with a lower immunosuppressive power with respect to fish oil [ ] . moreover, it improved systemic and gut immunity, in a piglet model with intrauterine growth retardation: increased plasma concentration of immunoglobulin g, decreased cd +cd + t lymphocytes, and the downregulation of genes expression for proinflammatory factors have been reported [ ] . regarding o. biennis, the administration of the seed oil (namely, evening primrose oil) in animal models enhanced pge synthesis in peritoneal macrophages, decreased pge amounts in granulocytes, and suppressed the natural killer (nk) cell activity and lymphocyte proliferation; moreover, it decreased the serum levels of interferon γ (ifn-γ) and mcp- , while stimulating tnf-α [ ] [ ] [ ] [ ] [ ] . furthermore, anti-inflammatory effects have been found to be involved in the immunomodulation by evening primrose oil [ ] . these effects were ascribed to the content of gla, whose t-regulatory cell activity in autoimmune disease models was highlighted [ ] . however, a contribution of la to the antinflammatory effects seems to be likely; indeed, la can itself modulate inflammation as it is metabolized by lox to hydroxyoctadecadienoic acids (hodes) and oxo-hodes, characterized by antinflammatory properties [ ] . similarly, the seed oil from b. officinalis seeds produced immonomodulating and antinflammatory effects, likely through its gla content [ ] . a chemotactic migration of monocytes to necrotic site that differentiate into macrophages is associated with the administration of this product. moreover, it is known to reduce the levels of proinflammatory cytokines, such as tnf-α, and to promote pge generation; a reduced expression of inflammatory genes, especially those of macrophages involved in atherosclerosis, has been reported too [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . clinical studies mainly focused on the effects of fatty acid-enriched diet on inflammation, although specific immune-based pathological conditions associated with inflammation were assessed too. regarding mufa, few studies are available, and results differ from those in animal models. indeed, a mufa-rich diet (with highly refined olive oil for weeks) did not alter the immune function in healthy subjects; such effects could be due to the high amounts administered in animal models [ ] . conversely, clinical evidence about the immunomodulatory power of punicic acid in healthy or sick subjects is lacking [ ] . the ω- pufa series and the relative enriched fish oils have been mainly evaluated for their immunomodulating and antinflammatory effects in humans. although preclinical evidence highlighted their ability to influence both innate and adaptive immunity, the clinical relevance of these results remains to be clarified, due to lacking or inconclusive data [ ] . inadequacy of clinical results should be due to the different doses used in preclinical studies, wherein often high fatty acids levels were administered; moreover, other factors such as genetic and epigenetic heterogeneity of the recruited subjects, diet diversity, nutritional habits and microbiome can be considered as additional confounding factors [ ] . although limitations of clinical studies require further confirmation, ω- pufa intake produced significant clinical benefits and reduction of the symptoms in patients with autoimmune disorders, especially rheumatic diseases and systemic lupus erythematosus [ ] [ ] [ ] . in support, low levels of pufas have been found in the serum of patients with rheumatic diseases [ ] . conversely, inconsistent results are reported for multiple sclerosis, thus the possible usefulness of these fatty acids as supportive therapy requires more clinical trials [ ] . regarding ω- pufa, although they are associated with possible increased inflammatory conditions, being arachidonic acid a presursor of proinflammatory prostanoids, such a risk is not confirmed by clinical evidence [ ] . indeed, studies in healthy human adults highlighted that an increased intake of these fatty acids did not induce inflammation; conversely, epidemiological evidence reported reduced inflammatory conditions [ ] . the antinflammatory effects of la have been reported too [ ] . similarly, increased la intake was found to be not related to increased amounts of ara and proinflammatory factors; however, an inverse correlation with epa and dha was reported [ ] . this suggests that the interaction between ω-e and ω- series is regulated by complex mechanisms that requires further clarifications. major clinical studies have been performed using evening primrose oil (from the seeds of o. biennis), as a source of la and gla, in inflammatory diseases associated with immune system disorders, including atopic dermatitis, psoriasis, multiple sclerosis and rheumatoid arthritis. standardized oils for the content in la and ala (for instance, efamol is titred to contain % la and % gla) were usually used [ ] . the treatment with evening primrose oil ( and weeks) produced clinical improvements in patients with atopic dermatitis, as revealed by measuring the scoring atopic dermatitis [ ] . some beneficial effects were also reported in multiple schlerosis patients, although the few available studies limited the evidence in this disorder. conversely, evening primrose oil in combination with fish oil and vitamin e (efamol marine) failed to improve the symptoms of psoriasiac patients but produced antinflammatory effects [ ] . similarly, in association with ω- fatty acids, it did not induce improvements in patients with rheumatoid arthritis [ ] . a cochrane revision highlighted moderate evidence for oils containing gla (i.e., evening primrose, borage, or blackcurrant seed oil) to produce benefit in rheumatoid arthritis [ , ] . evening primrose oil along with borage oil were not effective to treat eczema too [ ] . highly variable results were also obtained for borage oil in the treatment of atopic dermatitis, although, in all the studies, a moderate efficacy degree was displayed [ ] . regarding flaxseed oils, some clinical trials higlighted a significant improvement of inflammatory parameters in subjects with cardiovascular diseases non-associated with the immune system [ ] . reported studies, although performed in pathological conditions associated with immune system disfunction, did not give a direct measure of the immunomodulatory effects of the treatments. furthermore, specific and high-quality studies are required for better characterizing the possible usefuleness of these pufa-enriched oils as anti-inflammatory and immunomodulating treatments. labdane compounds have a molecular formula c h with an average mass of . da ( figure ). labdane-related molecules have a hydrocarbon skeleton, originated from dual biosynthetic cyclization and/or rearrangement reactions, produced through the biosynthetic pathway of gibberellin phytohormones by the diterpene cyclases. the labane diterpenoids belong to a superfamily of natural products, in which the hydrocarbon skeleton might serve as privileged scaffolds for their biological activity [ ] . labdane compounds have a molecular formula c h with an average mass of . da ( figure ). labdane-related molecules have a hydrocarbon skeleton, originated from dual biosynthetic cyclization and/or rearrangement reactions, produced through the biosynthetic pathway of gibberellin phytohormones by the diterpene cyclases. the labane diterpenoids belong to a superfamily of natural products, in which the hydrocarbon skeleton might serve as privileged scaffolds for their biological activity [ ] . labdane diterpenes have been found in the various matrix of vegetal origin (leaves, rizomes, fruits, etc.) of different plants. in table , some of them (where diterpenes have been found), their botanical family (in parentheses) and the part of plant of biological interest are reported. some labdane diterpenoids, isolated from plant matrix, include the following: andrographolide ( figure ) (from andrographis paniculata (burm.f.) nees) [ ] , labda- ( ), -diene- , -dial (from curcuma amada roxb) [ ] , podoimbricatin c (a , -cyclo-labdane diterpenoid from dacrycarpus imbricatus (blume) de laub) [ ] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [ ] , ( r, s, s, r)- -des-ethyl- -oxolabda- ( ), e-dien- -oic acid (from juniperus oblonga m. bieb) [ ] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [ ] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [ ] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [ ] . labdane diterpenes have been found in the various matrix of vegetal origin (leaves, rizomes, fruits, etc.) of different plants. in table , some of them (where diterpenes have been found), their botanical family (in parentheses) and the part of plant of biological interest are reported. some labdane diterpenoids, isolated from plant matrix, include the following: andrographolide ( figure ) (from andrographis paniculata (burm.f.) nees) [ ] , labda- ( ), -diene- , -dial (from curcuma amada roxb) [ ] , podoimbricatin c (a , -cyclo-labdane diterpenoid from dacrycarpus imbricatus (blume) de laub) [ ] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [ ] , ( r, s, s, r)- -des-ethyl- -oxolabda- ( ), e-dien- -oic acid (from juniperus oblonga m. bieb) [ ] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [ ] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [ ] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [ ] . imbricatus (blume) de laub) [ ] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [ ] , ( r, s, s, r)- -des-ethyl- -oxolabda- ( ), e-dien- -oic acid (from juniperus oblonga m. bieb) [ ] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [ ] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [ ] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [ ] . figure . chemical structure of andrographolide. this has been obtained using chemspider ® chemical structure database. the body's defense responses can be improved through various properties induced by plants. some of them, referring to the plants in table , are shown below. for each plants and plant-derived nutraceutical, only properties potentially attributable to the labdane skeleton and useful to improve the immune system are reported. as both inflammation (biological response of body tissues to harmful stimuli) and oxidative stress (imbalance between reactive oxygen species and a biological system's ability to detoxify/repair the resulting damage of the reactive intermediates) are the main self-defend methods to eliminate pathogens and protect living bodies, plants with antinflammatory and/or radical scavenger properties are considered too [ ] . indeed, labdane diterpenoids have recently gained greater attention from the scientific point of view, due to a wide range of biological activities, including the anti-inflammatory modulation of immune cell functions [ ] . a. paniculata exhibited, in vitro and in vivo, various pharmacological activities, including antihyperglycemic, antiplatelet aggregation, anti-microbial, anti-inflammatory, anti-hiv, anti-cancer, anti-nociceptive activity, etc. it has also been used for autoimmune encephalomyelitis and, in indian and chinese medicine, for respiratory tract infections [ , ] . more recently, a. paniculata has been used to stimulate the immune system and treat myocardial ischemia [ ] . a. paniculata inhibited interleukin (il)- , tnf-α mrna, lps-induced expression, and suppressed levels of tnf-α, il- β, jnk, c-reactive protein, and nf-κb [ ] . many labdane diterpenoids compounds have been found to act on the latter. the activation of the nf-κb pathway leads to several physiological responses, including inflammatory or innate immune response [ ] . in vitro, andrographolide (the main phytoconstituent of a. paniculata) can inhibit inflammation, by regulating protein expression (cytokines, chemokines) and by reducing immune cell infiltration. andrographolide was shown to inhibit also oxidative stress by binding to adenosine a a receptor, by inducing nuclear factor (erthroid-derived )-like (nrf ) translocation, and by increasing the expression of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase- [ ] . these effects can contribute to the immunoregulatory activity of this plant-derived nutraceutical, as it can modulate the innate and adaptive immune responses by regulating macrophage phenotypic polarization and antibody productions [ ] . moreover, it was found to exert cytotoxic/anticancer effects on almost all types of tumour cell lines (human leukemia, renal tubular epithelial cells, breast cancer cells, etc.), mainly by cell cycle arrest, autophagy, cell death, anti-inflammatory and immune system mediated effects [ ] . other preclinical studies have highlighted pharmacological properties of labdane diterpenoidscontaining plants. data are limited, and consequently also their preclinical evidence. some examples are reported below. c. amada, also known as mango ginger, and its labdane-diterpenoids have shown antiflammatory, antibacterial, insecticidal, antifungal, antipyretic, antioxidant, anticancer, and antitubercolar properties in preclinical trials [ ] . d. imbricatus displayed cytotoxic and anti-neuroinflammatory activities, but it had no cytotoxic activity against human tumour cell lines [ ] . e. macrophyllus, brazilian plant, also known as "leather hat", is used as a methanolic (which contains mainly labane diterpenoids, steroids, alcaloids, etc.) or aqueous extract (rich in flavonoids) of the aerial parts, leaves in particular. in folk medicine, e. macrophyllus is used for various illnesses (respiratoy and urinary diseases, rheumatoid arthritis, atherosclerosis, etc.), as it has been shown to possess tissues protective activity and immunosuppressive effects (impaired secretion and function of b and/or t cells), on humoral or cellular immune responses and on autoimmune rheumatic diseases [ ] . in in vitro/vivo studies, the aqueous extract of e. macrophyllus exhibited strong antinflammatory activities by decreasing rats paw edema, inflammatory exudates, infiltrate tissues, no production, ltb release, and neutrophil migration [ ] . in preclinical studies, the methanolic extract of e. macrophyllus was not cytotoxic, genotoxic, mutagenic, and no acute toxicity (up to the maximum dose of mg/kg b.w.) has been observed in tested animals [ ] . however, the extrapolation of animal experiments to clinical practice must be done with caution [ ] . compounds from j. oblonga have shown anti-tumor effects, through moderate cytotoxicity against human tumor cell lines obtained from various human tissues, including: hepatocellular carcinoma (hepg ), breast cancer (mcf- ), and cervical carcinoma cancer (hela) [ ] . the berries from j. oblonga also have antimicrobial activity and anti-inflammatory effects. labdane diterpenoid (e.g., leonurine), extracted from l. japonicus, exhibited cytotoxicity and cell cicle arrest against cancer cell lines and presented immunomodulatory and antinflammatory activities (suppresses tnf-α, nf-κb, and down-regulated expression of inos, cox , and conseguently peg and no levels) [ ] . marrubium spp. (aschersonii, globosum, etc.) have multiple actions, including antimicrobial and anti-inflammatory activities. marrubasch a-f and marrubenol, isolated from the ethanolic extract of m. aschersonii, exhibited weak reduction in inos activity and, consequentely, no production [ ] . marrulibanoside, obtained from the aerial parts of m. globosum, inhibited catalytic activity of inos and cox- enzymes, and consequentely, the peg and no production. v. limonifolia, in preclinical trial, have shown a strong antiviral activity against coxsackievirus b , human rhinovirus b, and enterovirus (ev ). all of them could be responsible for various illnesses, ranging from common cold, hand, foot, and mouth diseases, to acute flaccid paralysis [ ] . the clinical efficacy of the medicinal plants, and plant-derived nutraceuticals discussed above are almost totally lacking. only for a. paniculata there are some evidence in humans. andrographis extract (various and not standardized), andrographolide, and its derivatives have been studied in the treatment of various disease (multiple sclerosis, infection disease, gastro-intestinal upsets, respiratory ailments, pain), and in the maintenance of immune function. in this last context, it seemed to improve the response to cough and sore throat, shortening the sick leave/time to resolution [ ] . the most interesting activity is the increase of cd + lymphocyte levels, in hiv-positive patients [ ] . the increase in these lymphocytes testifies an improvement in the state of the immune system. moreover, a chinese product containing andrographolide improved the efficacy of glucocorticoids and immunoglobulin in patients with severe hand, food, and mouth disease. however, andrographolide is considered a hazard, as it is irritating, and its injectable use is limited because it could induce allergic reactions (erythema, pruritus, etc.), which are sometimes life-threatening [ ] . preclinical data suggested that andrographolide could be responsible of pharmacokinetics interactions, as it induced cyp a [ ] . the european medicines agency (ema) reports a possibility of causing reproductive toxicity of andrographis extracts (decreases in sperm motility and counts) [ ] . on the other hand, no major adverse effects have been reported for a. paniculata; only minor side effects, mainly gastrointestinal, are known [ ] . notably, even if a. paniculata presents numerous pharmacological properties, andrographolide possess poor solubility (principally in dmso), which severely limits the possibility of achieving a therapeutic effect (if not properly formulated). its better absorption could be achieved by nano-formulations (e.g., nano-emulsion, nano-capsules). immunomodulation by plant-based nutraceuticals represents an interesting tool to be exploited for the treatment and preventing purposes of immune system disorders, due to their multiple bioactivities, well tolerability and good patient compliance. however, as often reported for several herbal medicinal products, some points require being underlined to improve the research in the field and provide solid evidence to support their rational use. according to previous stated critical issues [ , ] , herbal products under study must be characterized for the phytochemical composition, using validated analytical methodologies, and for the extraction procedures; moreover, the starting material should be fully defined in terms of origin (country and region), cultivation conditions, botanical identity and plant part. the content of specific compounds, used as analytical or active markers, should be determined too. these requirements are needed to ensure reproducible pharmacological/clinical activity and to compare different studies. indeed, using nonstandardized phytocomplexes increases variability of the biological response, thus limiting the reliability and validity of the studies. furthermore, to assess the pharmacological activity of specific compounds, purity (at least %) and identity should be characterized. indeed, when assessed as mixtures, the subtle interactions which can be established among phytochemicals make it difficult to understand whether the observed benefits are attributable to a specific class or to the whole phytocomplex. for instance, both fatty acids and polyphenols can be involved in the immunomodulating effects of pufa-enriched plant oils. moreover, as found for both polysaccharides and fatty acids, among the same class, different subclasses can co-occur, thus contributing to the whole effects. regarding preclinical studies, detailed methodologies, including information about specific extraction process, the choice of the tested concentrations and experimental procedures, vehicle effects, and comparison with standard effective compounds (positive controls) should be reported. in order to validate the "goodness" of the treatment, promising results in preclinical studies should be confirmed by clinical evidence of efficacy and lack of toxicological concerns for both the isolated compounds and the whole phytocomplex. at last, possible interactions with diet constituents or possible pharmacological treatments, as reported for andrographolide, which is a cyp a inducer, should be considered. as highlighted for a number of natural products, clinical evidence is a major challenge for plant-based immunomodulating nutraceuticals too, due to limited specific studies. moreover, methodological quality of the available trials was overall poor, the studies often being not blinded, protocol unavailable and lacking the standardization of tested products, thus making the claimed effect difficult to be reproduced. at last, standardized methodologies for systematic reviews and meta-analyses, such as the prisma guidelines [ ] , would allow a rational interpretation of the results and suggestions for future research. medicinal plants are rich sources of bioactive phytochemicals, characterized by multiple and often pleiotropic activities, which can be exploited both therapeutically and as nutraceutical strategies for preventive purposes. among plant-based nutraceuticals, immunomodulators have been highlighted to be of interest as boosters of the immune system, to counteract infectious or exogenous injuries, immunosuppressor, to control the abnormal immune response occurring during autoimmune diseases, or as adjuvants, which contribute by modulating nonimmune targets. in this review, we highlighted the scientific evidence about the immunomodulating properties of three emerging classes of nutraceuticals, including polysaccharides, fatty acids and labdane diterpenes. some of them, especially polysaccharides and labdane diterpenes, act as immune system booster, while fatty acids (mufa and pufa) mainly act as immunosuppressor, although punicic acid (a conjugate pufa) exhibited immunostimulant properties. 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reviews and meta-analyses: the prisma statement this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license a.d.s. and s.d.g. fellowships were funded by grants from sapienza university (ateneo ) and regione lazio. the authors thank "enrico and enrica sovena" foundation (italy) for supporting the study. the authors declare no conflict of interest. key: cord- -k jssr authors: volk, aaron; hackbart, matthew; deng, xufang; cruz-pulido, yazmin; o’brien, amornrat; baker, susan c. title: coronavirus endoribonuclease and deubiquitinating interferon antagonists differentially modulate the host response during replication in macrophages date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: k jssr coronaviruses (covs) encode multiple interferon (ifn) antagonists that modulate the host response to virus replication. here, we evaluated the host transcriptional response to infection with murine coronaviruses encoding independent mutations in one of two different viral antagonists, the deubiquitinase (dub) within nonstructural protein or the endoribonuclease (endou) within nonstructural protein . we used transcriptomics approaches to compare the scope and kinetics of the host response to the wild-type (wt), dubmut, and endoumut viruses in infected macrophages. we found that the endoumut virus activates a focused response that predominantly involves type i interferons and interferon-related genes, whereas the wt and dubmut viruses more broadly stimulate upregulation of over , genes, including networks associated with activating the unfolded protein response (upr) and the proinflammatory response associated with viral pathogenesis. this study highlights the role of viral interferon antagonists in shaping the kinetics and magnitude of the host response during virus infection and demonstrates that inactivating a dominant viral antagonist, the coronavirus endoribonuclease, dramatically alters the host response in macrophages. importance macrophages are an important cell type during coronavirus infections because they “notice” the infection and respond by inducing type i interferons, which limits virus replication. in turn, coronaviruses encode proteins that mitigate the cell’s ability to signal an interferon response. here, we evaluated the host macrophage response to two independent mutant coronaviruses, one with reduced deubiquitinating activity (dubmut) and the other containing an inactivated endoribonuclease (endoumut). we observed a rapid, robust, and focused response to the endoumut virus, which was characterized by enhanced expression of interferon and interferon-related genes. in contrast, wild-type virus and the dubmut virus elicited a more limited interferon response and ultimately activated over , genes, including players in the unfolded protein response and proinflammatory pathways associated with progression of significant disease. this study reveals that endou activity substantially contributes to the ability of coronaviruses to evade the host innate response and to replicate in macrophages. responses or of later, adaptive responses that aim to limit virus replication. by understanding how these viral antagonists regulate the immune response, we can fine-tune the rational design of therapeutics and vaccines to control existing and emerging viral pathogens. coronaviruses (covs) are characterized in part by their large (ϳ kb), positive-sense single-stranded rna genomes that yield a nested set of subgenomic mrnas during viral replication ( , ) . these large genomes encode the replicase polyprotein, the canonical structural proteins (spike, envelope, membrane, and nucleocapsid), and, depending on the cov, a number of accessory genes. notably, many of these different genes (replicase, structural, and accessory) encode proteins that regulate the antiviral response, indicating that viral antagonists of host defenses play an important role during infection ( ) ( ) ( ) ( ) ( ) . in this report, we investigate the role(s) of two highly conserved enzymatic domains within the replicase polyprotein of mouse hepatitis virus (mhv)the viral protease/deubiquitinase (dub) and the endoribonuclease (endou)-in altering host immune responses. the replicase polyprotein machinery must be processed into its functional parts during viral replication, which, in the case of mhv, requires the activity of three proteases, two papain-like proteases (plp and plp ) and one chymotrypsin-like protease ( clpro, sometimes referred to as mpro). mhv plp is structurally similar to the single papain-like protease (termed plpro) encoded by severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) . previous studies demonstrated that cov plp s/plpros are multifunctional, with catalytic residues that mediate protease, deubiquitinase (dub), and deisgylating (deconjugating interferon-stimulated gene [isg ] molecule from modified substrates) activities ( ) ( ) ( ) ( ) ( ) . in our companion manuscript ( ) , we describe the x-ray structure-guided identification of a residue in mhv plp that is required for dub activity, but not protease activity. using reverse genetics, we generated an mhv that expressed the dub mutant enzyme, and we found that dubmut virus is mildly attenuated in mice. however, the role of dub activity during replication in macrophages, a cell type critical for virus replication and pathogenesis, was not known. we wanted to determine the host response to a dubmut virus and compare it to that against a virus expressing a mutant form of another recently identified antagonist, the coronavirus endoribonuclease (endou). endou, a highly conserved enzyme within the coronavirus family, was initially thought to play a role in coronavirus rna synthesis ( , ) . recent studies revealed that endou acts as an interferon (ifn) antagonist by preventing activation of host sensors by viral double-stranded rna (dsrna) ( , ) . viruses encoding a catalytically inactive endou undergo initial rna replication; however, viral dsrna intermediates accumulate during viral replication and are detected by the host sensors mda , pkr, and oas. activation of these dsrna sensors initiates the innate immune response, including activation of type i interferons, interferon-responsive genes, and apoptosispromoting caspases, all of which collectively limit virus replication. we previously reported that mhv-endou mutant viruses are profoundly attenuated in mice and elicit a protective immune response ( ) . in the current study, we use transcriptome profiling to evaluate the kinetics of host gene expression in macrophages upon infection with mhv wild-type (wt), dubmut, and endoumut viruses. our analyses of the respective transcriptional response to each virus reveal significant differences in the kinetics, magnitude, and breadth of host gene expression during infection and provide new information on the extent to which viral interferon antagonists manipulate the overall host response to viral infection. transcriptome profiling reveals differences in kinetics and magnitude of host responses to mutant viruses. we sought to determine if viruses that contain inactivating mutations in distinct interferon antagonists (dub versus endou) would exhibit unique host transcriptional signatures in response to virus infection. briefly, bone marrow-derived macrophages (bmdms) were infected with the designated virus at a multiplicity of infection (moi) of , and total rna was harvested for transcriptome profiling at , , , and h postinfection (h p.i.). rna was isolated from all cells in the well, both infected and uninfected bmdms. using the rna-sequencing (rna-seq) data, we identified differentially expressed genes by applying a cutoff of at least -fold elevated expression in wild-type-infected cells at h p.i. over mock (q Ͻ . ). we then utilized cluster . software to visualize overall patterns of gene expression between all groups across all time points (fig. ) . these analyses reveal striking differences in overall patterns of gene expression between the endoumut-infected cells and wild-type virusinfected cells, whereas the gene expression profile induced by the dubmut virus is remarkably similar to that in wild-type virus infection (fig. ). more specifically, , genes are significantly transcriptionally activated (Ͼ -fold; q Ͻ . ) in wt-and dubmut-infected macrophages by h p.i. compared to those in mock-infected macrophages. interestingly, our analyses also identified a subset of genes, indicated by the bracket in fig. , that were most highly upregulated in endoumut-infected cells. it is important to note that these genes were also induced during wt and dubmut infections, but not to the same magnitude as that detected in the endoumut-infected cells. we note a transient downregulation of a subset of genes related to transcriptional responses in the endoumut-infected cells at h p.i., which may be related to the apoptotic response of these cells ( ) . we next sought to functionally cluster the genes most highly upregulated by each mutant virus as a means of investigating the respective host transcriptional response to the different viruses. endoumut infection activates genes associated with an antiviral response. we used an online tool called database for annotation, visualization and integrated discovery (david) to cluster the genes highly upregulated by endoumut virus infection (bracket in fig. ) based on functional similarities ( , ) . we found that the protein products of these genes are predominantly involved in antiviral response and signaling pathways. notably, the david analyses reveal a subset of unique genes, including several interferon isoforms, which are clustered together into statistically significant pathways associated with the immune response and signaling cascades. interestingly, the heat map of these genes reveals a similar temporal trend in upregulation of ifn gene expression during infection with each of the three viruses; however, this expression profile is by far the most pronounced in the endoumut-infected cells ( fig. a) . in this heat map view, as in fig. , it is difficult to ascertain any difference in expression of ifn genes between wild-type-and dubmut-infected cells. therefore, to obtain more information on the kinetics of transcriptional activation between groups, we quantitated the reads associated with each gene and graphically displayed the normalized read counts (fig. b ). using this method of plotting the read counts over time, we detected statistically significant transcriptional upregulation of ifn gene expression in the dubmut-infected cells. however, the magnitude of dubmut-induced ifn gene expression was dwarfed by ifn activation in endoumut-infected cells. endoumutinfected macrophages exhibited markedly elevated levels of ifn transcripts as early as h p.i. by h p.i., expression of the ifn gene transcripts upon endoumut infection was significantly higher than that during wild-type or dubmut infection, with our assay detecting , to , more reads per gene in the endoumut-infected cells. this focused response to endoumut is successful in limiting virus replication in cultured macrophages, as demonstrated in our previous report ( ) . notably, we evaluated the transcriptional response during the first h p.i., prior to the onset of apoptosis, which we and others have shown to occur in bmdms upon endoumut infection ( , ) . we note that the differential expression of ifn genes in the endoumut-infected cells influence the row z-score in fig. a to directly compare ifn gene expression between wild-type and dubmut infections, we generated a heat map of the comparative response of type i ifns, with the row z-score calculations reflecting the relative expression between these two infections (fig. c) . overall, we observe a significant increase in expression of all of these ifns, particularly at h p.i., during dubmutinfection relative to that during wt-virus infection. interestingly, despite of this elevated ifn expression during dubmut infection, we did not observe a similar increase in isg mrna, which is upregulated by endoumut virus infection (fig. b) . dubmut-infected cells exhibit downregulation of proinflammatory cytokine and chemokine genes compared to wild-type virus-infected cells. although we observed an enhanced ifn response during dubmut infection of bmdms, this phenotype did not lead to dramatic differences in pathogenesis during infection of mice ( ) . to further investigate the differences between the host responses to dubmut versus wild-type virus infection, we reduced the cutoff stringency to at least -fold differential expression with a significant q value (q Ͻ . ) and a normalized read count of at least . we identified genes upregulated in dubmut-infected cells at and h p.i. compared to wild type-infected cells (fig. a) ifna ifnb ifna ifna ifna ifna ifna ifna ifna ifna ifnab ifna ifna ifna ifna row z-score color key infected cells compared to wt-infected cells (fig. b ). our analysis identified dysregulation relative to the wild type of multiple genes that encode proinflammatory cytokines and chemokines, including il , il ␤, and il ␣ (fig. c) ccl , cx cl , and cxcl , compared to wt-infected cells. we note that the host response to wild-type and dubmut virus infections trended in the same direction and that these cytokine and chemokine responses were much more robust compared to the response to the endoumut virus infection. the results of proinflammatory cytokines gene expression shown in fig. prompted us to evaluate secretion of proinflammatory cytokines in response to the wild-type, dubmut, and endoumut virus infection. to determine the concentrations of pro-and anti-inflammatory cytokines released during infection, we performed cytometric bead array (cba) analysis on supernatants collected from virus-infected bmdms at h p.i. we performed the cba at h p.i. to allow for the cytokine mrna to be translated into protein and the protein to be secreted from the cells into the supernatant. we report that dubmut-and endoumut-infected cells have reduced levels of the inflammatory cytokines tumor necrosis factor alpha (tnf-␣), interleukin (il- ), and macrophage inflammatory protein ␣ (mip- ␣) relative to those in wild-ϩ type-infected cells (fig. a) . we also found that endoumut-infected cells exhibit lower levels of kc, il- , and mip- ␤ proteins relative to those in wt-and dubmut-infected cells (fig. b) . endoumut viruses induce apoptosis in bmdms ( ) , which likely limits their ability to produce proinflammatory cytokines. the results of these analyses indicate that wild type-infected bmdms have a heightened proinflammatory profile compared to those of dubmut-and endoumut-infected cells. this proinflammatory cytokine response has been shown to contribute to increased immunopathogenesis during in vivo infections ( ) . wild-type and dubmut coronavirus replication activate the unfolded protein response and host response to cell damage. in contrast to the robust and specific antiviral gene expression response detected in endoumut-infected macrophages, we uncovered a more complex response involving , genes in both wild-type-and and ) . in addition to a similar proinflammatory signature ( fig. and ) , we found that wild type-and dubmut-infected cells also exhibit hallmarks of activation of the host unfolded protein response (upr). upr pathways are activated when unfolded proteins accumulate in the endoplasmic reticulum (er), at which point the host cell initiates measures to resolve this overload ( ) . in the context of virus infection, viral glycoproteins accumulate in the er, triggering the release of bip from three sensor proteins: ire , atf , and perk. activation of these sensors triggers signaling cascades resulting in transcriptional activation of genes encoding er chaperones and proteins involved in lipid synthesis and amino acid transport (fig. a ). our previous study documented the activation of these pathways after mhv infection of fibroblasts ( ) . this study provides new information on the early transcriptional response to mhv in macrophages. based on our analyses of differential data were subjected to statistical analysis using two-tailed student's t tests. *, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ; ****, p Ͻ . ; ns, not significant. data are presented as means Ϯ sd. gene expression, we report significant transcriptional upregulation of multiple genes associated with activation of er sensors ire , atf , and perk, such as edem , hspa (encoding bip protein), and ddit (encoding chop), in response to virus replication, with the most robust response detected in cells infected with the wild-type or dubmut virus infection (fig. b, c, and d) . to further evaluate the activation of ire- during virus infection, we analyzed the splicing of xbp- mrna by reverse transcription-pcr (rt-pcr) and visualized the products by electrophoresis on agarose gels. we found that the wt and dubmut viruses exhibit higher proportions of spliced xbp- mrna at h p.i. and h p.i. compared to that in endoumut-infected cells (fig. e) , consistent with activation of the ire- arm of the unfolded protein response. overall, these differential gene expression analyses in macrophages reveal similar host responses to the wild-type and dubmut viruses that include activation of upr pathways and proinflammatory genes, whereas a distinct transcriptional profile during infection with the endoumut virus is predominately defined by a focused, robust antiviral response. we report that inactivation of a coronaviral interferon antagonist, endou, profoundly alters the host response to viral replication in macrophages compared to the response to wild-type coronavirus infection. we find that the endoumut virus elicits a rapid, robust, and specific antiviral response that is effective in limiting virus replication. in contrast, our data show that the wild-type and dubmut viruses ultimately elicit very similar host responses that are both characteristic of an unfolded protein response and consistent with a proinflammatory profile that is associated with viral pathogenesis ( ) . our studies of the dubmut-infected macrophages indicate that mere induction of type i ifn is not a sufficient marker for attenuation of the virus. instead, these results suggest that the timing and the magnitude of the host antiviral response are critical for determining the outcome of infection in macrophages (fig. ) and for pathogenesis in infected animal ( ) . our observation that the endoumut virus induces an earlier and more profoundly elevated type i interferon response than even the dubmut virus implies that there is a threshold of ifn expression that must be breached before the cell mounts an effective antiviral response. a remarkable finding from this study is the distinct transcriptional profile elicited by the endoumut virus during infection of bone marrow-derived macrophages compared to the profiles of wild-type-and dubmut-infected cells (fig. ) . we detected elevated levels of interferon and interferon-responsive genes as early as to h p.i., with endoumut-infected cells exhibiting by far the highest expression levels of these genes. we and others found that an antiviral response to endoumut infection results in activation of apoptosis, which subsequently limits virus replication in cell culture and in infected mice ( , ) . the current study indicates that screening mhv mutants in interferon-responsive cells may be an effective approach to identifying mutations that stimulate a robust innate immune response, which may then restrict virus replication in animals. previous studies of coronavirus-encoded interferon antagonists focused on evaluating the host transcriptional response to infection at later time points (such as and h p.i.) and in a variety of cell types or the tissues of infected animals. for example, a study comparing infection of mice with wild-type sars-cov versus a virus containing a mutation in the interferon antagonist nsp , termed the dnsp cov, revealed that the transcriptional profile in the lungs of the dnsp -infected mice mirrored the response to wt virus ( ) . however, combining the dnsp mutation with a mutation in another interferon antagonist, exon, was shown to reduce disease in mice and elicit protective immunity. it would be interesting to determine if the transcriptional profile elicited by the double mutant sars-cov is altered compared to that of the wild-type virus, particularly at early times after infection. a study of mers-cov comparing the host response to wt virus with the response to a mutant virus containing deleted accessory open reading frames (orfs) (mers-cov dorf - ) provides evidence of significant differences in the early transcriptional responses to infection in calu b cells ( ) . this study revealed that mers-cov dorf - infection prompted earlier ( , , and h p.i.) and more robust type i and type iii interferon responses, and that the mutant virus was more sensitive than the wild type to pretreatment with interferon. the mers-cov interferon antagonist mutant virus was attenuated in mice and elicited a protective response against subsequent lethal challenge. one difficulty that arises when evaluating the role(s) of virally mediated modulation of the host response is that multiple viral proteins, including both structural and nonstructural proteins, have been shown to antagonize the innate immune response in vitro and/or in vivo. these include: nsp - =o mtase, nsp -exon, nsp , nsp , e protein, n protein, m protein, sars-cov-orf , mers-cov-orf - , mers-cov- a, and mers-cov- b ( , ( ) ( ) ( ) ( ) ( ) . each of these interferon antagonists may play either a cell-or tissue-type-specific role, or may act in concert with other factors during viral replication to mitigate the innate immune response. further studies are needed to fully understand if all or only a subset of these antagonists must be silenced to generate an effective live-attenuated vaccine. the results presented here, and from studies of the mers-cov dorf - mutant virus ( ) upon infection of a bmdm with endoumut, host double-stranded rna (dsrna) sensors (including mda , pkr, and oas) are activated, resulting in robust transcription of type i ifn genes and rapid induction of apoptosis, the latter of which precludes the development of a potent inflammatory response. viral replication is severely restricted in these apoptotic macrophages as early as h p.i. although dubmut induces significantly higher type i ifn during infection than wt-mhv, infection with either dubmut or the wt yields same outcome, accumulation of er stress and subsequent activation of the upr, which then likely leads to the establishment of a robust nf-b-mediated proinflammatory response. bmdms infected with either of these viruses acquire a potently activated inflammatory phenotype, are unable to attenuate viral replication, and likely contribute to severe immunopathology in vivo. and inactivating nsp in pedv ( ) , suggest that inactivating interferon antagonists and screening for an early and robust antiviral transcriptional profile may represent an efficient and informative approach to evaluating live-attenuated vaccine candidate strains for existing and emerging coronaviruses. the dysregulation of innate immune signaling in myeloid cells, such as macrophages, is a key component of coronavirus-associated immunopathogenesis ( ) ( ) ( ) . using viral infection of macrophages, we can identify and evaluate the viral factors involved in antagonizing innate immune pathways. we and others have found that endou is important for the inhibition of innate immune signaling in macrophages ( , ) . loss of endou activity significantly alters gene expression and cytokine production in macrophages, which results in the attenuation of endoumut viruses in vivo ( ) . in contrast, we report mild attenuation of the dubmut virus in mice ( ) and show here that dubmut and mhv-a generate similar patterns of transcriptional activation in macrophages. while macrophages have been documented to be important for controlling cov infections, it is also important to appreciate that covs infect multiple cell types, including as epithelial cells in the lung and intestines. viral dub activity may play a more significant role during infection of epithelial cells, and further studies are needed to provide further insights into the role of dub activity during coronavirus infection. our results demonstrating upregulation of the unfolded protein response (upr) in response to wild-type and dubmut coronavirus replication confirm and extend the work of earlier studies that documented activation of the er sensors perk, ire , and atf- during coronavirus infection ( , , ) . heavy utilization of the endoplasmic reticulum for generating coronavirus replication complexes and of the er-golgi intermediate compartment for assembling virus particles places a substantial load on the host translational machinery during infection. host sensors ire , atf- , and perk are situated in the er to sense and respond to such overload by prompting upregulated expression of genes encoding er chaperones, amino acid transporters, and activators of lipid biosynthesis. ultimately, many of these proteins may facilitate virus replication and assembly. notably, it has been demonstrated that upr pathways that promote apoptosis are blocked during coronavirus replication ( , ) . the ability of viruses to modulate the upr has important implications for the innate immune response to such viruses because the upr has been shown to attenuate antiviral defenses by way of degrading the type i interferon receptor ( ) . to our knowledge, the data presented here provide the first transcriptomic evidence of upr activation in coronavirus-infected macrophages, underlining an important role for upr pathways in the coronavirus life cycle. our observation that endoumut-infected macrophages exhibit significantly lower expression of several genes involved in upr pathways compared with wild type-and dubmut-infected cells is consistent with the reduced levels of virus replication detected in endoumut-infected macrophages. the notion of inactivating viral interferon antagonists as a means of generating live-attenuated vaccines is supported by recent reports of screening for inactivation of influenza a virus-encoded interferon antagonists ( ), as well as studies that revealed that the classic vaccine strain of yellow fever virus encodes an interferon antagonist in the ns protein ( ) . for coronaviruses, it is not yet clear if disabling a single interferon antagonist, such as the highly conserved endou, will be sufficient to attenuate viruses that infect different cell types in different species. promisingly, our studies of a coronavirus that causes lethal disease in piglets, porcine epidemic diarrhea virus (pedv), revealed that inactivation of endou activity is associated with attenuated disease ( ) . we believe the information gained from studying coronaviruses containing inactivated interferon antagonists can be directly applied to the recently emerged coronavirus designated severe acute respiratory syndrome coronavirus (sars-cov- ) ( ) ( ) ( ) . more research is needed to determine if inactivating multiple interferon antagonists, including endou, is an effective approach for generating safe and protective live-attenuated coronavirus vaccines. ethics statement. the harvesting of bone marrow for obtaining bone marrow-derived macrophages in this study was carried out in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the experimental protocol was reviewed and approved by the institutional animal care and use committee (iacuc) at loyola university chicago (iacuc no. - ). c bl/ female mice were purchased from the jackson laboratory and maintained in the comparative medicine facility of loyola university chicago. bmdms and viruses. bone marrow-derived macrophages (bmdms) were prepared and cultured as described previously ( ) . differentiated bmdms were maintained in bone marrow macrophage (bmm) media containing dulbecco's modified eagle's medium (dmem) (product no. - -cv; corning) supplemented with % l cell supernatant, % fetal bovine serum (fbs), % l-glutamine, % sodium pyruvate, and % penicillin-streptomycin. wild-type mhv strain a (genbank accession number ay ) and endoumut (h a) were previously generated by reverse genetics and confirmed by deep sequencing. dubmut (d a) was generated as described in the companion paper ( ) . rna-seq data analysis pipeline. rna-seq data have been deposited in the ncbi geo database (accession number gse ). raw rna-seq reads were subjected to analysis using galaxy's online platform to generate differential gene expression data between infection groups ( ) . reads were clipped to remove any residual unique barcode sequences that were added during preparation of each sample for sequencing. clipped reads were concatenated to combine multiple files per sample into a single file per sample. these files were groomed to ensure that all reads were in sanger fastq format. fastq reads were aligned to the grcm ensembl build of the c bl/ j mouse genome using the hisat aligner, which locates the region of the genome to which each read corresponds, resulting in an output bam file ( ) . all reads that did not align to the mouse genome (i.e., reads that originated from viral rna) were discarded. the bam files contained the alignment information for each read in that sample and were used as inputs into featurecounts, which quantifies the number of reads in each sample that corresponds to each gene in the mouse genome ( ) . finally, the output count data from featurecounts was used as the input for deseq to calculate differential expression for each gene across all samples and treatment groups. deseq was used to generate normalized count values for each gene in all samples ( ) . these normalized counts are intended to correct for size differences between samples that might otherwise skew differential expression calculations if some samples contained substantially different numbers of total reads. the normalized count values were plotted and visualized in heat maps, generated using r, and line graphs, generated using prism software. identifying differentially expressed genes. to identify and analyze differentially expressed genes (degs) between infection groups, we used as a starting point the list of degs between mock-and wild-type mhv-infected bmdms at h p.i. that was generated as an output by deseq . this list of genes was filtered based on the statistical significance associated with the fold change differential expression value. a q value (adjusted p value, calculated by deseq for each gene in each comparison using the benjamini-hochberg procedure) of Ͻ . was chosen as the cutoff for statistical significance; genes whose differential upregulation values did not meet this cutoff in wild-type mhv-infected bmdms at h p.i. compared with mock-infected cells were removed from the list ( ). next, a differential expression magnitude cutoff of Ͼ was applied to the remaining genes. genes that were not more highly expressed by at least -fold in wild-type mhv-infected cells at h p.i. compared to mock were removed from the list. after applying these cutoffs, , genes remained and were arranged in order of most to least highly upregulated in wild-type mhv-infected cells at h p.i. compared to mock. cluster . software was then used to apply mathematical clustering to the z-score-standardized log -normalized mean normalized count values associated with each gene at each time point and in each infection group ( ) . specifically, the default settings-the similarity metric "pearson correlation (uncentered)" and the clustering method "centroid linkage"-were applied to the list of , genes and the corresponding expression values for each gene across all samples to produce a hierarchically clustered gene list based on how similar or different the expression patterns were between groups of genes across all samples. this new list of clustered genes and their associated expression values were then visualized as a heat map using java treeview software. functional clustering analyses using david. the bracketed set of genes identified in fig. were subjected to david analyses. specifically, under the "gene ontology" category, the data from the chart associated with the "goterm_bp_direct" result were reported here. after excluding all functional cluster terms that were not statistically significant using a cutoff of q Ͻ . , we identified a total of unique genes that appeared in at least one cluster of statistical significance. expression values of these genes were plotted in heat map and line graph forms as described above. xbp- splice analysis. c bl/ bmdms were infected with wild-type-, dubmut-, or endoumut-mhv at an moi of . at designated time points, intracellular rna was isolated from cells with rneasy kit (catalog no. ; qiagen). isolated rna was reverse transcribed into cdna with an rt first strand kit (catalog no. ; qiagen). xbp- splicing was analyzed by pcr following the protocol from bechill et al. ( ) . briefly, pcr was performed with the forward primer =-gttgagaaccaggagttaag- = and the reverse primer, =-agagaaagggaggctggtaag- =. pcr conditions were (i) °c for min, (ii) °c for min, (iii) °c for min, and (iv) °c for min, repeating steps ii to iv for cycles. pcr products were separated on % polyacrylamide-tbe ( mm tris-borate and . mm edta [ph . ]) gel, stained with sybr green ii stain, and imaged. quantification of band density was performed with image lab software (bio-rad). cytometric bead array. c bl/ bmdms were infected with wild-type-, dubmut-, or endoumut-mhv at an moi of . at h p.i., supernatants were collected and centrifuged at , ϫ g for min at cba staining was performed according to the manufacturer's instructions (cba flex set mouse soluble proteins, catalog no. ; bd biosciences) molecular mechanisms of innate immune inhibition by non-segmented negativesense rna viruses vaccinia virus protein c inhibits type i ifn signalling in the nucleus and binds to the 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spliced aligner with low memory requirements featurecounts: an efficient general purpose program for assigning sequence reads to genomic features moderated estimation of fold change and dispersion for rna-seq data with deseq open source clustering software we thank qunfeng dong for assistance with the bioinformatic analyses. we also thank robert mettelman for assistance with quantitative pcr (qpcr) setups and help editing the manuscript. we thank francis alonzo iii and zack resko for assistance with the cytometric bead array analyses. key: cord- -b a hhw authors: bellingan, g title: leukocytes: friend or foe date: journal: intensive care med doi: . /s sha: doc_id: cord_uid: b a hhw leukocytes have a fundamental role in innate and adaptive immunity, wound healing, tumour surveillance and in tissue remodelling. it is their function in the inflammatory response however that is of most interest to us in the intensive care setting. over the last three decades we have gained significant insights into leukocyte activation, recruitment and mediator secretion and the contribution of these agents to both the onset and resolution of sepsis and inflammation.¶the body relies on the inflammatory response for protection. leukocytes occupy a pivotal position in this but to maintain these cells in a state of permanent activation would be unsustainable, with widespread microvascular plugging, uncontrolled free radical release and an excessive metabolic demand. leukocytes thus circulate in a quiescent state and are rapidly activated by invading pathogens and other stimuli. a direct consequence of this protective strategy is that the inflammatory response may be inadequate, with the risk of overwhelming sepsis, or excessive, leading to rampant systemic inflammation and consequent multiple organ damage.¶it is now becoming apparent however that in addition to leukocytes other cells have important roles both in defence against invading pathogens and in driving malignant inflammation. this review will focus on two new facets of the innate immune system, the toll family of proteins as the signal transduction element for endotoxin, and the antimicrobial peptides. these exemplify potential damaging and protective response elements but importantly neither are restricted to leukocytes. the capacity of cells and tissues other than the leukocytes to participate and even lead in the inflammatory responses will also be explored. leukocytes are essential for a functional immune response, for normal wound healing, tumour surveillance and during development and tissue remodelling, it is their role in the inflammatory response however that is of most interest to us in the intensive care setting [ ± ] . it is now well established that leukocyte activation can be a double edged sword; the body is critically dependent upon these cells for protection from pathogens and an ineffective inflammatory/immune response can be lethal. a balance needs to be struck however as an excessive inflammatory response can also kill through progressive inflammation and consequent multi-organ dysfunction [ ± ] . leukocytes normally circulate in a quiescent state but can be rapidly activated to repel invading pathogens. this system of leukocyte activation has evolved because it would be unsustainable to maintain these cells in a state of permanent activation. this would lead to widespread leukocyte plugging, uncontrolled free radical release and an unwarranted metabolic de- abstract leukocytes have a fundamental role in innate and adaptive immunity, wound healing, tumour surveillance and in tissue remodelling. it is their function in the inflammatory response however that is of most interest to us in the intensive care setting. over the last three decades we have gained significant insights into leukocyte activation, recruitment and mediator secretion and the contribution of these agents to both the onset and resolution of sepsis and inflammation. the body relies on the inflammatory response for protection. leukocytes occupy a pivotal position in this but to maintain these cells in a state of permanent activation would be unsustainable, with widespread microvascular plugging, uncontrolled free radical release and an excessive metabolic demand. leukocytes thus circulate in a quiescent state and are rapidly activated by invading pathogens and other stimuli. a direct consequence of this protective strategy is that the inflammatory response may be inadequate, with the risk of overwhelming sepsis, or excessive, leading to rampant systemic inflammation and consequent multiple organ damage. it is now becoming apparent however that in addition to leukocytes other cells have important roles both in defence against invading pathogens and in driving malignant inflammation. this review will focus on two new facets of the innate immune system, the toll family of proteins as the signal transduction element for endotoxin, and the antimicrobial peptides. these exemplify potential damaging and protective response elements but importantly neither are restricted to leukocytes. the capacity of cells and tissues other than the leukocytes to participate and even lead in the inflammatory responses will also be explored. mand with progressive tissue damage and multi-organ dysfunction syndrome (mods). a direct consequence of this protective strategy is that the inflammatory response may be either inadequate, with the risk of overwhelming sepsis, or excessive, leading to rampant systemic inflammation [ ] . recently further layers of complexity have been described including the endogenous compensatory anti-inflammatory response syndrome (cars) protecting against excessive inflammation [ ] . moreover the underlying insult can impair the immune response and aggressive or persisting inflammation can lead to a state of anergy and heightened risk of further infection [ ] . to address the role of leukocytes in the inflammatory process and the balance that needs to be struck between an ineffective and an excessive response we need to understand a little about the inflammatory response itself. this also needs to be put into context as the leukocytes are not the only players involved. the genetic regulation of these processes is also of increasing importance. serious infections are the most common cause for admission to intensive care and the clinical features are well described [ ] . it is now clear that other conditions such as multiple trauma and burns also result in a state of generalised inflammation in the absence of infection. this has led to the description of the systemic inflammatory response syndrome (sirs), a multi-system inflammatory state characterised by excessive immuno-inflammatory cascade activation with widespread reduction in cellular oxygen utilisation, atp depletion, cell injury and death [ ] . the most common agents initiating inflammation are bacterial cell wall components including teichoic acid and peptidoglycans from gram-positive bacteria and lipopolysaccharide (lps) from gram-negative bacteria [ ] . activation induces multiple mediator networks including the complement, kinin, coagulation and fibrinolytic cascades, synthesis of lipid mediators, chemokines, cytokines and release of soluble receptors, along with free radical synthesis and leukocyte degranulation with release of numerous enzymes, including elastase, myeloperoxidase, proteases, collagenase and plasminogen activator all occurring as part of an interrelated network [ , , , ] . importantly, many cells other than leukocytes are also activated during inflammation, including endothelial cells, mesothelial cells and fibroblasts [ ± ]. these cells are all able to elaborate multiple inflammatory agents and represent a huge reservoir for mediator synthesis that does not often receive due consideration. as most infections occur primarily in the tissue and not in the blood stream, extravasation of leukocytes is essential in bring inflammatory cells and foreign pathogens into contact. this requires both a chemotactic gradient and co-ordinated up-regulation of endothelial and inflammatory cell adhesion molecule expression. pro-inflammatory agents rapidly up regulate e and pselectins which, with l-selectin, mediate leukocyte rolling along the endothelium [ ]. the b integrins lfa- (cd a), mac- (cd b) and p / (cd c) are the main leukocyte adhesion molecules responsible for firm adhesion, their endothelial ligands are icam- (cd ), icam- (cd ) and vcam- (cd ). pe-cam and vla- are essential for transmigration. these adhesion molecules are also all closely regulated by activating signals [ ± ] . once initiated, the intensity and duration of the inflammatory response is closely regulated [ ] . numerous mechanisms are invoked in this including ªanti-inflammatoryº cytokines (e. g. il- ra, il- , il- ) and endogenous anti-endotoxin antibodies acting to damp down the inflammatory response while the leukocytes phagocytose and kill the offending pathogens. leukocytes have only a finite life-span at the inflamed site with neutrophils rapidly undergoing apoptosis to be cleared by inflammatory macrophages, which themselves emigrate from the inflamed site during the resolution phase [ ± ]. thus a successful inflammatory event requires not only appropriate activation of cells and mediators with phagocytosis and removal of the inciting stimulus but also consequent elimination of the inflammatory cells and debris to allow the tissues to return to normal architecture and function. it is quite clear that leukocytes are fundamental for survival as exemplified so devastatingly by neutropenia [ ] . in addition, defects in leukocyte function can be just as harmful as reduction in numbers, with clear examples ranging from leukocyte adhesion deficiency (lad) where the cells are functional but cannot get to the site of infection, to chronic granulomatous disease (cgd) where cells form collections at sites of infection but lack functional killing ability [ , ] . the list of such conditions is long and simply serves to demonstrate the relative importance of different functions on adhesion, phagocytosis, free radical generation, and killing. neutrophils and macrophages not only directly phagocytose and kill pathogens but are also key regulators of the inflammatory response as they have a powerful capacity to initiate and perpetuate the multiple inflammatory mediator cascades described above. although billions of dollars have been spent on the, as yet, fruitless search for immunomodulatory agents that will effectively damp this response it has become clear that an intact and robust inflammatory response is indeed crucial for survival. anti-tnf therapy provides an excellent example of how important the normal inflammatory response is as inhibition of tnf-a activity leads to increased not decreased mortality in a number of septic models including caecal ligation and puncture, listeria, and candida albicans infections [ ± ] . in other diseases such as malaria, tnf-a blockade may be beneficial or harmful depending upon the underlying susceptibility which may be under genetic regulation [ , ] . this highlights the importance of differentiating between live micro-organisms and non-infectious agents such as lps when interpreting results from inflammatory models [ ] . furthermore the method whereby neutralising antibodies are delivered may also be critical [ ] . this is even more neatly demonstrated by yersinia enterocolitica infection where this bacterium produces a protein called yopb which directly inhibits host tnf-a production [ ] . inhibition of yopb with an anti-yopb serum increases tnf-a production and reduces bacterial growth again demonstrating the key role of tnf-a in controlling infection. other yop proteins may also be involved [ ] . severe critical illness can depress the immune system which can increase mortality [ ] . for example, patients with blunt or penetrating trauma exhibit reduced responses to usual recall antigens, the greater the injury severity the longer the period of anergy [ ] . haemorrhage also depressed macrophage antigen presentation by % or more in a mouse model probably through reduced antigen catabolism rather than reduced presentation [ , ] . similarly prolonged critical illness leads to immune hypo-responsiveness, the cause of which is unclear but can be related to reduced leukocyte responsiveness and monocyte hla-dr expression has successfully been used as a surrogate marker of this [ ]. boosting leukocyte functional responses by interferon g have been associated with a significant improvement in outcome suggesting that a fully functional leukocyte response throughout the inflammatory response is vital to successful outcome. leukocyte stimulation with g-csf or gm-csf also demonstrates the therapeutic value of these cells both with leukopenia and also in the treatment of infection in the non-neutropenic patients [ ± ] . other leukocyte functions vital to recovery from critical illness include wound healing, tissue remodelling and generation of an adaptive immune response with memory. this is in addition to those functions not immediately relevant to the intensive care setting including tumour surveillance and their role in growth and development. it is thus abundantly clear that leukocytes are friends without which we would not have survived or evolved. problems such as transplant rejection, hypersensitivity and allergy and white cell malignancies will not be considered in this review, although these are leukocyte driven and can lead to the requirement for intensive care. instead the focus will be on the potential for leukocytes to damage the host as part of the systemic inflammatory response syndrome. the concept that leukocytes could directly damage the host is not new. as so eloquently described by lewis thomas ªit is the information carried by the bacteria that we cannot abide. the gramnegative bacteria are the best examples of this. they display lipopolysaccharide endotoxin in their walls, and these macromolecules are read by our tissues as the very worst of bad news. when we sense lipopolysaccharide, we are likely to turn on every defence at our disposalº [ ] . this is supported by many observations that leukocyte depletion could prevent host damage as shown in table . this emphasises however the essential balance between leukocytes as a powerful motor of tissue damage and their key role in prevention of infection. ards is a particularly important condition in which the neutrophils are believed to act as a prime driving force. these cells have the capacity to damage the host with the production of free radicals and release of enzymes into a protected local microenvironment. moreover the neutrophil can secrete the pro-inflammatory the leukocytes are not the only cells that can synthesise and release significant quantities of inflammatory agents. endothelial cells, mesothelial cells and fibroblasts are all highly metabolically active and are amongst many cells that can produce pro-inflammatory cytokines, free radicals and lipid mediators, some of these are listed in table [ ± ]. moreover endothelial cells and mesothelial cells can present antigen while fibroblasts can phagocytose apoptotic cells. thus consider the peritoneum in the absence of neutrophils and macrophages. infection can elicit a massive surge of pro-inflammatory cytokines and free radicals, the omentum can wall off and localise infection while mesothelial cells can present antigen to lymphocytes driving an adaptive immune response. similarly in the lung the endothelial and epithelial cells along with fibroblasts can reproduce much of the classic inflammatory response that we normally associate with the leukocytes. these include many cytokines such as il- and tgfb which are directly implicated in the pathogenesis of ards and consequent fibrosis [ ] . it is becoming clearer that a greater understanding of the involvement of stromal cells in the synthesis and release of pro-inflammatory mediators in response to infection is required especially in regard to inflammation limited to specific regions or organs. as noted earlier the response to lps is uniquely sensitive but it is only recently that the signal transduction pathway for this has been fully established. in an intricate pathway lps has been shown to complex with circulating lipopolysaccharide binding protein (lbp) which binds to the cd receptor leading to inflammatory cell activation [ ] . cd has no cytoplasmic tail hence the mechanism through which it led to activation of nfkb and pro-inflammatory cytokine synthesis was not clear [ ] . detailed experiments on drosophila melanogaster have led to a much greater understanding of the innate immune response. it is now recognised that there are multiple pattern recognition receptors expressed on leukocytes and also on other cells that respond to major groups of pathogens [ ] . through these receptors activation responses are elicited to membrane components such as teichoic acid, peptidoglycans and endotoxin. the toll gene was first described for its function in dorsoventral pattern formation in drosophila, but it also controls the fly's immune response to fungal infection [ ] . one of the other four drosophila toll-like receptor (tlr) proteins called w is also involved in immune responses as antibacterial responses are compromised if this protein is deleted [ ] . the cytoplasmic domain of toll was found to be closely homologous to the cytoplasmic domain of the il- receptor although the extracellular domains are unrelated. although the toll protein family was first identified in drosophila it has now been characterised as the signal transducing element for lps in man [ , ] . lps induced signalling though toll leads to activation of nfkb and pro-inflammatory cytokine synthesis. importantly, non-myeloid cells can be activated by lps in a cd /lbp dependent fashion and toll-like receptors are expressed on cells other than peripheral blood leukocytes. there are tlr described in man, it is unknown as yet whether other tlr recognise other major pattern recognition molecules such as peptidoglycan, teichoic acid. certainly drosophila, which do not have an adaptive immune response, can elicit relatively specific responses to the major classes of bacteria through innate pattern recognition molecules [ ] . furthermore the tlr are not restricted to the phagocytes, indeed with expression on intestinal cells for example, their distribution is quite broad. this provides multiple cells with the capacity to respond to lps (and possibly to other major classes of bacterial cell wall proteins). hence it may well be through innate immune defences such as toll that cells other than phagocytes participate in the inflammatory response. we should thus beware of focusing on the leukocytes as the effector cells of the immune system when it is clear that functional receptors may have a much wider distribution than previously thought. it is now known that the main lps detoxification systems are bactericidal permeability increasing protein (bpi), serum amyloid protein and the lipoprotein system, especially hdl [ ± ]. the main source of bpi are the phagocytes and bpi inhibits lps delivery to monocyte cd and appears to condense lps aggregates whilst lbp promotes lps delivery to monocyte cd and disaggregates lps [ ] . lbp does however promote the transfer of lps into phospholipid micelles and most endotoxin added to blood ends up in the lipoprotein fraction. moreover lipoprotein depletion increases tnf-a levels and mortality while increased lipoprotein levels improve mortality and this is due at least in part to reduced production of pro-inflammatory cytokines [ ] . hdl is the main lps binding lipoprotein, although some is found in ldl but very little in vldl/chylomicrons. it seems that with time hdl hands lps over to ldl which takes it to the liver where it is secreted in the bile. septic patients often have very low hdl and apo a- levels which may increase their sensitivity to lps. furthermore increasing hdl through the use of reconstituted rhdl discs decreases tnf-a levels [ ] . this is despite an increase in detectable lps due to retention in the circulation in relatively inaccessible aggregates in hdl. the binding of lps to cells is far faster however than hdl inactivation, with cell binding occurring within minutes while hdl inactivation occurring over to hours [ ] . reconstituted hdl is a powerful binder and neutraliser of lps but is similarly slow. thus the major method for lps detoxification is not through leukocytes but via the lipoprotein micelles. it is not just the leukocytes that limit the inflammatory response through direct antimicrobial inhibitory mechanisms. key components of the innate immune response are the antimicrobial peptides which are listed in table . these are small antimicrobial peptides with usually less than amino acid residues that commonly carry a positive changes and are widely distributed across body surfaces and in secretions. they are reviewed by lehrer and ganz [ ] . although many of these are produced by leukocytes their major site of production is by cells lining the respiratory, renal and reproductive tracts and the epithelial surfaces. the defensins are b pleated sheet peptides of to amino acids in size. there are six a-defensins, human defensins ± are restricted to neutrophils while defensins and are produced by epithelial cells and protect the intestinal and female reproductive tracts respectively. the b-defensins produced by epithelial cells protect the respiratory, renal and reproductive tracts. likewise the cathelicidins (hcap- and its c terminal domain active fragment ll- ) are found at surface epithelial cells and mucous glands of the respiratory tract. the histatins are salivary proteins with activity against fungi, including azole resistant organisms [ ] . secretory leukoprotease inhibitor is found in many secretions and on epithelial surfaces. it is a amino acid peptide with antiprotease activity at the carboxy-terminal domain and broad spectrum antimicrobial activity at the amino terminal end. it is clear that in the antimicrobial peptides we have a broad and effective system to protect the body against infection that is mainly dependent table the main classes of human antimicrobial peptides on non-leukocyte production, hence the body does not rely solely on leukocytes as the only safeguard against pathogens. it is clear that leukocytes are essential to surviving an infective challenge, but that they can also cause overwhelming damage to the host. moreover, inflammation is a continually evolving process and different aspects of leukocyte function will be paramount at different stages, while the invading pathogens themselves can further modulate the host's inflammatory response. it is also clear that the leukocytes are not the only cells involved in the inflammatory response. endothelial cells, mesothelial cells, fibroblasts and epithelial cells are also all involved not only with their capacity to drive the inflammatory response through mediator generation but also in innate immune defences including through the production of antimicrobial proteins. paracrine signals between these cells will contribute to ªregionsº of inflammation rather than total systemic inflammation. our ability to monitor local and regional inflammation is only in its infancy. furthermore, early in the inflammatory response it may be appropriate to block excessive cytokine production while during later states anergy can develop where it would be more appropriate to boost leukocyte function. we do not have clear methods as yet to determine which stage inflammation is at nor do have the necessary tools to selectively block or boost specific leukocyte functions. leukocytes are an essential component in a system involving nearly every cell and tissue in the body. while excessive leukocyte activation is a key feature of malignant inflammation, many other cells are capable of synthesising pro-inflammatory cytokines and driving a damaging response. the complexity, redundancy and plasticity of host defence mechanisms make it unlikely that a global panacea for the inflammatory response will be discovered. chemokine expression in endothelial cells and monocytes is differentially regulated neutrophilic differentiation induced by human upper airway fibroblast-derived granulocyte/macrophage colony-stimulating factor (gm-csf) elevated transforming growth factor-alpha levels in bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome activation of the adhesive capacity of cr on neutrophils by endotoxin: dependence on lipopolysaccharide binding protein and cd recognition of gram-negative bacteria and endotoxin by the innate immune system innate immunity: the virtues of a nonclonal system of recognition the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults the toll-receptor family and control of innate immunity a human homologue of the drosophila toll protein signals activation of adaptive immunity toll-like receptor- mediates lipopolysaccharide-induced cellular signalling interchangeable endotoxinbinding domains in proteins with opposite lipopolysaccharide-dependent activities lipopolysaccharide (lps)-binding synthetic peptides derived from serum amyloid p component neutralize lps effects of specific antibodies, hormones, and lipoproteins on bacterial lipopolysaccharides injected into the rat lipopolysaccharide (lps)-binding proteins bpi and lbp form different types of complexes with lps lowdensity lipoprotein receptor-deficient mice are protected against lethal endotoxemia and severe gram-negative infections reconstituted high-density lipoprotein neutralizes gram-negative bacterial lipopolysaccharides in human whole blood bacterial lipopolysaccharide binds and stimulates cytokine-producing cells before neutralization by endogenous lipoproteins can occur antimicrobial peptides in mammalian and insect host defence candidacidal activity of salivary histatins. identification of a histatin -binding protein on candida albicans key: cord- -d gxew authors: grimble, robert f. title: the interaction between nutrition and inflammatory stress throughout the life cycle date: journal: nutrients, stress, and medical disorders doi: . / - - - : sha: doc_id: cord_uid: d gxew the human race inhabits a world in which it is surrounded by a myriad of different microorganisms—yeasts, bacteria, protozoa, and viruses. most of these are benign, and some, such as the normal gut flora, play an important part in promoting health via the synthesis of vitamins and stimulation of normal function of gut epithelia. approximately . % of microbes in our environment have catastrophic effects if they penetrate the epithelial surfaces of the body (bryson, ). history reveals many instances in which armies have been defeated and civilizations have collapsed because of encounters between humans and such microorganisms (diamond, ). the human race inhabits a world in which it is surrounded by a myriad of different microorganisms--yeasts, bacteria, protozoa, and viruses. most of these are benign, and some, such as the normal gut flora, play an important part in promoting health via the synthesis of vitamins and stimulation of normal function of gut epithelia. approximately . % of microbes in our environment have catastrophic effects if they penetrate the epithelial surfaces of the body (bryson, ) . history reveals many instances in which armies have been defeated and civilizations have collapsed because of encounters between humans and such microorganisms (diamond, ) . humans, like all mammals, have evolved with a complex immune system, which is present as specialized organs (spleen, thymus) or cell types (lymphocytes, macrophages, and mast cells) throughout the body. the system can detect and destroy any cell or particle that is not"self," i.e., a normal component of the body. a complex series of events follows from contact between components of the immune system and microbes invading the body ( fig. ) . the response can be divided into two main categories. the first is the acquired immune response, in which the immune system recognizes specific chemical motifs on the invader and "remembers" the encounter so that a more rapid, specific, and intense response can be produced at any future meeting. the second category is the nonspeciflc response in which the response to each encounter is similar for all invaders of the body. the process of inflammation is a central part of the second category of response. the immune response is also activated by a wide range of adverse events, such as surgery, bums, and trauma. the primary purposes of the response are to kill pathogens and initiate the curative processes that will restore body function to normal. the first purpose is achieved by creating a hostile tissue environment through production of oxidant molecules and activation of t and b lymphocytes. part of the response ensures a supply of substrate, from endogenous sources, for supporting the activity of t and b lymphocytes and enhancement of antioxidant defenses. the latter event is important for protecting healthy tissue from the oxidants produced as part of the inflammatory response (grimble, a) . the response exerts considerable biological demands and stress on the body. a central part of substrate provision is the release of amino acids into the blood from the breakdown of proteins in skeletal muscle, skin, and bone matrix, and fatty acids released from triglycerides stored in adipose tissue. enhanced gluconeogenesis, catabolic hormone production, and decreased insulin sensitivity occurs to facilitate this redistribution of tissue infective or cellular fig. . diseases and conditions in which inappropriate or excessive amounts of pro-inflammatory cytokines exert adverse or lethal effects on the host. components (fig. ) . the animal loses the desire to carry out many day-to-day activities. physical weakness ensues, exploratory activity declines, appetite is decreased, and apathy and sleep may occur. the response thus exerts physiological and mental stress upon the body. inflammation comes under the control of signaling proteins (cytokines) that possess hormone-like actions. the pro-inflammatory cytokines interleukin (il)- , il- , and tumor necrosis factor (tnf)-t~, are major activators and modulators of the events described above. to modulate the degree of stress imposed on the body, in achieving the essential functions of inflammation, the response comes under the control of powerful anti-inflammatory mechanisms. these will impose their biological effects with increasing vigor as the original stimulus for the inflammatory response (infection, injury) declines in intensity. heat-shock proteins, endorphins, glucocorticoid hormones, and cytokine receptor antagonists are important components of this anti-inflammatory system.this system is essential for closing down the inflammatory response once it has achieved its primary purposes because of the high biological cost it imposes on the body (grimble, a) . although cytokines play an important role in the response to infection and injury, they can exert damaging and lethal effects on the host. many studies have shown that excessive or prolonged production of cytokines is associated with increased morbidity and mortality in a wide range of acute and chronic inflammatory conditions (fig. ) . these include sepsis, adult respiratory distress syndrome, malaria, meningitis, cancer, cystic fibrosis, systemic lupus erythematosus, inflammatory bowel disease, rheumatoid arthritis, and asthma. events similar to those seen in the inflammatory response to injury and infection can be observed during the course of overt inflammatory diseases such as rheumatoid arthritis and crohn's disease and in diseases that have a covert inflammatory basis, for example, atherosclerosis and diabetes mellitus (fig. ) . clearly the inflammatory response in these situations does not have a purposeful nature and contribute to the disease process. recent studies indicate that low-intensity inflammation occurs in elderly and obese individuals (grimble (grimble , . thus, the inflammatory response, which has evolved to allow humankind to survive infection and injury, is indiscriminate in both its triggers and targets. as a result, the process is a two-edged sword capable of both defending and damaging its bearer. during the remainder of this chapter we will be exploring the biological and nutritional factors that determine the intensity of, and outcome from, the inflammatory process. various components of the inflammatory response interact to modulate its intensity. predominant among these interactions are the relative amounts of pro-and anti-inflammatory cytokines produced during the response to microbes and injury and the effect of oxidant molecules on cytokine production. early work on cytokines and the response to infection linked excessive pro-inflammatory cytokine production with increased morbidity and mortality in a wide range of conditions, such as malaria, meningitis, and sepsis. however, research in the last yr has shown that the balance in production between pro-and anti-inflammatory cytokines has a more direct bearing on the outcome of infection and injury. for example, in sepsis, plasma il- concentrations were higher and il- concentrations were lower in patients who died than in those who survived (arnalich et al., ; taniguchi et al., ) . a survey of over patients admitted to hospital in the netherlands with fever showed that, independently of how the patients were clinically classified (positive blood cultures, presence of endotoxin), those who subsequently died had a higher plasma il- :tnf-o~ ratio than patients who survived (van dissell, van langervelde, westendorp, kwappenberg, & frolich, ) . powerful oxidant molecules (e.g., superoxide, hydrogen peroxide, hypochlorous acid) are produced as part of the inflammatory response. their biological purpose is to destroy invading microbes. however, these molecules also have the capacity to damage host tissues and to increase the intensity of the inflammatory response. clearly both of these biological events can have adverse effects upon the host. the oxidant molecules activate at least two important families of proteins in the host that are sensitive to changes in cellular redox state. the families are nuclear transcription factor k b (nf-rd ) and activator protein (ap ). these transcription factors act as "control switches" for biological processes, not all of which are of advantage to the individual. nf-rd is present in the cytosol in an inactive form, by virtue of being bound to an inhibitory unit i-r,b. phosphorylation and dissociation of i-r,b renders the remaining nf-~cb dimer active. the dissociated i-rd is degraded, and the active nf-r,b is translocated to the nucleus, where it binds to response elements in the promoter regions of genes. a similar translocation of ap , a transcription factor composed of the protooncogenes c-fos and c-jun, from cytosol to nucleus, also occurs in the presence of oxidant stress. binding of the transcription factors is implicated in activation of a wide range of genes associated with inflammation and the immune response, including those encoding cytokines, cytokine receptors, cell adhesion molecules, acute-phase proteins, and growth factors (schreck, rieber, & baeurerle, ) (fig. ) . activation of nf-rd can be brought about by a wide range of stimuli including pro-inflammatory cytokines, hydrogen peroxide, mitogens, bacteria and viruses and their related products, and ultraviolet (uv) and ionizing radiations. the extent of activation of nf-rd will depend in part upon the strength and efficiency of the antioxidant defenses of the body. these comprise endogenous components such as glutathione (gsh) and enzymatic components of antioxidant defenses, such as catalase, superoxide dismutase (sod), and gsh peroxidase, and dietary components that have antioxidant properties (e.g., vitamins c and e and polyphenolic compounds). the influence of modulation of inflammation by these dietary factors are dealt with later. an unfortunate side effect of activation of nf-r,b arises from the ability of the transcription factor to activate transcription of the genes of some viruses, such as human immunodeficiency virus (hiv) (fig. ) . this sequence of events, in the case of hiv, accounts for the ability of minor infections to speed the progression of individuals who are infected with hiv towards acquired immunodeficiency syndrome (aids). thus, if antioxidant defenses are poor, each encounter with general infections results in cytokine and oxidant production, nf-~zb activation, and an increase in hiv replication. it is thus unfortunate that reduced cellular concentrations of gsh are a common feature of infections, including that from the hiv (staal, ela, & roederer, ) . oxidant damage to cells will indirectly create a pro-inflammatory effect by the production of lipid peroxides. this situation may also lead to upregulation of nf-tcb activity. as will be seen in later sections, genetic and dietary factors change the intensity of the inflammatory response. thus, although the inflammatory response has evolved to ensure the survival of the human species, individuals may die as a result primarily of the response to invasion rather than from the invasive agent itself. it has recently become apparent that single base changes (single-nucleotide polymorphisms [snps]), usually in the promoter region of genes responsible for producing the molecules involved in the inflammatory process, exert a modulatory effect on the intensity of inflammation. in vitro production of tnf-o~ by peripheral blood mononuclear cells (pbmcs) from healthy and diseased subjects stimulated with inflammatory agents shows remarkable individual constancy in males and postmenopausal females (jacob et al., ) . this constancy suggests that genetic factors exert a strong influence. a number of studies have shown that snps in the promoter regions for the tnf-o~ and lymphotoxin table single nucleotide polymorphisms (snps) in cytokine genes associated with altered levels of cytokine production # gene and location of polymorphism in promoter region genotype associated with raised cytokine production and~or altered clinical outcome to inflammation b pro-inflammatory cytokines tnf-t~ - lt-t~ + il-l~- il- - anti-inflammatory cytokines il- - c tgf- [~ + (arg- -pro) c tnf-ct - a allele (tnf ) lt-t~ + aa (tnfb : ) ct or tt g allelle gg gg tnf, tumor necrosis factor; lt, lymphotoxin; il, interleukin; tgf, transforming growth factor; c, cytosine; g, guanosine; t, thymidine, a, adenine. athe location of the polymorphism is indicated by the nucleotide position in the promotor region. bpoor clinical outcome for pro-inflammatory cytokines. clmproved clinical outcome for anti-inflammatory cytokines. (lt)-o~ genes are associated with differential tnf-t~ production (allen, ; messer et al., ; wilson et al., ) . the tnf (a) and tnfb (a) alleles (at - and + for the tnf-o~ and lt-t~ genes, respectively) are linked to high tnf production, particularly in homozygous individuals. the snp in the lt-ot gene (+ ) is found in linkage disequilibrium with major histocompatibility molecules hla-a , b , dr (messer et al., ; wilson et al., ) . this genotype has also been reported to define a tnf "high expresser" haplotype (warzocha et al., ) , in addition to modifying expression of lt-o~ itself (messer et al., ) . a large body of research has indicated that snps occur in the upstream regulatory (promoter) regions of many cytokine genes (bidwell et al., ) . many of these genetic variations influence the level of expression of genes and the outcome from the inflammatory response. both pro-and anti-inflammatory cytokines are influenced by the differences in genotype (allen ; turner, williams, & sankeran, ) . a number of snps that have been implicated in the outcome of inflammatory stress are shown in table . nf-lcb is activated by oxidants and switches on many of the genes involved in the inflammatory response (cytokines, adhesion molecules, and acute-phase proteins). enhancement of antioxidant defenses is important in protecting healthy tissues and in preventing excessive activation of nf-~zb by the oxidative cellular environment during inflammation (schreck et al., ) . nf-rd upregulates cytokine and adhesion molecule expression, increasing the risk of host damage (jersmann, hii, ferrante, & ferrante, ) . genetic factors also influence the propensity of individuals to produce oxidant molecules and thereby influence nf-rd activation. natural resistance-associated macrophage protein (nramp ) has effects on macrophage functions, including tnf-ct production and activation of inducible nitric oxide synthase (inos), which occurs by cooperation between the nramp , tnf-o~, and lt-o~ genes (ables et al., ) . there are four variations in the nramp gene, resulting in different basal levels of activity and differential sensitivity to stimulation by inflammatory agents. alleles , , and are poor promoters, whereas allele causes high gene expression. hyperactivity of macrophages, associated with allele , is linked to autoimmune disease susceptibility and high resistance to infection, whereas allele increases susceptibility to infection and protects against autoimmune disease (searle & blackwell, ) . as indicated earlier, a number of molecules suppress production of pro-inflammatory cytokines and exert an anti-inflammatory influence. these include antioxidant defenses and il- (chernoff et al., ; espevik et al., ) . production is modulated by genetic factors. there are at least three polymorphic sites (- , - , - ) in the il-l promoter that influence production (perrey, pravice, sinnott, & hutchinson, ) . snps also occur in genes encoding enzymatic components of antioxidant defenses, such as catalase, sod, and gsh peroxidase, which influence levels of activity (chorazy, schumacher, & edlind, ; forsberg, lyrenas, de faire, & morgenstern, ; mitrunen et al., ) . there is circumstantial evidence, that at an individual level, an inflammatory genotype exists that can adversely effect the host. in a study of inflammatory lung disease caused by exposure to coal dust, the tnf (lt-a+ a) allele was almost twice as common in miners with the disease than in those who were healthy (zhai, jetten, schins, franssen, & borm, ) . development of farmer's lung from exposure to hay dust was % greater in individuals with the tnf allele than in those without the allele (schaaf, seitzer, pravica, aries, & zabel, ) . the tnf allele was also twice as common in smokers who developed chronic obstructive pulmonary disease than in those who remained disease-free (sakao et al., ) . in addition to disease progression, genetic factors have important effects on mortality and morbidity in infectious and inflammatory disease. during malaria, children who were homozygous for tnf had a sevenfold greater risk of death or serious pathology than children who were homozygous for the tnf allele (mcguire, hill, allsopp, greenwood, & kwiatkowski, ) . in intensive-care patients the occurrence of "g high-producing allele for il- was present in those who developed multiorgan failure with a frequency of one-fifth of that of the normal population (reid, hutchinson, campbell, & little, ) . in sepsis, patients possessing the tnf allele had a . -fold greater risk of death than those without the allele, and patients who were homozygous for the lt-~ + a allele had twice the mortality rate and higher peak plasma tnf-ot concentrations than heterozygotic individuals (mira et al., ; stuber, peterson, bokelmann, & schade, ) . the tnf allele also been found in increased frequencies in systemic lupus erythromatosus, dermatitis hepetiformis, and insulin-dependent diabetes mellitus and noninsulin-dependent diabetes mellitus (niddm) (jacob et al., , wilson, clay, & crane, wilson, gordon, & di giovine, ) . thus, it now appears that each individual possesses combinations of snps in their genes associated with inflammation corresponding to inflammatory drives of differing intensities when microbes or tissue injury are encountered. at an individual level this may express itself as differing degrees of morbidity and mortality (fig. ) . the strength of the genomic influence on the inflammatory process may affect the chances of an individual developing inflammatory disease, particularly if their antioxidant defenses are poor. in addition to disease progression, genetic factors have important effects on mortality and morbidity in infectious and inflammatory disease and following injury (paolini-giacobino, grimble, & pichard, ) . there are sex-linked differences in the influence of genotype on the inflammatory processes. in general, males are more sensitive to the genomic influences on the strength of the inflammatory process than females. in a study on lt-a genotype and mortality from sepsis, it was found that men possessing a tnfb (lt-o~+ aa) genotype had a mortality of % compared with men who were tnfb (lt-o~+ gg), who had a % mortality rate. in female patients the mortalities for the two genotypes were % and %, respectively (schroder, kahlke, book, & stuber, ) . in a study on patients undergoing surgery for gastrointestinal cancer, it was found that postoperative c-reactive protein (crp) and il- concentrations were higher in men than in women. multivariate analysis showed that males possessing the tnf (tnf-o~- a) allele had greater responses than men without it. the genomic influence was not seen in females (table ) (grimble, thorell, et al., ) . furthermore, possession of the il- - t allele was associated with a % greater length of stay in hospital in old men admitted for geriatric care (table ) (grimble, anderson, et al., ) . women were unaffected by these genetic influences. paradoxically, with improvements in hygiene and vaccination programs against infectious diseases, two major changes in public health and population characteristics have led to a general increase in inflammatory stress in populations of industrialized countries in the last half century. these are, respectively, an increase in the number of overweight and obese subjects and an increase in longevity. we will now examine the mechanisms underlying this phenomenon. it has been recognized for many years that there is a strong link between the "diseases of affluence"--obesity, insulin sensitivity, and atherosclerosis. however, it is only quite recently that the realization came that inflammation provided a link between the three ( ) tnf, tumor necrosis factor; il, interleukin; crp, c-reactive protein. a d postoperatively. bl d postoperatively. *significantly different from females with same genotype by multivariate analysis allowing for longer operation time and greater blood loss; p = . andp = . for crp and il- , respectively. means + sd, values in parentheses are the number of patients. ( ) + ( ) + ( ) + ( )* + ( ) tnf, tumor necrosis factor; il, interleukin; c, cytosine; t, thymidine; a, adenine; g, guanine. athe location of the polymorphism is indicated by the nucleotide position in the il- ~ and lt-t~ genes, tnfb (gg), tnfb (ag), tnfb (aa). *significantly different from value for same sex possessing the other genotype; p < . using mann-whitney test. means + sd, values in parentheses are the number of patients. biological phenomena (fig. ) . many studies have shown a clear link between obesity, oxidant stress, and inflammation (grimble ) . the link may lie in the ability of adipose tissue to produce pro-inflammatory cytokines, particularly tnf-t~. there is a positive relationship between adiposity and tnf production. a positive correlation ,i, plasma triglyceride concentrations fig. . interaction between leptin and tumor necrosis factor (tnf) with adipose tissue mass, lipid metabolism, and inflammation. tnf and leptin stimulate the immune system and adipose tissue, respectively. both also act on lipid metabolism and plasma triglyceride concentrations. between serum tnf-~ production and body mass index (bmi) has been noted in niddm patients and healthy women (nilsson, jovinge, niemann, reneland, & lithell, ; yaqoob, newsholme, & calder, ) . leptin has been shown to influence proinflammatory cytokine production ( fig. ). thus, plasma triglycerides, body fat mass, and inflammation may be loosely associated because of these endocrine relationships. a number of population studies have been conducted to explore the extent and nature of the relationship of inflammation to these diseases of affluence. the studies have examined populations in which there is a high incidence of insulin insensitivity, such as pima indians and individuals with a south asian background. tnf-o~ is overexpressed in adipose and muscle tissues of obese subjects compared with tissues from lean individuals (hotamisligl & spiegelman. ) . in a study of a group of nondiabetic pima indians, employing the hyperinsulinemic euglycemic clamp to assess insulin action, strong evidence of the links between inflammation, insulin insensitivity, and obesity emerged. plasma il- was found to be related positively to adiposity and negatively to insulin sensitivity. the investigators concluded that the relationship between il- and insulin action appeared to be mediated through adiposity (vozarova, weyer, & hanson, ) . a number of studies have looked at the extent of the interaction between insulin insensitivity and inflammation by studying the extreme form of diabetes, type diabetes mellitus. a study assessed endothelial cell perturbation by measurement of von willebrand factor and tissue-plasminogen activator (t-pa), in type diabetics who had had the disease for < or > yr. compared with normal subjects, children with diabetes for <lyear had the highest concentrations of von willebrand factor, indicating that endothelial perturbation represents an early event in type diabetes (romano, pomilio, & vigneri, ) . studies that have attempted either to remove the cause of inflammation, to lower plasma lipids, or improve insulin sensitivity have supported the hypothesis that inflammation, insulin sensitivity, and atherosclerosis are intimately interlinked. when a study of the potential anti-inflammatory effect of weight loss was conducted in obese women, it was found that a yr weight-reduction program resulted in lowering of plasma il- , tnf-cx, and adhesion molecule concentrations (ziccardi, nappo, & giugliano, ) . since the s large population studies have indicated that inflammation plays a key role in cardiovascular disease (cvd) (grimble, ; ross, ) . periodontal disease and other low-grade infections, such as chlamydia pneumoniae infection, have been linked closely with atherosclerosis. development of atherosclerotic plaques to which macrophages have already been recruited occurs by cytokines inducing hypertriglyceridemia and hypercholesterolemia (kol, sukhova, lichtman, & libby, ; saldeen & rand ) . chlamydial infection induces production of tnf-~. the cytokine inhibits the action of lipoprotein lipase, leading to changed lipid metabolism, elevation of serum triglycerides, and a decrease in serum high-density lipoprotein (hdl) cholesterol, thereby exerting an atherogenic influence (arrnitage, ) . in the bruneck study, raised plasma bacterial lipopolysaccharide (lps) was associated with an increased rate of thickening of the coronary artery intima. smoking, a further inflammatory stress, exacerbated this effect (williet & kiechl, ) . lps binds in human serum to both low-density lipoprotein (ldl) and hdl cholesterol and makes ldl cholesterol immunogenic or toxic to endothelial cells. thus, the link between specific infections and atherosclerosis may be a nonspecific effect of chronic inflammation on the atherosclerotic process. it is well recognized that alterations in plasma protein concentrations invariably occur among the many metabolic changes that occur during inflammation. proteins that increase during inflammation (positive acute-phase proteins, e.g., crp and fibrinogen) and those that decrease (negative acute-phase proteins, e.g., serum albumin and retinolbinding protein) are used to diagnose inflammation in population nutritional surveys. the early indications that inflammation was involved in atherosclerosis came from the findings that there were links between concentrations of positive and negative acute-phase proteins in blood and cvd (grimble, ). in the bruneck study of a group of to -yr-old italians, it was found that impaired glucose tolerance and, to a greater extent, type diabetes were strong independent predictors of advanced atherosclerosis (measured by high-resolution ultrasound of the carotid artery) (bonora, kiechl, & oberhollenzer, ) . a cross-sectional study of obesity, plasma crp, fibrinogen, and carotid artery intima media thickness (an index of atherosclerosis) in more than multiethnic subjects showed a positive relationship between plasma crp and body fat. intima media thickness was related to crp and fibrinogen in men. the relationship was attenuated by adjustment for bmi (festa, d'agostino, & williams, ) . a cross-sectional study of more than men and women examined the link between elevated plasma crp concentrations and prevalent cvd, ankle/brachial blood pressure, and carotid artery intima media thickness. after adjustment for age and family type, there was a weak association between crp and intima media thickness in both sexes and with prevalent heart disease in women (folsom, pankow, & tracy, ) . a study on a turkish population of individuals with low cholesterol concentration but a high prevalence of other risk factors for coronary heart disease investigated whether crp acted as a predictor of coronary heart disease. among the risk factors, only crp and systolic blood pressure were independent risk factors for cvd (onat, sansoy, & yildirim, ) . in a study in which crp concentrations and conventional risk factors for cvd in healthy indian asians were compared with values in a similar number of healthy european white subjects, crp and cvd risk factors were higher in the former group. however, differences were eliminated when adjustment was made for central obesity and insulin resistance score (chambers, eda, & bassett, ) . a study on a brazilian population found that markers of inflammation correlated with components of the metabolic syndrome---cardiovascular and diabetes risk factors, insulin resistance, and central obesity (duncan & schmidt, ) . in a study of healthy elderly subjects in the netherlands, acute-phase proteins, soluble adhesion molecules, il- , and insulin were measured and associated with cholesterol and obesity. the association between insulin, obesity, and cholesterol was as strong as between insulin, acute-phase proteins, and adhesion molecules (hak, pols, & stehouwer, ) . insulin insensitivity occurs as part of the normal inflammatory response to pathogens. during inflammation the secretion of catabolic hormones, which enhances muscle protein breakdown and glutamine release, will oppose insulin action. paradoxically, however, although insulin insensitivity may initially exert a beneficial effect during the response to infection and injury, it has an adverse influence in chronic disease processes. glucose and glutamine act as major fuels for immune cells during the normal response to infection. large increases in glucose and glutamine utilization by immune cells occur during the response to infection and injury (spitzer, bagby, meszaros, & lang, , . studies in rats given lps and observations in patients with sepsis show that the flow of amino acids into the circulation increases and gluconeogenesis is enhanced under the influence of pro-inflammatory cytokines. an insulin-insensitive state will reduce glucose uptake by tissues in which the process is insulin dependent (muscle), thereby increasing availability to tissues in which the process is not insulin dependent (immune tissue). a study in the united states investigated whether elevated plasma il- and crp was associated with the development of type diabetes mellitus in more than , healthy women. in the -yr follow-up period, women developed type diabetes. for these women, baseline il- and crp were higher than in controls. the relative risk of future type diabetes in women between the highest and lowest quartiles of these inflammatory markers was . for il- and . for crp (pradhan, manson, & rifai, ) these data suggest a possible role for inflammation in diabetogenesis. furthermore, data collected from the third national health and nutrition examination survey (nhanes iii) in the united states provide further evidence for a possible role of inflammation in insulin resistance and glucose intolerance. more than men and women were studied for associations between plasma crp, fasting insulin, glucose, and glycosylated hemoglobin (hba c). elevated crp was associated with higher insulin and hba c in both sexes and with raised glucose in women (wu, dorn, & donahue, ) . a study on the link between crp, central adiposity, and fasting glucose and insulin in more than healthy italian women showed an independent relationship of adiposity to insulin resistance and crp concentrations (pannacciulli, cantatore, & minenna, ) . in a review, nishimura and murayama ( ) discussed the possibility that treatment of periodontal infection may improve insulin sensitivity. their conclusions about the efficacious effect of an anti-infective approach are supported by a study in which type diabetic patients with periodontal disease were given antimicrobial treatment with minocycline. blood tnf-t~ concentrations and glycosylated hemoglobin decreased (iwamoto, nishimura, & nakagawa, ) . conversely, improvement in insulin sensitivity exerted an anti-inflammatory effect. a group of hyperlipidemic patients and normolipidemic controls who were insulin resistant and hypertriglyceridemic with low hdl cholesterol concentrations and raised tnf-o~ production and plasma il- and fibrinogen concentrations were studied. all subjects were treated with the lipid-lowering drug bezafibrate. the drug normalized all parameters.the drug thus exerts an anti-inflammatory effect associated with its ability to normalize lipid metabolism and insulin sensitivity (jonkers, mohrschladt, & westendorp, ) . in an in vitro study on a lung epithelial cell line, the oral hypoglycemic agent thiazolidinedione exerted an anti-inflammatory influence by suppressing production of monocyte chemoattractant protein (mcp- ) (momoi, murao, & imachi, ). it can be concluded from the studies reported in the above sections that inflammation is closely linked to obesity, cvd, insulin insensitivity, and diabetes mellitus. although this interaction is a relatively novel concept, it has been known for many years that these diseases are interlinked and that insulin insensitivity is a common factor in their pathology. a number of recent studies have examined the mechanisms underlying the link between inflammation and the conditions and diseases in which insulin insensitivity plays a part (fig. ) . a difficult question to address is whether chronic inflammation leads to a condition of insulin insensitivity and associated diseases, or whether insulin insensitivity, which is associated with obesity, diabetes, and atherosclerosis, brings about a condition of chronic inflammatory stress. many studies suggest that the former is more likely to be the case. reviews have highlighted the risk of inflammation and infection of atherosclerotic plaques associated with poor diabetic control and the importance of elevated nonesterified fatty acid concentrations, glucocorticoids, and low-grade inflammation as causative agents in atherosclerosis and insulin insensitivity (bell, ; corry & tuck, ) . a study on type diabetes mellitus patients determined the extent to which pbmcs contributed to oxidative stress and inflammation. a linear correlation between hbalc and superoxide release was found. the authors concluded that type diabetes mellitus is accompanied by priming of pbmcs and increased self-necrosis. the necrosis may start a chain of events that results ultimately in oxidant stress and endothelial dysfunction (shurtz-swirski, sela, & herskovitis, ) . in a study on pima indians, who are characterized by high incidence of obesity and insulin resistance but not atherosclerotic disease, crp, icam- , and secretory phospholipase a are correlated with body fat but not e-selectin and von willebrand factor. in addition to showing that markers of inflammation increase with adiposity, the study showed that markers of endothelial dysfunction increase in proportion to insulin resistance and inflammation (weyer, yudkin, & stehouwer, ) . a further study on pima indians examined whether a raised white blood cell count predicted a worsening of insulin action, insulin secretory function, and the development of type diabetes. a high white blood cell count predicted the development of diabetes with a relative risk of . when adjusted for age and sex (vozarova, weyer, & lindsay, ) . tnf-o~ has been shown to play a key role in mediating insulin resistance as a result of obesity in patients and in numerous rodent models of obesity---diabetes syndromes (hotamiligil & spiegelman, ) . multiple mechanisms have been suggested to account for these metabolic effects of tnf-ot. these include the downregulation of genes that are required for normal insulin action, direct effects on insulin signaling, induction of elevated plasma free fatty acids via stimulation of lipolysis, and negative regulation of ppar- , an important insulin-sensitizing nuclear receptor (moiler, ) . the induction of insulin resistance is mediated through its ability to produce serine phosphorylation of insulin receptor substrate (irs)- , decreasing the tyrosine kinase activity of the insulin receptor (hotamisligil, budavari, murray, & spiegelman, ) . neutralization of tnf-t~ in obese fa/fa rats by intravenous administration of a soluble tnf receptor immunoglobulin g chimeric protein substantially improved insulin sensitivity and restored the tyrosine kinase activity in fat and muscle (hotamisligil et al., ) . in an in vitro study using t -l adipocytes, tnf-t~ was shown to induce sustained suppressor of cytokine signaling protein (socs- ) production. socs- has been shown to decrease insulin-induced irs tyrosine phosphorylation and its association with the p regulatory subunit of phosphatidylinositol- kinase (emanuelli, peraldi, & filloux, ) . these observations therefore suggest that socs- may be a key mediator in the development of insulin sensitivity during inflammation. obesity is associated with insulin resistance, particularly when body fat has a central distribution. while elevated plasma leptin concentrations are associated with obesity, some studies have suggested that insulin sensitivity is an additional determinant of circulating leptin concentrations. leptin is produced by adipose tissue in proportion to adipose tissue mass and is a pleiotropic molecule. in addition to playing a role in appetite and adipose tissue regulation, leptin influences immune functions. leptin concentrations increase acutely during inflammation and regulate t-cell responses, polarizing t-helper (th) cells toward a th phenotype. thus, increased leptin production during obesity, may exert a pro-inflammatory influence (faggioni, feingold, & grunfeld, ) . further complexity is added to the concept of a link between insulin sensitivity, inflammation, and obesity by the results of a study of individuals selected from the health professionals follow-up study in the united states (chu, spiegelman, & hotamisligil, ) . in the study plasma insulin, leptin, and soluble tnf receptor (stnf-r, an index of tnf-o~ production) concentrations were measured and correlated with bmi and the cvd risk factors insulin, triglyceride, t-pa antigen levels, and apolipoprotein (apo)-a . in a multivariate regression model controlling for age, smoking, alcohol intake, physical activity, and diet, bmi was inversely associated with hdl cholesterol and apo-a and positively associated with trigyceride, apo-b, and t-pa antigen levels. the associations between bmi and these cvd risk factors were only slightly changed after adjusting for leptin and/or stnf-r, but were substantially attenuated after controlling for insulin levels. these data suggest that the association between obesity and biological predictors of cvd may be mediated through changes in plasma insulin, rather than leptin or stnf-r levels, and that insulin may be exerting an anti-inflammatory effect. (cnop, landchild, & vidal, ) . genetic factors may play a part in the interaction between inflammation, insulin insensitivity, and obesity. nf-~:b is an important mediator of inflammation by increasing transcription of a range of genes central to the inflammatory process (cytokines, adhesion molecules, acute-phase proteins). snps in the nf-r,~b gene were investigated in a group of type diabetic patients and compared with gene frequencies in normal controls. it was found that there was a higher frequency of allele bp (a ) (high bioactivity) and a lower frequency of allele bp (a ) (less bioactive) in diabetics than in controls. genotype may thus contribute to inflammatory stress in diabetes mellitus (hegazy, o'reilly, & yang, ) . nf-~:b may also provide an important focus for ppar- action, because it has been shown in a number of studies that ppar-~/in combination with retinoid x receptor is able to inhibit nf-~:b activation (wada, nakajima, & blumberg, ; fruchart, staels, & durlez, ; debril, renaud, fajas, & auwerx, ) . it is interesting to note that the n- polyunsaturated fatty acids (pufas), found in abundance in fish oil, are ppar-y agonists, raising the possibility that the oil may exert its anti-inflammatory effects partly via this mechanism. in an investigation of cytokine production in healthy males, the author found that in the study population as a whole there were no statistically significant relationships between bmi, plasma fasting triglycerides, and the ability of pbmcs to produce tnf-a. however, individuals with the lt-c~+ aa genotype (associated with raised tnf production) showed significant relationships between tnf production and bmi and fasting triglycerldes (fig. ) . thus, despite the study population being comprised of healthy subjects, within that population were individuals with a genotype that resulted in an "aged" phenotype as far as plasma lipids, bmi, and inflammation were concerned (paolini-giacobino et al., ) . it is well known that the incidence of diseases of affluence and recognizable inflammatory diseases, such as rheumatoid arthritis, increase with aging. are these phenomena an unfortunate side effect of maturity, or is there a common mechanism that determines the appearance of these diseases patterns in the elderly.'? paradoxically, aging is associated with a decline in t-lymphocyte function and an increase in inflammatory stress. a number of elements of the chronic inflammatory response are apparent in otherwise healthy elderly subjects. the elements of the response include muscle protein loss, a rise in plasma acute-phase protein concentrations, and a decrease in plasma zinc. an age-related increase in il- concentration has been found in serum, plasma, and supernatants of mononuclear blood cell cultures from apparently healthy elderly people and centenarians (fagiolo et al., ; baggio, donnazzan, monti, & marl, ; ershler & kerller, ) . because il- is a pleiotropic cytokine capable of regulating proliferation, differentiation, and activity of a variety of cell types (ershler & kerller, ) and plays a pivotal role in neuroendocrlne and immune system homeostasis, it is not surprising that the rise in production of pro-inflammatory cytokines might have long-term pathological effects (bethin, vogt, & muglia, fasting triglycerides fig. . relationships between tnf production, bmi and fasting triglycerides, and the ability of pbmcs to produce tnf-o~ in the study populations: (a) all subjects irrespective of genotype, (b) individuals with the lt-ct+ aa genotype, associated with raised tnf production. ns, nonsignificant relationship; bmi, body mass index; pbmcs, peripheral blood mononuclear cells. the number of subjects is shown in parentheses; the correlations were examined by spearman' s rank correlation. levels of this cytokine have also been found as early as at - yr of age (mysliwska, bryl, foerster, & mysliwski, ) , particularly in men (young, skibinski, mason, & james, ) . population studies have shown that the magnitude of increase in the concentration of il- is a reliable marker for functional disability and a predictor of mortality in the elderly (ferrucci, harris, guralnik, & tracy, ; harris, ferrucci, tracy, & corti, ) . antioxidant status may decline with age (nuttall, dunne, kendall, & martin, ) and may thus be linked to increased tnf-~ production (kudoh, katagai, takazawa, & matsuki, ; rink, cakman, & kirchner, ). an enhanced capacity for the release of pro-inflammatory cytokines by white blood cells may contribute to the pathogenesis of ischemic stroke. grau et al. ( ) investigated the lps-induced release of il- [ , il- , il- , and tnf- ~ in whole blood from patients with a history of ischemic stroke under the age of and age-and sex-matched healthy control subjects. release ofll- was significantly higher in young stroke patients than in control subjects (grau et al., ) . the question of whether aging is associated with chronic elevation of cytokine production or whether an increased capacity for cytokine production following the normal inflammatory challenges of life develops during aging is an interesting one to consider. insight into this issue can be gained from the response to surgery where an inflammatory stimulus is applied at a defined moment in time, making it easy to follow the subsequent response. ono, aosasa, tsujimoto, ueno, and mochizuki ( ) investigated the agerelated changes in the inflammatory response in patients with gastric cancer undergoing distal gastrectomy. patients were divided into two groups: > yr of age (elderly group) and -< years of age (young group). serum il- concentrations, tnf-ct production and cd b/cd expression by monocytes, and the postoperative clinical course were compared between the two groups to assess the inflammatory response to surgery. tnf-~ production by lps-stimulated monocytes and cd l b/cd expression on monocytes were significantly higher in the elderly than in the young group. moreover, serum il- concentrations on the first postoperative day in the elderly group were significantly higher than those in the young group. paradoxically, both loss of body weight and lean tissue and obesity are found in elderly populations. is there, therefore, a link between this phenomenon and increased levels of inflammation? the loss of muscle mass and strength that occurs with aging is described clinically as sarcopenia (rosenberg, ; roubenoff, ) . it is an important contributor to the development of frailty and functional impairment during aging. it is well established that aging is associated with a significant decline in muscle strength that becomes functionally important by the seventh decade of life. the relationship between chronic inflammation owing to disease during aging and the prevalence of low body mass are well illustrated in rheumatoid arthritis. in a study on patients with rheumatoid arthritis, the loss of body mass was greater for lean tissue than fat, with over % of the rheumatoid group falling into the lowest th percentile of a reference population for skeletal muscle mass assessed from the upper arm muscle area. in female patients there was a significant correlation between reduced fat-free mass and two indicators of inflammatory stress---erythrocyte sedimentation rate and plasma crp concentration (munro & capell, ) . clinical and animal studies show a relationship between raised plasma cytokine concentrations and low muscle mass. visser et al. ( ) investigated whether markers of inflammation are associated with muscle mass and strength over a time course of several years in over healthy well-functioning black and white elderly persons ( - yr). mid-thigh muscle cross-sectional area, appendicular muscle mass, and muscle strength were assessed. plasma concentrations of il- and tnf-~ were also measured. higher cytokine concentrations were associated with lower muscle mass and lower muscle strength. the most consistent relationship across the gender and race groups was observed for il- and grip strength. when an overall indicator of elevated cytokine production was created by combining the concentrations of il- and tnf-o~, with the exception of white men, elderly persons having high concentrations of il- (> . pg/ml) as well as high levels of tnf-~ (> . pg/ml) had a smaller muscle area, less appendicular muscle mass, and lower muscle strength compared to those with low levels of both cytokines. thus, raised plasma concentrations of il- and tnf-~ are associated with lower muscle mass and lower muscle strength in well-functioning older men and women as well as those suffering frank inflammatory disease. nutrient intake is clearly another important determinant of lean body weight and fat mass and may play a part in the decline in lean tissue with age as well as an increase in inflammatory stress. a recent survey of , subjects in communities in nhanes iii in the united states also included a survey of about elderly people ranging in age from to yr, to yr, and + yr (marwick, ) . the report indicated that the median intake of total energy was in general lower than the recommended kcal for men and kcal for women (marwick, ) . chronic inflammation is either a causative agent or a closely associated process in the pathology of obesity, insulin insensitivity, and atherosclerosis.the incidence of these conditions increases with aging. a fundamental question is which precedes the other-the general increase in inflammation or the development of diseases with overt and covert inflammatory bases? this "chicken-and-egg" question is difficult to answer. however, examination of data from studies conducted in elderly populations may throw some light on the answer to this conumdrum. there are at least two potential mechanisms for the higher level of chronic inflammation observed in elderly than in younger subjects. the first of these is that the elderly are experiencing a higher level of asymptomatic urinary infection. this possibility was studied in consecutive patients ( - yr) admitted to the hospital for functional disability. patients were examined for the presence or absence of bacteria in the urine. twenty subjects had a positive urine culture, and sex-and age-matched subjects had a negative urine culture. inclusion criteria were temperature < . °c, no clinical signs of infection, and no current antibiotic treatment. patients with asymptomatic bacteriuria had significantly increased levels of tnf receptors and a higher number of neutrophils in the blood compared to the group without bacteriuria. thus, the study provides some support for the hypothesis that asymptomatic urinary infections are associated with low-grade inflammatory activity in frail, elderly subjects (prio, bruunsgaard, roge, & pedersen, ) . a second potential mechanism resides in endocrine changes during aging. in aging, dysregulation of secretion of hormones that come under the regulation of the hypothalamic-pituitary-adrenal axis may occur. this may have an effect on the regulation of cortisol secretion, as mentioned earlier. cortisol is important as an anti-inflammatory agent. the effect of aging on glucocorticoid sensitivity of pro-inflammatory cytokine production was examined in elderly men, testosterone-treated elderly men, and young controls. stress-induced increases in cortisol did not differ significantly between experimental groups, but glucocorticoid sensitivity increased significantly in young controls and testosterone-treated elderly men, whereas a decrease was found in untreated elderly men. as the increase in glucocorticoid sensitivity after stress serves to protect the individual from detrimental increases of pro-inflammatory cytokines, the disturbed mechanism in elderly men may result in an increase in inflammatory stress (rohleder, kudielka, hellhammer, wolf, & kirschbaum, ) . there is now a large body of evidence suggesting that the decline in ovarian function with menopause is associated with spontaneous increases in pro-inflammatory cytokine production. as mentioned earlier, studies in men and postmenopausal women indicate a remarkable individual constancy in the ability of pbmcs to produce tnf-cz ex vivo, and genetic determinants underlie this constancy. however in premenopausal women production is highly variable at an individual level, indicating how ovarian hormones are able to override the influence of genotype (jacob et al., ) . the exact mechanisms by which estrogen interferes with cytokine activity are still incompletely known but may include interactions of the estrogen receptor with other transcription factors, modulation of nitric oxide activity, antioxidative effects, plasma membrane actions, and changes in immune cell function. experimental and clinical studies also strongly support a link between the increased state of pro-inflammatory cytokine activity and postmenopausal bone loss (pfeilschifter et al., ) . recent evidence indicates the presence of snps, associated with the strength of the inflammatory response, affects longevity. human longevity may be directly correlated with optimal functioning of the immune system. therefore, it is likely that one of the genetic determinants of longevity resides in polymorphisms for genes influencing the activity of the immune system. it has been estimated that up to variations in the genome contribute to life span (martin, ) . those contributing to loss of muscle and bone mass during aging are related to the inflammatory process and include pro-and anti-inflammatory cytokines and their receptors. studies in mice have shown that the genes controlling the major histocompatibility complex (mhc), known to control a variety of immune functions, are associated with differences in the life span of different strains of mice, but a major difference between observations in mice and humans is that the latter have a lifetime experience of exposure to pathogens, whereas for laboratory animals this exposure is kept to a minimum. thus, although hla studies in mice of different genotypes may be interpreted to support studies of mhc effects on longevity in humans, in mice the association may be by way of altered susceptibility to lymphomas, whereas in human beings the effect on longevity is likely to be via an altered response to pathogens and susceptibility to infectious disease. a number of cross-sectional studies have examined the role of hla genes on human longevity by comparing hla antigen frequencies between groups of young and elderly persons. conflicting findings have been obtained. when this topic was reviewed (caruso et al., ) , it was concluded that in humans there may be an association between longevity and some hla-dr alleles or the hla-bs,dr haplotype. these genotypes are involved in the antigen nonspecific control of immune response, in other words, the component of immune function associated with inflammation and cytokine biology. recent evidence indicates that presence of snps in certain pro-and anti-inflammatory cytokine genes influences life span. when individuals between and yr of age were studied, it was noted that not only was plasma il- concentration positively related to age but individuals with a snp in the promoter region of the il- gene, which predisposes to high levels of production of the cytokine (- gg), decreased in frequency with age. the effect was seen in men but not in women (bonafe, olivieri, & cavallone, ) . although men with snps made up % of the -to -yr-old age group, the percentage fell to % in subjects < years of age. conversely, one of three snps in the il- gene (- gg), which is closely linked to higher production of the anti-inflammatory cytokine il- (hutchinson, pravica, hajeer, & sinnott, ; turner et al., ) , was found in higher proportions in male centenarians than in younger controls ( vs %). in females this genotype exerted no effect upon longevity (lio et al., ) . thus, it would appear that genetic characteristics that might influence the balance between proand anti-inflammatory cytokines influence mortality in men but not in women (franceschi et al., ) . a study on snps that influence interferon (ifn)-~t production further reinforces the concept that possession of a genotype that predisposes to a raised pro-inflammatory status is not compatible with a long life span (lio et al., ) . in women, possession of the a allele, which is associated with low production of ifn-'~, significantly increased the possibility of reaching old age. it might be concluded that possession of high-producing alleles of the il- is universally protective against morbidity as well as mortality. possession of a genotype that results in low levels of il- production (- aa) increases the risk of developing inflammatory diseases (hajeer, lazanes, & turner, ; huizinga, keijsers, & yanni, ; tagore, gonsalkorale, & pravica, ) . however, as already mentioned, in a large survey of hospitai admission in the netherlands, patients with raised il- :il- ratios had higher mortality rates (van dissel et al., ) . not all studies implicate cytokine gene snp in longevity. cytokine gene polymorphisms at il- o~, il- ~, il- ra, il- , il- , and tnf-o~ were measured in finnish nonagenarians ( men and women) and in healthy blood donors ( - yr) used as controls. no statistically significant differences were found in the distribution of genotype, allelic frequencies, and a + carrier status between nonagenarians and younger controls (wang, hurme, jylha, & hervonen, ) . in a review on the different impact of genetic factors on the probability of reaching old age, franceschi et al. ( ) concluded from studies conducted in italy that emerging evidence (regarding mtdna haplogroups, thyrosine hydroxylase, and il- genes) suggests that female longevity is less dependent on genetics than male longevity and that female centenarians are more likely to have had a healthier lifestyle and more favorable environmental conditions than males. however, a recent study conducted by our group suggests that although a pro-inflammatory genotype may be disadvantageous to elderly males, it may confer a survival benefit in females. subsequent survival was studied in elderly geriatric patients ( + yr) after a period of hospitalization for a range of conditions necessitating geriatric care. although women possessing a pro-inflammatory genotype (tnf-o~- a allele or il- - gg) had improved yr survival rates, men possessing pro-inflammatory genotypes (il-l~- t allele or lt-ct + aa) had shortened survival rates (grimble, thorell, et al., ) (table ) . as outlined in the preceding sections, the inflammatory response, although essential for survival in the presence of pathogens, can exert deleterious effects on the host.the clear need to find ways of modulating cytokine production and other aspects of inflammation has fostered the research area of immunonutrition. in a clinical context the purpose of immunonutrition is to find nutritional means of altering the patient' s inflammatory response to infection and injury, from the detrimental to the beneficial side of the pivot on which an individual undergoing a response is positioned. while inflammation may be exerting deleterious effects most obviously in patients, people on the borderline of health and disease living in the general population table nutrients commonly used in immunonutrient supplements and their potential mode of action • n- polyunsaturated fatty acids: act as anti-inflammatory agents and reverse immunosuppression • sulfur amino acids and their precursors: enhance antioxidant status via gsh synthesis • glutamine: nutrient for immune cells, improves gut barrier function, precursor for gsh • arginine: stimulates nitric oxide and growth hormone production, improves helper t-cell numbers • nucleotides: rna and dna precursors, improve t-cell function may also require nutritional modulation of ongoing inflammatory processes. during the last years the pace of evolution ofimmunomodulatory feeds and intravenous solutions has accelerated. these products contain combinations of a number of components to which various functional attributes are ascribed them (table ) (grimble, a) . many studies have indicated that n- polyunsaturated fatty acids (pufas), glutamine, arginine, sulfur amino acids, and nucleotides are all potentially capable of shifting the balance from a disadvantageous to an advantageous response to infection and injury. the examples used here are illustrative rather than comprehensive. a number of studies indicate that improvement of antioxidant status is associated with an increase in cellular aspects of immune function. meta-analyses have been conducted on the efficacy of immunonutrients that influence antioxidant status. in clinical trials, indices such as infection rates, mortality rates, and length of stay are often measured in the absence of functional and biochemical aspects of the response, such as t-cell function, cytokine production, and antioxidant status, and vice versa, giving a rather incomplete picture of the mechanisms of any observed effects of immunonutrition. however, beale, bryg, & bihari ( ) , in a meta-analysis of studies containing more than patients receiving enteral immunonutrition, observed that although there was no effect upon mortality, there were significant reductions in infection rates, time spent on a ventilator, and length of hospital stay. while this finding indicates that immunonutrition may be useful in modulating the inflammatory process in patients experiencing severe inflammation, the consistency of the effects observed was disappointing. there are at least three major reasons why it is difficult to demonstrate a consistent effect. first, patients used as the subjects of clinical trials of immunonutrients will constitute a diverse population--different ages, at different stages of a disease process, and undergoing complex clinical treatment in addition to nutrient therapy. second, patients will have differing genetic backgrounds that will influence the intensity of the inflammatory and immune responses they are undergoing. this issue is dealt with below. third, nutrients may exert paradoxical effects, as illustrated by the findings of the first observations of the effects of fish oil on cytokine production in healthy subjects. the findings of endres et al. ( ) that a daily supplement of g/d of fish oil given to nine young men for wk was able to reduce ex vivo production ofll- and tnf-~ by lps-stimulated pbmcs aroused great interest in fish oil as an anti-inflammatory nutrient. this perception was supported by a large amount of animal data. however, endres' data showed a wide variability in the effect of the fish oil supplements. the standard deviations of the mean for il- and tnf-cx production were and %, respectively. this indicates that cytokine production could have risen or fallen as a consequence of taking the supplement. the effects of supplementing healthy young men with g/d offish oil for wk, on tnf-ct production by pbmcs stimulated with endotoxin have been studied in the author' s laboratory. it was found that % of subjects experienced a fall in production and % a rise. although the ability of fish oil to increase tnf-t~ production is at first sight paradoxical, earlier work of dinarello, bishai, rosenwasser, and coceani ( ) and kunkel, remick, spengler, and chensue ( ) indicated that fish oil could potentially change cytokine production in either direction. what mechanisms could result in this divergent effect? inflammation will result in activation of phospholipase a , which releases arachidonic acid (aa) (c : n- ) from the cell membrane for prostaglandin e (pge ) or leukotriene b (lt b ) synthesis. the in vitro studies (kunkel et al., ) showed that pge suppressed tnf-t~ production, whereas ltb had the opposite effect (dinarello et al., ) .. fish oil is rich in eicosapentaenoic acid (c : n- ), which will replace aa in the cell membrane and results in the production of pge and lt b . pge and lt b are considerably less potent than the corresponding compounds produced from aa, and thus dietary fish oil may lessen the inhibitory influence of pge or the stimulatory influence of ltb on tnf-o~ production, resulting in a potential increase or decrease, respectively, in production of the cytokine. fish oil could thus result in an inflammatory cytokine response, which could fall on either side of the pivot. the response to bacterial invasion of the body, or injury, contains a paradox. although the inflammatory response and the t-cell response both play a part in defeating the invader, the inflammatory response may in some clinical circumstances exert an inhibitory influence on t-cell function. in severely infected or traumatized patients, an enhanced inflammatory state occurs, which is associated with immunosuppression. in vitro studies support this inverse relationship. pbmcs taken from healthy young subjects and incubated with gsh show decreased pge and ltb production (reduced inflammation) and an increase in mitogenic index and il- production (enhanced immune function) (wu, meydani, sastre, hayek, & meydani, ) . thus, enhancement of antioxidant defenses reduces the likelihood of the inflammatory response suppressing t-cell function (grimble, (grimble, , b . although all antioxidants are important owing to the linked nature of antioxidant defense (fig. ) , gsh plays a pivotal role as it acts directly as an antioxidant and maintains other components of defense in a reduced state through enzymic conversion between the oxidized and reduced states. various compounds can be used to increase gsh synthesis (fig. ). n-acetyl cysteine (nac) and the gsh prodrug oxothiazalidine- -carboxylate (procysteine) have been used in a number of clinical studies. tissue gsh content is also influenced by protein and sulfur amino acid intake. unfortunately, surgery, a wide range of diseases that have an inflammatory component, and aging and protein energy malnutrition decrease gsh concentration in blood and other tissues (boya et al., ; loguercio et al., ; luo, hammarqvist, anderson, & wernerman, ; micke, beeh, schlaak, & buhl, ; nuttal et al., ; reid et al., ) (table ). within h of elective abdominal surgery, muscle gsh content falls by > %. values return to normal h postoperatively. a smaller perturbation in blood gsh occurs over a shorter time course. modification of the gsh content of liver, lung, spleen, and thymus in young rats by feeding diets containing a range of casein (a protein with a low sulfur amino acid content) concentrations changed immune cell numbers in lung (hunter & grimble, found that in unstressed animals the number of lung neutrophils decreased as dietary protein intake and tissue gsh content fell. however, in animals given an inflammatory challenge (endotoxin), liver and lung gsh concentrations increased directly in relation to dietary protein intake. lung neutrophils, however, became related inversely with tissue gsh content. addition of methionine to the protein-deficient diets normalized tissue gsh content and restored lung neutrophil numbers to those seen in unstressed animals fed a diet with adequate protein content (fig. ) . why does tissue gsh content have differing effects on immune cell populations depending on whether or not an inflammatory response is occurring? a partial explanation may come from an in vitro study using hela cells and cells from human embryonic kidney. in the study, both tnf-o~ and hydrogen peroxide resulted in activation of nf-~cb and ap (wesselborg, bauer, vogt, schmitz, & schulze-osthoff, ) . addition of the antioxidant sorbitol to the medium suppressed nf-~fl activation as expected, but unexpectedly activated ap . thus, the antioxidant environment of the cell might exert opposite effects upon transcription factors closely associated with inflammation (e.g., nf-~fl ) and cellular proliferation (e.g., ap ). evidence for this biphasic effect was seen when gsh was incubated with immune cells from young adults (wu et al., ) . a rise in cellular gsh content was accompanied by an increase in il- production and lymphocyte proliferation (enhancement of t-cell function) and a decrease in production of the inflammatory mediators pge and ltb (anti-inflammatory influence). without doubt, a decline in antioxidant status in the presence of oxidant stress will increase inflammatory stress. the interaction between oxidant stress and an impaired ability to synthesize gsh, a situation that stimulates inflammation, is clearly seen in cirrhosis, a disease that results in high levels of oxidative stress and an impaired ability to synthesize gsh . in pena an inverse relationship between gsh concentration and the ability of monocytes to produce il- , il- , and tnf-t~ was observed. treatment of cirrhotic patients with the procysteine increased monocyte gsh content and reduced il- , il- , and tnf-t~ production. septic patients given an infusion of nac ( mg/kg bolus followed by infusion of mg/kg over -h) showed a decrease in plasma il- and soluble tnf receptor p , had a reduced requirement for ventilator support, and spent fewer days in intensive care than patients not receiving nac (spapen et al., ). de rosa et al. ( showed that nac was able to restore tissue gsh concentrations in individuals with hiv infection. in a study on hiv-positive patients, brietkreutz et al. ( ) showed that a dose of mg/d of nac for mo resulted in a decrease in plasma il- (decreased inflammation), an increase in natural killer cell activity, and increased responsiveness of t lymphocytes to tetanus toxin stimulation (improved lymphocyte function). antioxidants might act to prevent nf-r,b activation by quenching oxidants. however, nf-rd and ap may not respond to changes in cell redox state in the same way. when rats were subjected to depletion of effective tissue gsh pools by administration of diethyl maleate, there was a significant reduction in lymphocyte proliferation in spleen and mesenteric lymph nodes (robinson et al., ) . an increase in inflammatory stress would be expected in this study. thus, it can be hypothesized that antioxidants exert an immunoenhancing effect by activating transcription factors that are strongly associated with cell proliferation (e.g., ap ) and an anti-inflammatory effect by preventing activation of nf-r,b by oxidants produced during the inflammatory response (dr&ouml; ge et al., ) .thus, inclusion of antioxidants or substances that increase gsh synthesis in immunonutrient mixes would seem to be beneficial. improvement of antioxidant defenses is also possible by feeding other components of antioxidant defenses. supplementation of the diet of healthy subjects and smokers with iu/d t~-tocopherol for wk suppressed the ability of pbmcs to produce tnf-t~ (mol, de rijke, demacher, & stalenhoef, ) . the same dose given to healthy elderly subjects for d increased delayed-type hypersensitivity and raised antibody titers to hepatitis b (meydani et al., ). an enteral feed enriched with vitamin e, vitamin c, and taurine given to intensive-care patients decreased total lymphocyte and neutrophil content in bronchioalveolar lavage fluid (decreased inflammation) and resulted in a reduction in organ failure rate, a reduced requirement for artificial ventilation, and a reduction of d in intensive-care stay (gadek et al., ) . a number of roles have been ascribed to glutamine as an immunonutrient: (a) as an essential nutrient for immune cells, (b) as an important modulator of gut barrier function, and (c) as a substrate for gsh synthesis. a number of reviews have been written about the first two of these roles (newsholme, crabtree, salleh, & ardawi, ; elia, ) ; we will consider the last one here. could glutamine be exerting an anti-inflammatory influence via an effect on gsh that enhances immune function? in a study in rats, glutamine supplementation resulted in an increased production of gsh by the gut (cao, feng, hoos, & klimberg, ) , and total parenteral nutrition (tpn) with glutamine raised plasma gsh concentrations in these animals (denno, rounds, faris, halejko, & wilmore, ) . in randomized controlled trials the administration of glutamine, either as a dipeptide during tpn to surgical patients or as a glutamine-enriched enteral feed to trauma patients, resulted, respectively, in improved nitrogen retention (less tissue protein depletion) and a . -d reduction in hospital stay, a concomitant suppression of the rise in plasma-soluble tnf receptors (reduced inflammation), and a lower incidence ofbacteremia, pneumonia, and sepsis (improved immune function) (houdijk et al., ; morlion et al., ) in the previous section the influence of antioxidants on severe inflammation was considered. do the general findings from this type of study also apply to modulation of low-grade chronic inflammation, such as has been observed in the elderly and obese? because aging is so closely associated with increased oxidative stress, which might both result from and contribute to a stimulation in the level of inflammation in the elderly, antioxidant therapy could produce beneficial effects. the effects would be seen in a decrease in oxidant damage, downregulation of inflammation, and, because of the inverse link between inflammation and immune function, an improvement in t-lymphocyte function. meydani, meydani, & verdon ( ) reported that supplementation of aged mice ( mo old) with dietary vitamin e ( ppm) improved several indices of the immune system to levels comparable to those seen in young animals. supplementation of aged mice with this vitamin also increased clearance of influenza virus from the lung to that observed in animals supplemented with other antioxidants such as melathonine, gsh, or strawberry extract, which contains a high level of flavonoids with antioxidant activity . in a double-blind, placebo-controlled study, meydani and colleagues (meydani, barklund, & lui, ; meydani et al., ) also reported that supplementation of elderly subjects with vitamin e for a short ( mo) or long ( . too) period of time also improved several in vitro and in vivo indices of immune response. the optimal immune response was observed with iu of vitamin e per day in the long-term study. it is worth noting that this level of vitamin e has also been reported to be the optimal level for reducing plasma f -isoprostane, a reliable index of lipid peroxidation (dillon, vita, & leeuwenburgh, ) . improving the immune response in the elderly may result in a lower incidence of infections, which are prevalent among the elderly, and thus may contribute to a longer and healthier life. many observational and clinical trials have also indicated that a high intake or high plasma level of this vitamin is associated with a low risk of cardiovascular disease. the vitamin may be operating at two levels; first, by protecting ldl from peroxidation, thereby reducing its atherogenicity, and second, by lowering the level of chronic inflammation by downregulation of nf-r,b. a reduction in platelet aggregability may also arise out of this action (huang et al., ; tanus-santos et al., ) . indeed, several lines of evidence indicated that supplements of vitamin e may prevent cardiovascular disease by reducing the susceptibility of ldls to oxidation (jailal, fuller, & huet, ) , reducing the expression of chemokines, adhesion molecule expression, and monocyte adhesion (wu, koga, martin, & meydani, ) , decreasing smooth muscle proliferation (azzi, boscoboinik, & marilley, ) , and decreasing platelet aggregation (steiner ) . another anti-inflammatory approach using nutrients would be to supplement diets of the elderly with n- pufas. supplementation with n- pufas from fish oil, however, has been reported to suppress the immune response (meydani, endres, & woods, ; meydani, ) , which hampers enthusiasm for the use of n- pufas for their benefits in cvd. however, the latter concern could be addressed by including a vitamin e supplement along with fish oil supplements. in a recent study it was found that supplementing elderly persons with (n- ) fatty acids of fish oil in combination with vitamin e while maintaining the anti-inflammatory properties of (n- ) pufas did not reduce immune indices in the elderly . fish oil supplementation is not universally efficacious in the treatment of inflammatory disease (grimble, ) . rheumatoid arthritis and inflammatory bowel disease have been the most successfully treated of all inflammatory diseases (calder, ) . the antiinflammatory mechanism may be through suppression of tnf-tx production. endres et al. ( ) reported that large doses ( g/d for wk) of oil in nine healthy volunteers resulted in a small but statistically significant reduction in tnf-o~ and il- [ production from pbmcs. subsequently, fewer than half of similar small intervention studies were able to demonstrate a statistically significant reduction in cytokine production. to understand the differences in response more closely, the author's laboratory conducted a study on young men fed g fish oil daily for wk and measured tnf-tx production by pbmcs before and after supplementation in relation to the snp at - in the tnf-o~ and at + in the lt-tx genes. no significant effect of fish oil on cytokine production was noted in the group as a whole. however, when data were examined according to tertile of tnf-ct production prior to supplementation, homozygosity for tnfb (lt-o~+ a) was . times more frequent in the highest than in the lowest tertile of production. the percentage of individuals in whom fish oil suppressed production was lowest ( %) in the lowest tertile and doubled with each ascending tertile. in the highest tertile, mean values were decreased by % (p < . ). in the lowest tertile, mean values were increased by % (p < . ). tnfb (lt-cz+ aa) homozygotes were strongly represented among unresponsive individuals in the lowest tertile of tnf-cz production prior to supplementation. in this lowest tertile, only tnfb l/b (lt-o~+ ga) heterozygous subjects were responsive to the suppressive effects of fish oil. in the medium tertile, this genotype was six times more frequent than other lt-t~ genotypes among responsive individuals. no relationship between possession of tnf or (tnf-ot- g or a) alleles and responsiveness to fish oil was found. clearly, although the level of inflammation determines whether fish oil will exert an anti-inflammatory influence or not and is influenced by the tnfb (lt-ct+ a) allele, the precise genomic mechanism for an anti-inflammatory effect is unclear at present . antioxidant intake also modifies cytokine production. in a study on healthy men and women and smokers, dietary supplementation with iu/d ct-tocopherol for mo suppressed the ability of pbmcs to produce tnf-t~. production was reduced by and % in nonsmokers and smokers, respectively (mol et al., ) . in a similar dietary intervention study on normolipaemic and hypertriglyceridaemic subjects given iu/ d of o~-tocopherol for wk, reduced tnf-o~, il- , and il- production by lps-stimulated blood mononuclear cells occurred (mol et al., ; van tits, demacker, de graaf, hak-lemmers, & stalenhoef, ) . a similar effect of cx-tocopherol was noted in a study on normal subjects and type diabetics (devaraj & jialal, ) . however, there were large standard deviations in the data from these studies, indicating major intraindividual variability in the ability of vitamin e to suppress production of the cytokine. although a number of studies have shown that o~-tocopherol suppresses superoxide production, the situation with regard to nitric oxide is less clear (mol et al., ; van tits et al., ) n the a-tocopherol derivative pentamethyl-hydroxychromane inhibited lps-stimulated nf-gb and inos activation in cultured j macrophages (hattori, ) . at present it is not known whether antioxidants interact differently with snps in the genes associated with oxidant stress and inflammation than they do with the other anti-inflammatory nutrient, n- pufa. this topic is currently an area of active research at the author's laboratory. proteomic studies have shown that inos and sod are both influenced by the nramp gene (kovorova, necasova, porkertova, radzoich, & macela, ) . the production of oxidant molecules enhancing pro-inflammatory cytokine production via high levels of nf-kb activation may thus be under a genomic influence owing to the aforementioned variations in the nramp gene (formica, roach, & blackwell, ) . a better understanding of this interaction and of the interaction of n- pufas and antioxidants with genotype may allow the better design of nutrient products for the treatment of inflammatory disease. it is clear from the current understanding about the purpose and functioning of the immune system throughout the life cycle that it is a powerful biological entity that profoundly alters body function while it is carrying out its prime purpose of defending the body against invasion by pathogens. however, within the response lie the seeds of disaster at an individual level, for the inflammatory component of the response can turn against the body, particularly as the body ages or becomes obese. the response, which can be devastating when directed against microbes entering the body, also sows the seeds of atherosclerosis, degradation of brain function, and insulin insensitivity and hastens the passage of hiv-infected individuals towards full-blown aids. along with the insights arising from the unraveling of the human genome has come evidence that the inflammatory response is able to protect the human species from invasion by pathogens but not all individuals within the species from ill health. the differing ability of humans, particularly the male of the species, at an individual level to mount an inflammatory response of different levels of intensity owing to genotype can result in widely contrasting outcomes of invasion of the body by pathogens. on the one hand, individuals may effectively fight off invasion provided the immune response follows a normal pathway, whereas other individuals within the same community encoutering the same pathogens will die from the strength and nature of the response rather than from the direct effects of the invader. insights gained from the genomic influences on cytokine production and the response to malaria suggest that the retention of alleles in pro-inflammatory cytokine genes that resulted in enhanced cytokine production within the human gene pool over generations could be a result of the heterozygotes' better capacity for 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in combination with different levels of vitamin e on immune response in healthy elderly human subjects in vitro glutathione supplementation enhances interleukin- production and mitogenic responses in peripheral blood mononuclear cells from young and old subjects associations of serum c-reactive protein with fasting insulin, glucose, and glycosylated hemoglobin: the third national health and nutrition examination survey production of tumour necrosis factor--alpha increases with age and bmi in healthy women the influence of age and gender on serum dehydroepiaandrosterone sulphate (dhea-s), il- , il- soluble receptor (il- sr) and transforming growth factor beta (tgf-beta ) levels in normal healthy blood donors polymorphisms in the promoter of the tumor necrosis factor-alpha gene in coal miners reduction of inflammatory cytokine concentrations and improvement of endothelial functions in obese women after weight loss over one year the author is grateful to the bbsrc for funding much of the work reported in this chapter. the author is also grateful to collegues in the united kingdom, sweden, and switzerland for scientific collaboration and advice. key: cord- -y zpvw authors: tan, kai sen; andiappan, anand kumar; lee, bernett; yan, yan; liu, jing; tang, see aik; lum, josephine; he, ting ting; ong, yew kwang; thong, mark; lim, hui fang; choi, hyung won; rotzschke, olaf; chow, vincent t; wang, de yun title: rna sequencing of h n influenza virus-infected human nasal epithelial cells from multiple subjects reveals molecular pathways associated with tissue injury and complications date: - - journal: cells doi: . /cells sha: doc_id: cord_uid: y zpvw the human nasal epithelium is the primary site of exposure to influenza virus, the initiator of host responses to influenza and the resultant pathologies. influenza virus may cause serious respiratory infection resulting in major complications, as well as severe impairment of the airways. here, we elucidated the global transcriptomic changes during h n infection of human nasal epithelial cells from multiple individuals. using rna sequencing, we characterized the differentially-expressed genes and pathways associated with changes occurring at the nasal epithelium following infection. we used in vitro differentiated human nasal epithelial cell culture model derived from seven different donors who had no concurrent history of viral infections. statistical analysis highlighted strong transcriptomic signatures significantly associated with and h after infection, but not at the earlier -h time point. in particular, we found that the influenza infection induced in the nasal epithelium early and altered responses in interferon gamma signaling, b-cell signaling, apoptosis, necrosis, smooth muscle proliferation, and metabolic alterations. these molecular events initiated at the infected nasal epithelium may potentially adversely impact the airway, and thus the genes we identified could serve as potential diagnostic biomarkers or therapeutic targets for influenza infection and associated disease management. the global burden of inter-pandemic influenza is high. it is estimated to affect billion people annually, with - million severe cases requiring hospitalization or intensive care treatment, resulting in approximately . million deaths [ ] . worryingly, drug-resistant influenza strains are emerging at a rapid rate that will severely hamper the ability of our healthcare systems to contain influenza outbreaks [ ] . therefore, alternative strategies are needed against severe influenza infections during both seasonal and pandemic influenza outbreaks. the normal human airway epithelium is a pseudo-stratified layer of ciliated and non-ciliated columnar cells, goblet cells, club cells, and basal cells [ ] . the airway epithelium protects against airway infection via efficient mucociliary clearance (mcc), the production of inflammatory mediators and chemokines against viruses, and the recruitment of immune cells [ ] . when the influenza virus breaches the defense of the human airway epithelium, it causes a myriad of innate responses by the infected host in response to viral invasion [ , ] . among these changes are critical factors that can determine disease severity, and which may lead to the development of diagnostic, prognostic prediction markers, or anti-influenza therapies [ ] [ ] [ ] [ ] . however, few studies have hitherto been performed in relevant models [ ] and human models of influenza are not feasible due to potential severity of the infection. therefore, the mechanistic study of viral-induced airway changes using relevant models can lead to better understanding of the development of severe complications. additionally, we need greater clarity on the different immune responses in view of the rising prevalence of chronic diseases such as diabetes mellitus and asthma. patients with these disorders are especially susceptible to severe influenza complications compared to healthy subjects [ ] . thus, the establishment of a baseline response against influenza infection of "healthy" tissue is beneficial to facilitate future comparative studies to better manage influenza in patients with co-morbidities. although the study of host responses in influenza infection is not new, current in vitro cell lines cannot accurately represent human airway infection due to the lack of key mucociliary features [ ] . hence, we have previously developed an air-liquid interface (ali) human airway epithelial cell culture that is able to sustain influenza infection [ , ] . we have also further compared the transcriptomic responses of our infected human nasal epithelial cells (hnecs) with other in vitro and in vivo influenza infection transcriptomic studies [ ] . the comparison revealed that at their peak responses against influenza, the differential transcriptome signature in hnecs was highly similar to the signatures from other influenza infection models [ ] . interestingly, compared to the homogenous cell lines tested, our heterogenous hnec model exhibited a more comparable response to the clinical influenza studies, indicating that most responses were initiated at the nasal epithelium [ ] . therefore, in this study, we aim to further utilize the hnec model as a physiologically relevant in vitro model to clarify the nasal epithelial responses against influenza h n infection, which would then facilitate the identification of the key host factors that are significant for future studies. to establish host factors that are significantly altered in the nasal epithelium as a reference of early innate responses against influenza, the dynamic expression of the genes needs to be clearly elucidated. while there are many studies that utilize microarray analysis to identify the host responses against influenza, the limitation of the microarray is its inability to determine the full extent of gene changes due to its hybridization-based protocol [ ] . the aim of this study was to utilize rna sequencing (rnaseq) technology to not only reveal the hnec responses (from multiple individuals) against influenza infection, but also to identify those genes with high magnitude changes to serve as potential reference markers of the innate responses of influenza infection. given that rnaseq functions by reading virtually all the rnas present in the samples tested, we can also discern the magnitude of each rna change and mark them as the canonical responses. in addition, as rnaseq is not constrained by probe usage as in microarrays, they are therefore more reliable in detecting novel interactions during influenza infections of hnecs. hence, rnaseq analysis will further augment the transcriptomic data established previously by microarray analysis. the augmented baseline can then be applied to future clinical studies and practice against influenza infection, especially for comparison against patients with other underlying co-morbidities that may be affected by more severe disease. approval to conduct this study was obtained from the national healthcare group domain-specific board of singapore (dsrb ref: d/ / ) and the institutional review board of the national university of singapore (irb ref: . written consent was obtained from donors prior to the collection of the tissue biopsies. at the time of collection, all subjects were free of symptoms of urti. the medical backgrounds of the subjects are summarized intable s . the hnespcs were isolated and enriched from the tissue biopsies according to a previously standardized protocol [ , ] , which normalized the hnespcs to a baseline state that differentiates into hnecs resembling healthy tissues if they pass the quality control checks for their differentiation [ ] . following enrichment, the hnespcs were expanded further and subjected to ali culture in transwells for in vitro differentiation according to previous protocol as well [ , ] . briefly, primary cells were subjected to isolation for selection of hnespcs, which were enriched and expanded with dulbecco's modified eagle medium: nutrient mixture f- (dmem/f ) (gibco-invitrogen, carlsbad, ca, usa) containing ng/ml of human epithelial growth factor (egf, gibco-invitrogen, carlsbad, ca, usa), µg/ml of insulin (sigma, st. louis, mo, usa), . nm of cholera toxin (sigma, st. louis, mo, usa), . µg/ml of hydrocortisone (sigma, st. louis, mo, usa), nm/ml of , , -triiodo-l-thyronine (t ) (sigma, st. louis, mo, usa), µl/ml of an n- supplement (gibco-invitrogen) and iu/ml of antibiotic-antimycotic (gibco-invitrogen, carlsbad, ca, usa). the expanded hnespcs were then transferred onto -well . µm transwell inserts (corning, corning, ny, usa). once confluent, growth medium was discarded and µl of pneumacult™-ali medium with inducer supplements (stemcell technologies inc., vancouver, canada) was added to the basal chamber to establish ali conditions. the cells were cultured in ali culture for weeks, with media change every - days. after - weeks of differentiation, hnecs from a total of seven donors were then subjected to influenza h n virus infection. the influenza a strain used in this study is of the h n subtype (a/aichi/ / ) (atcc, manassas, va, usa). the virus was propagated using embryonated egg culture and used for all the infection in the hnecs. prior to infection, fully differentiated hnecs were washed with × dpbs and infected with the h n influenza virus at a multiplicity of infection (moi) of . and incubated for h at • c. after the h incubation, the viral inoculum was removed and the hnecs were incubated back in • c. the control hnecs were harvested for apical wash and rna prior to the infection at h post-infection (hpi). the infected hnecs were then harvested for the apical wash and rna following , , and hpi incubation at • c. at each infection time point, µl of x dpbs was added and incubated in the apical chamber for min at • c to recover progeny viruses as the apical wash. the plaque assay for viral quantification was performed using overnight mdck cultures (atcc, manassas, va, usa) at - % confluence in -well plates. the mdck cells were incubated with µl of serial dilutions (from − to − ) of virus from apical washes at • c for h, where plates were rocked every min to ensure equal viral distribution. after incubation, the inocula were removed and replaced with ml of avicel (fmc biopolymer, philadelphia, pa, usa) overlay, and incubated at • c for - h. after incubation, avicel overlay were removed, and cells were fixed with % formaldehyde in × pbs for h. formaldehyde was then removed, and cells were washed with × pbs prior to staining with % crystal violet for min before washing the stain away. the plaque-forming units (pfu) were calculated as follows: number of plaques × dilution factor = number of pfu per µl. at each time point after the collection of apical wash, the hnecs were lysed using rna lysis buffer. total rna was then extracted from the lysate using mirvana mirna isolation kit (life technologies, grand island, ny, usa). the extracted total rna was first subjected to nanodrop analysis to first ensure the rna quality, before being submitted for rnaseq analysis. then, ng from the remaining rna was subjected to cdna synthesis using maxima first-strand cdna synthesis kit (thermoscientific, pittsburgh pa, usa). after this, qpcr analysis was performed to evaluate the transcriptional levels of host response genes selected based on previous microarray analysis using pre-designed primers (sigma aldrich). each qpcr reaction was performed in duplicate using gotaq-qpcr master mix kit (promega, san luis obispo, ca, usa), and relative gene expression was calculated using the comparative method of -∆∆ct normalized to the housekeeping gene pgk . relative gene expression levels were presented as median values and interquartile ranges, while statistical significance was determined using the wilcoxon signed-rank test. all human rnas were analyzed on the agilent bioanalyzer (agilent, santa clara, ca, usa) or the perkin elmer labchip gx system (perkin elmer, waltham, ma, usa) for quality assessment with rna integrity number (rin) or rna quality score range from . - . and median of . . cdna libraries were prepared using ng of total rna and µl of a : , dilution of ercc rna spike in controls (ambion ® thermo fisher scientific, waltham, ma, usa) using smartseq v protocol [ ] , except for the following modifications: fastq files were mapped to the human genome build hg using star. gene counts were computed using featurecounts (part of the subread package) using annotations from gencode version . differential gene expression analysis was performed using edger in a paired fashion under r version . . . multiple testing correction was done using the method of benjamini and hochberg and p-values (false discovery rate; fdr) less than . was deemed to be significant. geneset enrichment analysis using data from gene ontology (go) was performed using the bioconductor package topgo, while the analysis using reactome pathway was performed using the vioconductor package reactomepa. both analyses were run in r version . . using multiple testing-corrected significant differentially-expressed genes. tnf-α, tnf-β, vegf, eotaxin/ccl , and pdgf-aa. samples and standards were incubated with fluorescent-coded magnetic beads which had been pre-coated with the respective capture antibodies. after an overnight incubation at • c, plates were washed twice. biotinylated detection antibodies were incubated with the complex for h, and streptavidin-pe was then added and incubated for another min. plates were washed twice again, then beads were re-suspended with sheath fluid before acquiring on the flexmap ® d (luminex) using xponent ® . (luminex) acquisition software. data analysis was done on bio-plex manager™ . . (bio-rad). standard curves were generated with a -pl ( -parameter logistic) algorithm, reporting values for both mfi and concentration data. results were then expressed as mean fold change compared with uninfected control, and p-values (fdr) of less than . were considered significant. prior to analysis, the responses of all seven hnecs donors following influenza infection were plotted on a principal component analysis (pca) plot. the pca plot indicated a degree of variability in the responses between donors and time points (figure ). nonetheless, the responses were clustered tightly enough following infection to signify their consistency of infection for further transcriptomic analysis-similar to those observed in our previous study [ ] . the respective capture antibodies. after an overnight incubation at °c, plates were washed twice. biotinylated detection antibodies were incubated with the complex for h, and streptavidin-pe was then added and incubated for another min. plates were washed twice again, then beads were resuspended with sheath fluid before acquiring on the flexmap ® d (luminex) using xponent ® . (luminex) acquisition software. data analysis was done on bio-plex manager™ . . (bio-rad). standard curves were generated with a -pl ( -parameter logistic) algorithm, reporting values for both mfi and concentration data. results were then expressed as mean fold change compared with uninfected control, and p-values (fdr) of less than . were considered significant. prior to analysis, the responses of all seven hnecs donors following influenza infection were plotted on a principal component analysis (pca) plot. the pca plot indicated a degree of variability in the responses between donors and time points ( figure ). nonetheless, the responses were clustered tightly enough following infection to signify their consistency of infection for further transcriptomic analysis-similar to those observed in our previous study [ ] . significant gene expression changes (fdr < . ) of infected hnecs were detected as early as hpi, and further increased at and hpi (table ; figure a ). also, the number of genes decreased in a linear fashion as the fold change in expression increased, as seen in the x fold change genes significant gene expression changes (fdr < . ) of infected hnecs were detected as early as hpi, and further increased at and hpi (table ; figure a ). also, the number of genes decreased in a linear fashion as the fold change in expression increased, as seen in the x fold change genes indicated in figure b , where about % of the significantly altered genes remained. at hpi, there were upregulated genes and downregulated genes. the major upregulated genes were the antiviral sensors and early response genes such as ifns, ifits, and ifis. interestingly enough, interferon lambda (ifnλ) gene ifnls was the earliest response interferon of infected hnecs, as opposed to interferons alpha or beta, at hpi. at later time points, the number of gene expression changes increased substantially, with upregulation of and genes, and downregulation of and genes at and hpi, respectively. there was augmented expression of antiviral effectors and inflammatory genes at both time points. ifnl remained the interferon gene with highest expression at both time points, while a marked elevation of cytokines such as cxcl and cxcl was also observed. considering downregulated genes, proliferative and transcriptomic functions appeared to be suppressed, with diminished expression of genes such as fmo , klk , and fosb. genes associated with metabolism, cell cycle, and dna repair were further suppressed following infection at and hpi. tables s -s list the complete set of significant gene expression changes, arranged according to their fold change (log fc). in addition, we have also verified that the genes showing major expression changes by rnaseq generally concurred with rt-qpcr analyses. of the genes tested by qpcr at hpi, all of them showed the same directional changes in expression as observed by rnaseq. hence, seven of these genes showed a p-value of < . (il i , ifnl (il ), cxcl , tnfsf , ifi , ccl , and cyp a ), one gene had a p-value of < . (ctgf), while only two genes were not statistically significant (tgfa and ano ) ( figure s ). indicated in figure b , where about % of the significantly altered genes remained. at hpi, there were upregulated genes and downregulated genes. the major upregulated genes were the antiviral sensors and early response genes such as ifns, ifits, and ifis. interestingly enough, interferon lambda (ifnλ) gene ifnls was the earliest response interferon of infected hnecs, as opposed to interferons alpha or beta, at hpi. at later time points, the number of gene expression changes increased substantially, with upregulation of and genes, and downregulation of and genes at and hpi, respectively. there was augmented expression of antiviral effectors and inflammatory genes at both time points. ifnl remained the interferon gene with highest expression at both time points, while a marked elevation of cytokines such as cxcl and cxcl was also observed. considering downregulated genes, proliferative and transcriptomic functions appeared to be suppressed, with diminished expression of genes such as fmo , klk , and fosb. genes associated with metabolism, cell cycle, and dna repair were further suppressed following infection at and hpi. tables s - list the complete set of significant gene expression changes, arranged according to their fold change (log fc). in addition, we have also verified that the genes showing major expression changes by rnaseq generally concurred with rt-qpcr analyses. of the genes tested by qpcr at hpi, all of them showed the same directional changes in expression as observed by rnaseq. hence, seven of these genes showed a p-value of < . (il i , ifnl (il ), cxcl , tnfsf , ifi , ccl , and cyp a ), one gene had a p-value of < . (ctgf), while only two genes were not statistically significant (tgfa and ano ) ( figure s ). we then further compared the transcriptomic alterations in the hnecs over time, following influenza h n infection. the number of gene expression changes mirrored the viral titer changes, which peaked at hpi, and were consistent between donors ( figure a ). approximately two thirds of genes at and hpi overlapped with other time points, while about one third of genes at hpi overlapped ( figure b ). the overlapping genes displayed similar directional consistency at the significant time points. in addition, congruent with the consistent viral titer with most gene expression changes at hpi, we also noted the most consistent alterations in expression of genes across donors. this is highlighted in figure c , which portrays the heatmaps of the top genes with the smallest p-value, together with their direction and magnitude of change. based on these analyses, we proposed that hpi represents the optimal time point for the subsequent pathway analysis to ascertain influenza-specific pathway changes. we then further compared the transcriptomic alterations in the hnecs over time, following influenza h n infection. the number of gene expression changes mirrored the viral titer changes, which peaked at hpi, and were consistent between donors ( figure a ). approximately two thirds of genes at and hpi overlapped with other time points, while about one third of genes at hpi overlapped ( figure b ). the overlapping genes displayed similar directional consistency at the significant time points. in addition, congruent with the consistent viral titer with most gene expression changes at hpi, we also noted the most consistent alterations in expression of genes across donors. this is highlighted in figure c , which portrays the heatmaps of the top genes with the smallest p-value, together with their direction and magnitude of change. based on these analyses, we proposed that hpi represents the optimal time point for the subsequent pathway analysis to ascertain influenza-specific pathway changes. we then further subjected the significant gene changes to gene set enrichment using both go and reactome databases. at time points , , and hpi, there were , , and significant (adjusted p-value < . ) go biological processes (table s ) and , , and significant (adjusted pvalue < . ) reactome pathways (table s ) , respectively. at the early time of hpi, interferon- we then further subjected the significant gene changes to gene set enrichment using both go and reactome databases. at time points , , and hpi, there were , , and significant (adjusted p-value < . ) go biological processes (table s ) and , , and significant (adjusted p-value < . ) reactome pathways (table s ) , respectively. at the early time of hpi, interferon-mediated antiviral responses were elevated as expected. at hpi, the pathways appeared to be more stabilized and consistent for both go and reactome analyses, despite displaying more gene expression changes at this time point. responses to influenza virus skewing towards type i immunity were predominant in the go analysis. the expected interferon-mediated functions by the epithelium validated the authenticity of our model, where we found enriched type i interferon (go) and rig-i (reactome) pathways with upregulation of nearly all significant gene members (data not shown). besides the interferon and antiviral pathways, we identified several functions of interest initiated by the nasal epithelium that may contribute to the pathology and pathogenesis of influenza. at hpi, go pathway enrichment analysis revealed that the nasal epithelium was actively involved in initial ifnγ signaling (go: ), despite not directly producing ifnγ. we also observed enriched function in apoptosis and necroptosis (go: and go: ), immune evasion (go: ), and other pathways that may lead to complication events such as smooth muscle proliferation (go: ) and response to fatty acid (go: ) ( table ). for the reactome pathway analysis, we selected pathways that were enriched with more than significant genes present in the enriched pathway, and these were generally in agreement with the go analysis (table ). in addition to ifnγ signaling ( ) and apoptosis ( ), it also revealed changes in epithelial-initiated b cell receptor signaling ( and ) and amino acid metabolism ( ) following influenza infection. it is noteworthy that these pathways were initiated at the epithelial level without the participation of immune cells, thus highlighting the relevant genes of interest for future studies. given that rnaseq analysis facilitates more accurate expression changes following infection compared to hybridization technology such as microarray, we conducted further analysis on the levels of gene expression changes to enable more stringent and accurate transcriptomic analyses for future studies. by comparing these results to a previous study that identified influenza-specific signatures, we verified that these genes were all expressed in infected nasal epithelium later at hpi, but not at hpi. furthermore, at both and hpi, all but one of the gene signatures exhibited elevated expression of > . -fold change (> . log fc) compared to uninfected control hnecs (table ). when we applied the higher fold change cutoff, the number of significant genes decreased by approximately % (figure a) , which was also congruent with the linear association observed earlier. therefore, future studies on early transcriptional alterations could consider adopting the . -fold change in expression as a more stringent threshold, which may be more feasible, especially for large transcriptomic studies that yield large numbers of data points. in addition, when compared to the previous microarray study on a similar hnec model [ ] , both rnaseq and microarray shared a high degree of overlap, with about one third and half of total genes from rnaseq and microarray overlapping, respectively ( figure b ). the overlap was generally observed in genes with highly altered expression, such as cxcl , cxcl , and rsad , which were changed to a similar magnitude in both rnaseq and microarray (table s ). when we compared the influenza signature genes, rnaseq revealed a more consistent increase in magnitude, i.e., at hpi, the magnitude of the gene change was generally higher than that of the microarray (table ). in addition, rnaseq was also able to detect novel genes with expression changes of high magnitude that were generally higher than those found by microarray only ( genes versus genes with elevated expression greater than . -fold). genes such as heatr , pdcd , il i , art , and kcnh were altered to a higher magnitude than the . -fold threshold. hence, rnaseq-based transcriptomic analysis may augment transcriptomic findings to identify novel gene responses against influenza in the future. from rnaseq and microarray overlapping, respectively ( figure b ). the overlap was generally observed in genes with highly altered expression, such as cxcl , cxcl , and rsad , which were changed to a similar magnitude in both rnaseq and microarray (table s ). when we compared the influenza signature genes, rnaseq revealed a more consistent increase in magnitude, i.e., at hpi, the magnitude of the gene change was generally higher than that of the microarray (table ). in addition, rnaseq was also able to detect novel genes with expression changes of high magnitude that were generally higher than those found by microarray only ( genes versus genes with elevated expression greater than . -fold). genes such as heatr , pdcd , il i , art , and kcnh were altered to a higher magnitude than the . -fold threshold. hence, rnaseq-based transcriptomic analysis may augment transcriptomic findings to identify novel gene responses against influenza in the future. after deriving the transcriptomes by rnaseq, we then further investigated whether the changes in expression of genes resulted in alterations in secretory cytokines and chemokines early in the infection of hnecs. initially, we detected significant reductions in multiple cytokines at hpi, with the exception of il- which was increased ( figure s ). this may reflect the initial immune suppression during influenza infection. however, at and hpi, less significant changes were observed, i.e., only increase in tnf-a and decrease in mdc and pdgf-aa were noted at hpi. this was followed by increase in ip- (cxcl ) and tgf-a and decrease in pdgf-aa seen at hpi. this analysis highlights changes in ip- , tgf-a, and pdgf-aa to be significant early responses in secretory cytokines/chemokines following influenza infection. our study has identified epithelium-initiated host responses which are found to be involved in both innate and adaptive responses. the finding is significant as we can now focus on the primary point of contact of influenza-the nasal epithelium in the study of early host responses for identifying host factors that can be utilized for diagnostic and therapeutic purposes [ ] . in addition, our study also showed that it is important for reference databases to use relevant human models like the hnecs model, which contains the mucociliary component of the airways, in order to provide closely representative host responses. while there exists a high number of microarray studies that showed the host responses using similar hnec models, there are only a small number of equivalent rnaseq studies. compared to microarrays, rnaseq analysis can provide a more comprehensive picture of the transcriptomic landscape, and is not limited by the hybrid library variant and concentrations [ ] . hence, in order to derive accurate magnitude of gene expression changes, we performed an rnaseq analysis of h n infection using the hnecs model. h n influenza virus was selected, given that it is a major circulating subtype over long periods of time. in addition, relatively lower efficacy of vaccines against this subtype prompted us to study its interactions with the primary host target to elucidate the immune responses and association with adaptive immunity [ , ] . this model has been previously evaluated to be a highly clinically-relevant model that can facilitate controlled infection of nasal cells from multiple individuals. in addition, we have also previously shown-by microarray analysis-that the nasal epithelium is responsible for the initiation of host responses following influenza infection [ ] . this renders the hnecs to serve as a valuable tool to analyze transcriptomics from different individuals infected under the same conditions to ensure consistent and relevant responses in humans. once the magnitude of gene expression changes was considered, several interesting findings emerged. firstly, the infected hnecs were observed with strong activation of antiviral genes and early inflammatory genes leading to type i immune responses. a large number of gene expression changes were of magnitude of over -fold difference (log to log fold change). most of the genes with high-magnitude expression changes were verified by qpcr, with statistical significance congruent with the rnaseq analysis. secondly, despite the absence of immune cells, the infected hnecs were able to generate strong type i responses that may likely aid the recruitment of cytotoxic cells to clear the infected cells. thirdly, in early responses of the hnecs, ifnλ genes, which represent type iii interferons, were more strongly induced than the more frequently observed type i interferons (ifnα and ifnβ), while type ii interferons were not produced by hnecs, in agreement with previous studies [ , ] . the induction of type iii interferons may reflect an important event within the hnecs where ifnλ, the initial responders against the infection, may be more beneficial in the antiviral response [ , ] . moreover, we also observed notable suppression of expression of certain genes following influenza infection, including suppression of proliferation and dna repair genes, which may contribute to the pathology and pathogenesis of influenza [ ] . finally, rnaseq also unraveled expression changes of certain newly-discovered genes in response to influenza infection of the upper airway cells. genes such as heatr [ ] , il i [ ] , tnfsf b (baff) [ ] , and pdcd (pd- ) [ ] are recently implicated in influenza pathogenesis and mucosal defense, thereby signifying the role of the nasal epithelium against influenza infection. furthermore, rnaseq identified altered expression of art and kcnh genes that were not previously detected in influenza transcriptomes. these findings hence further reiterate the value of rnaseq in enhancing data on influenza transcriptomes for reference in future studies. via pathway enrichment analysis, we have identified known antiviral pathways to validate the hnecs responses against influenza. in addition, we have also documented the potential pathways initiated by the nasal epithelium that may contribute to influenza pathogenesis as represented by the gene expression changes listed in tables and . by analyses using literature-inferred go and reactome databases, we have demonstrated that the nasal epithelium can play a role in the main antiviral signaling, i.e., ifnγ responses despite not being a direct producer of ifnγ. the pathway enrichment indicated that hnecs may serve as important regulators of type ii interferons. even though the effects of ifnγ are vital to the robust clearance of influenza viruses [ ] , there are reports of unregulated ifnγ being a contributor to inflammatory damage [ , ] . therefore, the over-production of ifnγ response factors such as icam and cd may contribute to inflammatory damage of the epithelium. hence, production of factors such as stat [ ] by the hnecs is also crucial in ensuring appropriate regulation of ifnγ-mediated expression of influenza response genes to modulate inflammation and to minimize damage. the primary contact of influenza virus with the nasal epithelium may subsequently lead to damage to the airway epithelium as well. this is apparent with the clear enrichment of the pathways of apoptosis, mitochondrial apoptotic processes, and necroptosis that contribute to cell death and mechanical barrier loss during infection [ , ] . genes such as ifi , bak , caps , tnfsf , and fas suggest active apoptotic cell death that not only destroys cells in the epithelial barrier, but may also serve to propagate the virus and to perpetuate the damage [ ] [ ] [ ] . furthermore, during virus infection, aberrant regulation of apoptosis may also lead to further injury to the epithelium and surrounding tissues [ ] . on the other hand, necroptosis pathways have also been observed to be enriched in influenza-infected hnecs. compared to apoptosis, the study of necroptosis in influenza infection is relatively new with contradicting findings [ ] . ripk /necroptosis studies appear to generate contradictory results as to whether necroptosis protects against or is detrimental during influenza infection [ , ] . hence, its increased expression during infection of hnecs warrants further investigation on its role in influenza-induced damage. in addition, we also noted enrichment of b-cell signaling pathways by the infected hnecs which may be vital for b-cell responses during the adaptive immune response [ ] . we noted that most genes enriched in the b-cell pathways were related to antigen recognition such as proteasome subunits (psme , psmb , psma , etc.) and b-cell receptor-associated genes such as dapp and card [ ] . however, changes in expression of certain growth factors (including ereg and fgfs) following influenza infection may lead to complications involving airway remodeling and recruitment [ , ] . further, the effects of the growth factors were further confirmed by the enrichment of pathways related to the proliferation of smooth muscle cells also induced by the infection. changes to airway smooth muscle cells are usually implicated in airway remodeling [ ] [ ] [ ] , and may also contribute to post-influenza complications. hence, the genes found in this study may be crucial for elucidating the nasal-initiated responses that may contribute to the pathology and pathogenesis of influenza infection of the airways. finally, another interesting pathway that may contribute to epithelial damage is the negative regulation of innate immune responses. these genes may serve as proviral factors and aid in immune evasion. for example, adar is a proviral factor that works in synergy with influenza ns to enhance viral replication [ ] . trafd is a negative regulator of toll-like receptor signaling which is upregulated in influenza-infected hnecs [ ] . dhx is a negative regulator of rig-i/mda signaling pathway [ ] . ceacam is involved in regulation of liver inflammation [ ] and its expression appears to exert antiviral effects on influenza virus [ ] . nmi binds to influenza virus ns and inhibits irf -mediated interferon signaling [ , ] . therefore, aberrant expression of genes in this signaling pathway may directly contribute to immune evasion of influenza, culminating in viral propagation and increased epithelial damage. we summarized the identified pathways (listed in tables and ) that alluded to immune evasion (negative regulation of innate immune responses), antigen processing (metabolism of amino acids and derivatives), and immunomodulation (interferon gamma signaling, b cell receptor signaling, and response to fatty acid) that may contribute to severity of influenza. there was evidence of direct pathway enrichment of potential influenza evasion strategies and/or immunomodulation with accompanying transcriptomic changes. the genes in the pathway may be analyzed for their immunomodulatory activity and whether their expression is beneficial to the virus (immune evasion) or the host (preventing cytokine storm). in addition, the infected hnecs also revealed modified responses associated with fatty acid, with many lipid signaling molecules such as leukotrienes that mediate antiviral responses and subsequent inflammation of the airway [ , ] . such modified responses may also determine the afforded in the airway and the severity of airway inflammation and damage. in addition, the modification may also affect the lower airway responses to inflammatory mediators; hence, the changes in these pathways may also suggest a potential mechanistic link to the pathogenesis of viral-induced exacerbation of chronic inflammatory diseases. lastly, we also noted enrichment of pathways related to amino acid metabolism, which is important in antigen processing and proteasomal degradation of foreign protein. the changes in these genes at the hnecs, the target site of influenza infection, may determine the effectiveness of antiviral responses mounted and may therefore influenza disease severity. in addition, we also compared our rnaseq analysis against previously reported influenza-specific signatures in order to improve future transcriptomic analysis [ ] . in vitro transcriptomic analysis yields a large number of differentially-expressed genes that would require additional criteria to identify functionally significant genes. by means of this comparison, we discovered that almost all influenza-specific signatures exhibited differences in expression of above . -fold. hence, we propose applying fold change of > . as a threshold for future in vitro transcriptomic systems analyses, in order to increase the stringency in detecting functionally significant gene changes. finally, we observed that, unlike the transcriptome, there were notably fewer cytokines that were readily secreted during the acute phase of infection. expression of cytokines was reduced at hpi, except for il- , which interestingly is implicated in influenza-induced acute lung injury [ ] . even fewer cytokines showed altered expression at later time points. among them, only tgf-a, ip- (cxcl ), and pdgf-aa were significantly altered at and hpi. these may be significant markers that can be detected in the secretion of influenza-infected mucosal surface that may influence the severity of influenza. ip- is a well-established ifnγ response gene, and serves as a useful marker for response against influenza [ , ] . tgf-a represents an important factor involved in the secretion of il- in response to influenza, and may determine the early appropriate innate responses to prevent severe disease [ ] . on the other hand, it is also involved in pulmonary fibrosis as a ligand of epidermal growth factor receptor (egfr) and may contribute to complications in the lower airway [ ] . pdgf-aa was found to be elevated in the cerebrospinal fluid of influenza-associated encephalopathy [ ] , but was consistently reduced in hnec secretory fluid, thus warranting further investigation into its role in the infected nasal mucosa. the establishment of a reference transcriptome based on early responses of the human nasal epithelium model serves a key role in research on critical host factors involved in influenza. as the primary host contact with the virus, not only are immune responses against influenza important, but also the alterations in non-immune functions such as metabolism, cell content, and cell cycle, which may contribute to disease severity. in terms of translational potential, the model system identified gene expression changes of significant magnitude and pathways that impact responses against influenza and its severity. these genes may represent novel targets for future diagnostic and therapeutic development. under controlled conditions, the hnecs clinically establish the baseline for "normal" innate immune responses of the nasal epithelium against influenza viral infection. such a baseline can be particularly crucial when studying the changes in innate immune responses against influenza, especially in patients with underlying chronic diseases who may have aberrant airway responses against influenza. their antiviral responses may differ from "normal" subjects, and this study thus provides the basis for comparing the differential responses that culminate in more severe infections in patients with co-morbidities such as diabetes and chronic airway inflammatory diseases. such comparative clinical studies can potentially enhance the management of influenza viral infection in patients with chronic diseases. in conclusion, rnaseq technology allowed us to accurately quantify the magnitude of gene expression changes, as well as the relevant enriched pathways during h n influenza virus infection of hnecs, which can serve as a baseline for future clinical studies. the establishment of this baseline under controlled condition elucidated the antiviral innate response by the infected nasal epithelium, and highlighted the molecular factors and abnormalities in the upper airway that may contribute to influenza severity. furthermore, this study also culminated in the identification of novel gene signatures and host factors that may be harnessed for future research to develop influenza diagnostic markers and therapeutic targets. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s . real-time quantitative pcr validation of genes selected from rnaseq analyses. pcr data are expressed as log fold change using median and interquartile range. statistical significance was determined using wilcoxon signed-rank test. * p < . , # p < . ; figure s . luminex assay of secreted cytokines/chemokines in the apical supernatant. luminex data are expressed as mean fold change from uninfected control. statistical significance was determined using fdr. * p < . ; table s . information of seven donors of hnecs; table s . significant enriched pathways based on reactome pathway database analysis; table s . list of significant genes with altered expression at hpi of influenza h n infection of hnecs analyzed by rnaseq and microarray [ ] . oseltamivir resistance-disabling our influenza defenses role of il- ralpha in modulating il- -induced muc ac and ciliary changes in healthy and crswnp mucosa epithelial damage and response human nasal epithelial cells derived from multiple individuals exhibit differential responses to h n influenza virus infection in vitro comparative transcriptomic and metagenomic analyses of influenza virus-infected nasal epithelial cells from multiple individuals reveal specific nasal-initiated signatures systems-biology approaches to discover anti-viral effectors of the human innate immune response uncovering the global host cell requirements for influenza virus replication via rnai screening. microbes infect cellular networks involved in the influenza virus life cycle cd , a novel host factor of nuclear export signaling in influenza virus infection propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures distinction between rhinovirus-induced acute asthma and asthma-augmented influenza infection a novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells comparing bioinformatic gene expression profiling methods: microarray and rna-seq the use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro full-length rna-seq from single cells using smart-seq predicting clinical severity based on substitutions near epitope a of influenza a/h n effectiveness of seasonal influenza vaccinations against laboratory-confirmed influenza-associated infections among singapore military personnel in - . influenza other respir in vitro model of fully differentiated human nasal epithelial cells infected with rhinovirus reveals epithelium-initiated immune responses ifnlambda is a potent anti-influenza therapeutic without the inflammatory side effects of ifnalpha treatment interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness influenza infection induces host dna damage and dynamic dna damage responses during tissue regeneration heatr is upregulated during influenza virus infection in lung alveolar epithelial cells the il i enzyme: a new player in the immunosuppressive tumor microenvironment. cells cigarette smoke inhibits baff expression and mucosal immunoglobulin a responses in the lung during influenza virus infection highly pathological influenza a virus infection is associated with augmented expression of pd- by functionally compromised virus-specific cd + t cells new fronts emerge in the influenza cytokine storm inflammatory impact of ifn-gamma in cd + t cell-mediated lung injury is mediated by both stat -dependent and -independent pathways production of interferon-gamma by influenza hemagglutinin-specific cd effector t cells influences the development of pulmonary immunopathology h n influenza virus infection enhances oncostatin m expression in human nasal epithelium nf-kappab-dependent induction of tumor necrosis factor fas/fasl is crucial for efficient influenza virus propagation nucleoprotein of influenza a virus negatively impacts antiapoptotic protein api to enhance e f -dependent apoptosis and virus replication influenza a virus enhances its propagation through the modulation of annexin-a dependent endosomal trafficking and apoptosis programmed cell death in the pathogenesis of influenza dai senses influenza a virus genomic rna and activates ripk -dependent cell death zbp /dai is an innate sensor of influenza virus triggering the nlrp inflammasome and programmed cell death pathways the multifaceted b cell response to influenza virus influenza virus-induced type i interferon leads to polyclonal b-cell activation but does not break down b-cell tolerance neutrophils induce smooth muscle hyperplasia via neutrophil elastase-induced fgf- in a mouse model of asthma with mixed inflammation respiratory syncytial virus infection provokes airway remodelling in allergen-exposed mice in absence of prior allergen sensitization cd , a laminin receptor showing increased expression in asthma, contributes to airway hyperresponsiveness through calcium signaling regulation of human airway smooth muscle cell migration and relevance to asthma airway smooth muscle in asthma: phenotype plasticity and function. pulm pharm the interactomes of influenza virus ns and ns proteins identify new host factors and provide insights for adar playing a supportive role in virus replication yoshimura, a. fln , a novel interferon-and lps-inducible gene acting as a negative regulator of toll-like receptor signaling rna-and virus-independent inhibition of antiviral signaling by rna helicase lgp ceacam in liver injury, metabolic and immune regulation ceacam -mediated inhibition of virus production subcellular proteomic analysis of human host cells infected with h n swine influenza virus negative regulation of nmi on virus-triggered type i ifn production by targeting irf mast cells and influenza a virus: association with allergic responses and beyond leukotriene b enhances nod -dependent innate response against influenza virus infection multi-cohort analysis identifies conserved transcriptional signatures across multiple respiratory viruses interleukin- is critical in the pathogenesis of influenza a virus-induced acute lung injury influenza induces il- and gm-csf secretion by human alveolar epithelial cells through hgf/c-met and tgf-alpha/egfr signaling overactive epidermal growth factor receptor signaling leads to increased fibrosis after severe acute respiratory syndrome coronavirus infection vascular endothelial growth factor (vegf) and platelet-derived growth factor (pdgf) levels in the cerebrospinal fluid of children with influenza-associated encephalopathy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the surgeons and staff in the department of otolaryngology, national university hospital, singapore. we thank h.h. ong and t.t. he for the subject selection and recording. we thank m.c. phoon and s.h. lau for technical assistance in viral experiments. the authors would like to acknowledge the staff of the immunomonitoring platform at sign. the authors declare no conflicts of interest. key: cord- -lldbjm authors: soni, dheeraj; bobbala, sharan; li, sophia; scott, evan a.; dowling, david j. title: the sixth revolution in pediatric vaccinology: immunoengineering and delivery systems date: - - journal: pediatr res doi: . /s - - -y sha: doc_id: cord_uid: lldbjm abstract: infection is the predominant cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. however, very young infants fail to respond optimally to most vaccines currently in use, especially neonates. in , stanley plotkin proposed that new delivery systems would spur a new revolution in pediatric vaccinology, just as attenuation, inactivation, cell culture of viruses, genetic engineering, and adjuvantation had done in preceding decades. recent advances in the field of immunoengineering, which is evolving alongside vaccinology, have begun to increasingly influence vaccine formulation design. historically, the particulate nature of materials used in many vaccine formulations was empiric, often because of the need to stabilize antigens or reduce endotoxin levels. however, present vaccine delivery systems are rationally engineered to mimic the size, shape, and surface chemistry of pathogens, and are therefore often referred to as “pathogen-like particles”. more than a decade from his original assessment, we re-assess plotkin’s prediction. in addition, we highlight how immunoengineering and advanced delivery systems may be uniquely capable of enhancing vaccine responses in vulnerable populations, such as infants. impact: immunoengineering and advanced delivery systems are leading to new developments in pediatric vaccinology. summarizes delivery systems currently in use and development, and prospects for the future. broad overview of immunoengineering’s impact on vaccinology, catering to pediatric clinicians and immunologists. the impact of vaccination on the health of the world's peoples is hard to exaggerate. with the exception of safe water, no other modality has had such a major effect on mortality reduction and population growth (dr. stanley a. plotkin, md, vaccines, ) . so what is my choice for the sixth revolution?… i suggest that it will be new delivery systems (dr. stanley a. plotkin, md, pediatric academic societies meeting, may, ). the goal of vaccination is to trigger an immune response that reduces the risk of infection and prevents disease. initially, delivery systems were redundant since the majority of vaccines employed live attenuated organisms, which were often particulate in nature and inherently carried the abundant and necessary immune-stimulating signals. however, as vaccinology moved towards the development of defined antigens such as inactivated, subunit, and purified recombinant proteins and peptides, which are inadequate to trigger an immune response alone, the use of novel delivery systems became crucial. historically, antigen stabilization (i.e., adsorption onto alum) guided vaccine formulation design. therefore, the particulate nature of materials used in many early vaccine formulations was empiric. in this context, inclusion of alum adjuvants has been key to the acceptable efficacy of these subunit vaccine formulations, even though alumadjuvanted vaccines usually require multiple doses for optimal protection. second-generation efforts employed more characterized materials, such as the biodegradable synthetic polymer poly (d,l-lactic-co-glycolic acid) (plga), which is a widely investigated nanoparticle adjuvant for controlled and effective delivery of vaccine antigens, including synthetic peptides. these are typically produced as solid-core nanoparticles ranging from to nm in size, with antigens entrapped or adsorbed on the surface of the particles. more recently, advances in the field of immunoengineering, which are developing alongside vaccinology, have begun to greatly influence vaccine formulation design. , due to the wide differences in mechanisms of action of various non-adjuvanted and adjuvanted vaccines, vaccine formulation development has become a major consideration for vaccinologists and pharmaceutical companies. these considerations include ( ) physicochemical characteristics of the formulation, ( ) adjuvant chemical structure, ( ) proposed route of administration, and ( ) short-and long-term formulation stability. specifically, vaccine delivery systems can now be engineered to mimic the size, shape, and surface chemistry of pathogens, which are often referred to as "pathogen-like particles". nanoparticle formulations can now be manufactured to target subsets of immune cells and specific subcellular compartments. [ ] [ ] [ ] one aspect consistent across current and future novel vaccines is the need to determine their ability to instruct adaptive immunity through the manipulation of antigen-presenting cells (apcs). as a part of their function as professional apcs, dendritic cells (dcs) can integrate information from extrinsic stimuli (e.g., components of pathogens or vaccines) and orchestrate these signals into appropriately regulated adaptive immune responses. as such, immunoengineering, adjuvant and antigen discovery, vaccine delivery, and increased knowledge of human immune responses are fueling a revolution in vaccinology. more than years ago, stanley plotkin identified the five crucial technical advances that revolutionized the field of vaccinology. these were attenuation ( s; e.g., live attenuated rabies), inactivation ( s onwards; e.g., killed vaccines for typhoid and cholera), cell culture of viruses ( s; cultured poliovirus), genetic engineering ( s; recombinant proteinbased hepatitis b vaccine), and the induction of cell-mediated immunity (i.e., via adjuvantation) (fig. ). dr. plotkin identified six candidate areas that may start a possible sixth revolution (fig. ) . these ranged from the combinatorial employment of older vaccine strategies to newer methods such as reverse vaccinology. ultimately, the pioneering and burgeoning science driving the use of "delivery systems" was his choice to lead the way. interestingly, there has also been remarkable progress in vaccine design and technologies including structure-guided antigen design, broadly neutralizing antibodies and promising novel vaccine platforms (dna, mrna) (fig. ) . many of these advances are reliant, at least in part, on optimized delivery. after nearly a decade and a half, in this review we reevaluate dr. plotkin's vision to assess whether his prediction has been validated or is yet unanswered. recent advances in the nascent field of immunoengineering may allow for the design of vaccine delivery systems that can potentially accelerate the development of novel and effective early life vaccination strategies. immunoengineering may guide vaccine design , by enabling the targeting of dcs, specifically via synthetic vaccine delivery systems that mimic the size, shape, and surface chemistry of pathogens. from an immunoengineering stance, the ideal vaccine formulation is (a) customizable, (b) based upon a scalable synthetic vaccine platform with (c) tunable release kinetics and can (d) stably encapsulate controllable amounts of molecules possessing diverse chemistry and water solubility. such macro-and nanoscale-theranostic (i.e. therapeutic and diagnostic) materials would enable sustained and targeted delivery of complex antigen/adjuvant combinations via nanocarriers for hours, days, weeks, or months. by providing control over intracellular delivery of antigens and adjuvants to apcs, immunoengineering can more effectively activate innate immunity and moreover apcs to boost the adaptive immune response. nanoparticle delivery systems can increase intracellular delivery to apcs by mimicking the morphology of viruses and by s; hepatitis b vaccine (hbv), the st recombinant-antigen-based vaccine, incorporated the viral surface proteins, derived from molecular biology production. combination vaccines: simultaneous administration of vaccines to target multiple diseases. the adjuvant toolbox: ranging from small-molecule adjuvants to combination adjuvants. vaccines for non-infectious diseases: new treatments for tumors, allergy, or noninfectious disorders (e.g., prevention of drug overdose). systems vaccinology: systems biology approaches to identify predictors of vaccine efficacy and explore new insights about protective immunity. reverse vaccinology: bioinformatics aided vaccine design from pathogenic genetics. immunoengineering and delivery systems: delivering precise materials for specific activation of immune system (right time, right place, right size, right shape, etc.). incorporating targeting ligands. , in addition, the size, charge, and morphology of nanoparticles also affect lymph node trafficking. [ ] [ ] [ ] control of nanoparticle delivery to intracellular compartments can be used to modulate the immune response post-vaccination. delivery of antigens to the cytosolic or endocytic compartments respectively leads to antigen presentation via major histocompatibility complex (mhc) class i or mhc class ii to prime distinct t cell subsets. this can be achieved by engineering antigen-loaded nanoparticles to induce endosomal or lysosomal escape, leading to antigen cross-presentation. , furthermore, the targeting of nanoparticles carrying toll-like receptor (tlr) agonists to endosomes leads to activation of endosomal tlrs, promoting strong th -type responses. [ ] [ ] [ ] activation of the inflammasome through cytosolic nod-like receptors (nlrs) can lead to a self-adjuvant effect by the nanoparticle vehicle itself that promotes adaptive immunity. [ ] [ ] [ ] lastly, nanoparticles containing vaccines can be used to control the duration of delivery through synchronous and sustained release of antigens and adjuvants. , delivery kinetics can be further modified using strategies such as incorporating nanoparticles into hydrogels or intradermal microneedle delivery systems. [ ] [ ] [ ] these sustained release systems can allow for the development of single-dose vaccines. thus, pathogen-mimicking nanoparticles can be engineered to enhance the immune response by controlling when and where vaccine components are delivered intracellularly to apcs. a plethora of particulate delivery systems for immunoengineering have been developed, which are summarized further in this review. liposomes have been considered a dominant vaccine delivery platform because of their superior adjuvant properties and versatility in accommodating vaccine components. , liposomes are spherical vesicle structures comprising a phospholipid bilayer shell and aqueous lumen with sizes ranging from a few nanometers to several microns. , these vesicles have been engineered to optimize their immunological role using several approaches including modulation of size and charge, , surface decoration with immune cell recognition epitopes, , and pegylation to enhance in vivo circulation times. an adjuvanted-liposomal vaccine formulation as b is marketed by glaxosmithkline as a component of shingles vaccine (shingrix) and is also in human clinical trials for malaria and hiv vaccines. of note, cationic liposomes have shown high adjuvanticity, and a cationic liposomal formulation cfa is currently being tested in human clinical trials for hiv and tuberculosis vaccines. virosomes virosomes are nanosized phospholipid vesicles with membranes incorporating viral envelope proteins, typically produced from reconstituted empty envelopes of influenza viruses. they are utilized as both a vaccine carrier system and as an adjuvant, where the antigen of interest is either adsorbed or encapsulated within the lumen. , virosomes have been reported to induce both cellular and humoral immunity through efficient presentation of antigen via both mhc class i and ii proteins; however, their exact mode of action is still unclear. virosome-based influenza vaccines are licensed in europe (as inflexal) and as adjuvants for hepatitis a vaccine (as epaxal). immune-stimulating complexes (iscoms) are spherical cage-like nanoparticles (~ nm) formed via self-assembly of a mixture of the saponin adjuvant quil a, cholesterol, phospholipids, and antigens. iscoms in the absence of an antigen are called iscomatrix and can be mixed with any antigen of interest. iscoms have been reported to stimulate enhanced cellular responses with lower antigen doses through enhanced antigen cross-presentation. however, the role of individual components in generating these immune responses is unknown. at present, iscom technology has been approved only for veterinary vaccines; however human clinical trials are currently ongoing for the development of melanoma vaccines. modern-day vaccine research is highly dependent on flexible engineering strategies such as tunable immune cell recognition epitopes, morphological diversity, and precise intracellular delivery. polymer-based systems have been considered frontrunners for incorporating these strategies as compared to their lipid-based counterparts. polymeric delivery vehicles are mainly classified as solid-core or self-assembled. these delivery vehicles have an additional advantage of greater physicochemical and in vivo stability as compared to lipid-based systems. solid-core particles plga micro-and nanoparticles have been widely explored for vaccine delivery applications because of their ease of fabrication, amenability to surface modification, encapsulation efficiency of both hydrophilic and hydrophobic vaccine components, and modifiable release properties. that the depot forming ability of plga microspheres at the injection site could be a potential single-shot vaccination strategy. , the ability of plga particulate vaccines to generate strong cytotoxic responses make them great candidates for the development of vaccines against infectious diseases , and cancer. another interesting solid-core vaccine delivery vehicle, pluronicstabilized polypropylene sulfide (pps) nanoparticles, have been developed. , these particles contain a hydrophobic pps core, which becomes hydrophilic under oxidative conditions of cell endosomes, promoting intracellular release. reddy et al. showed highly efficient transportation of the pps nanoparticles ( nm) through lymphatic capillaries to target half of the lymph node resident dcs and unveiled the role of pps surface chemistry in activating the complement cascade. further, pps nanoparticles conjugated with a model protein antigen ova stimulated both cellular and humoral responses. , self-assembled particles the self-assembly of polymeric structures is often achieved using block copolymers, which comprise linked hydrophobic and hydrophilic polymer blocks. the application of diverse block copolymer chemistries and self-assembled morphologies has been evaluated for immunomodulation. , the simplest morphology that can be attained is a micelle, which contains a hydrophobic core and a hydrophilic corona. polylactide (pla)-based block copolymer micelles have been widely explored for vaccine delivery. specifically, polyethylene glycol (peg)-b-pla block copolymer micelles have been reported to show excellent biocompatibility, immunomodulatory ability, and success in inducing cellular immunity. recently, environment-responsive micelles consisting of ph- or oxidation-responsive block copolymers have shown great promise in releasing vaccine components inside cell lysosomes. there is also great interest in developing polymersomes, which are self-assembled polymeric vesicles analogous to liposomes, for delivering vaccines and inducing cellular immunity. peg-b-pps, an oxidation-responsive block copolymer, has been reported to self-assemble into monodisperse polymersomes and encapsulate a wide range of antigens and adjuvants. , stano et al. reported that peg-b-pps polymersomes loaded with antigen (ova) and adjuvant (cpg) induced enhanced cd + t cell responses in the spleen and lymph nodes. furthermore, peg-b-pps block copolymers have been engineered to self-assemble into other diverse morphological structures. these block copolymers with peg weight fractions (f peg ) of . , . , . , and . selfassemble to form bicontinuous nanospheres (polymeric cubosomes), vesicles (polymersomes), cylindrical micelles (filomicelles), and micelles, respectively. recent studies revealed that these structures have differential organ biodistributions and immune cell uptake in vivo, , which makes them strong contenders for the development of new rationally designed immunotherapies and vaccines. aluminum salts (alum) including aluminum hydroxide and aluminum phosphate are the most commonly used adjuvants in human vaccines. in aluminum salt-based vaccine formulations, antigens are adsorbed to the highly charged aluminum hydroxide or aluminum phosphate gel. the molecular mechanisms by which alum interacts with the human immune system continues to be studied and involves multiple pathways, both direct and indirect. the majority of studies suggest that the adjuvant action of aluminum salts is mediated by activation of the nlrp inflammasome. alum may directly or indirectly trigger innate immunity via activation of inflammasome complexes, required for the processing of il- family pro-inflammatory cytokines. this process is most likely nlr-mediated, since the adjuvant effects of alum are not impaired in the absence of key tlr-dependent signal transduction adaptor molecules myd and trif in knockout mice. secondly, alum enhances delivery of antigen to apcs as particulate vaccine formulations more readily interact with dcs and macrophages than soluble formulations of antigens alone. crystalline alum binds lipids on the surface of dcs and triggers a cellular activation cascade leading to initiation of an immune response, but without itself being internalized by the cells. thirdly, alum-induced cell death seems to modulate the local milieu in favor of enhanced adaptive immune stimulation. the release of damage-associated molecular patterns, such as uric acid and dsdna, act as autologously derived auto-adjuvants. induction of humoral immunity is a hallmark feature of aluminum-containing adjuvants. for instance, alum-adjuvanted vaccines often require multiple doses for induced protection, and drive th -over th -polarized immunity. presently, there are multiple licensed pediatric vaccines, such as diphtheria, tetanus, and hepatitis vaccines, listing alum as essential to produce effective antibody (ab) titers. several new strategies have been considered to modify these alum particles for induction of cellular responses. for example, liu et al. encapsulated alum colloid inside a yeast-derived β-glucan particle, which induced greater cellular immune responses. another group, wang et al., made phospholipid bilayer-coated aluminum nanoparticles that were readily taken up by the apcs and stimulated antigen-specific cellular and humoral responses in vivo. however, there are also vaccinal antigens for which addition of alum may not be necessary for effective immunogenicity. for example, alum was excluded from the recent vaccine menveo (menacwy) due to failure of alum to enhance improvement in serum bactericidal ab titers during infant clinical trials. gold nanoparticles are versatile systems that can be easily synthesized into different morphologies and are able to accommodate a diverse range of antigens and adjuvants onto their surface. in several studies, gold particles have been reported to have efficient accumulation in dcs and b cells, playing a critical role in generating cellular and humoral responses. , recent findings on gold nanoparticle-based cancer vaccines demonstrated that the photothermal ablation ability of gold particles alongside cellular cytotoxic responses can have a synergistic impact in cancer treatment. additionally, inorganic particles like carbon nanotubes, mesoporous silica, and iron oxide nanoparticles have been widely explored as vaccine delivery vehicles. however, more understanding of their toxicity and safety profiles may be required for the advancement of these nanoparticles. diverse water-in-oil emulsions, of which incomplete freund's adjuvant is the best-known, were originally evaluated in human trials during the mid-twentieth century. muscle after intramuscular injection, the mechanism of action of which is tlr-independent. for example, the adjuvant mf does not directly mature apcs, but rather induces the production of chemokines and immune modulatory proteins from monocytes, macrophages, granulocytes, and muscle cells that indirectly leads to increased migration of apcs to/from the site of injection and into draining lymph nodes. this cell recruitment is greater than that induced by alum. mf may also instruct peripheral site monocyte differentiation into dcs and possibly induce the release of endogenous tlr agonists. therefore, one major advantage of o/w adjuvants is the antigen dose-sparing potential. since vaccine development pipelines rarely tailor formulations (adjuvants, delivery systems, etc.) rationally for use in early life, it is critical to understand the optimization of vaccine efficacy by taking into account early life immune ontogeny. [ ] [ ] [ ] for example, early life vaccination against intracellular pathogens has proven difficult. due to functionally distinct and delayed t cell-mediated immunity, newborns and young infants are highly susceptible to infection with intracellular pathogens, including bacteria such as listeria spp. and salmonella spp., viruses such as herpes simplex virus and respiratory syncytial virus, and intracellular pathogens of global significance such as hiv, tuberculosis, and malaria. here, nanoparticle-based formulations may hold great promise for pediatric vaccine development. targeting the key deficiencies of newborn dcs relative to adult dcs may enable development of age-specific vaccine formulations to overcome sub-optimal immunization responses. recently, we have combined such engineering and rational vaccine design approaches to develop a nanoparticle-based adjuvant and antigen-delivery system designed to be active in human newborns and infants. the design mimicked, and in some instances, exceeded the immunostimulatory effect of live attenuated vaccines. we employed peg-b-pps polymersomes, which are significantly more stable than liposomes, as the effective adjuvantand antigen-delivery system. these polymersomes can be effectively engineered for bioresponsive intracellular payload delivery, making them highly advantageous for the specific targeting of endosomal receptors. this is notable, since the activation of endosomal pattern recognition receptors (prrs), as compared to surface prrs (e.g. tlr and tlr ), instructs more adult-like innate immune responses in newborn dcs. smallmolecule tlr agonists (e.g. imidazoquinoline) robustly activate newborn dcs but can result in systemic responses when delivered in soluble form. to overcome this and minimize off-target effects, we developed tlr -agonist-encapsulating polymersomes, which demonstrated a preference for uptake by dcs relative to other cell populations following subcutaneous administration. furthermore, such formulations hold substantial potential for early life immunization by serving as a dual antigen/adjuvant delivery system that mimics the enhanced neonatal innate and adaptive immune responses elicited by the live bacille calmette-guerin (bcg) vaccine. strikingly, when co-loaded with the mycobacterium tuberculosis antigen b peptide , the tlr -agonist containing polymersomes were comparable to bcg in inducing antigenspecific immune responses in human tlr -expressing neonatal mice in vivo. this is promising, since bcg reduces the risk of disseminated early life tuberculosis by safely eliciting th -type neonatal immune responses and requires only a single dose at or shortly after the time of birth. another promising approach is the use of microstructures designed for controlled release of vaccine formulations in vivo. mchugh et al. recently developed a microstructure called stamped assembly of polymer layers (seal). seals are small (≤ - μm) plga-based polymeric microdevices with complex geometries, which can be filled with soluble drug solutions and then completely sealed. such structures can be rationally designed with additive manufacturing processes (i.e., threedimensional ( d) printing). importantly, depending on the formulation and chemical composition, these materials can be tuned to achieve continuous or delayed/pulsatile release kinetics. this was achieved by tuning the degradation of the materials (i.e., the copolymer ratio). as opposed to solidparticulate delivery systems formulated to deliver antigen continuously, which demonstrate an initial burst and then slow release thereafter, such "pulsatile vaccines" more closely resemble traditional vaccine schedules, which are based around the concept of priming followed by multi-booster injections, but with the advantage of requiring only a single immunization. this may be especially promising for combinatorial vaccination approaches that require multiple boosters, as commonly used in the pediatric setting, or induction of protective immunity that may rely on persistent pathogenic antigen exposure. these and other such technologies may ultimately open the way for single-injection vaccine strategies. , reassessing vaccination schedules and expanding vaccine target populations most vaccination schedules throughout the world are designed for the pediatric age group. notably, most schedules recommend children to be immunized against hepatitis b virus (and often tuberculosis through the bcg vaccine) starting in the neonatal period. immunization to rotavirus, diphtheria, tetanus, pertussis, h. influenzae type b, streptococcus pneumoniae, poliovirus, influenza, measles, mumps, rubella, varicella, hepatitis a virus, meningococcal, and human papillomavirus extend from months of age up to adolescence. as the twenty-first century progresses, the current immunization schedule should remain flexible to incorporate newly invented vaccines as they become available with emerging and reemerging infectious diseases, such as meningococcus, influenza virus, group a streptococcus, helicobacter pylori, and respiratory syncytial virus (possibly the most important infant vaccine still missing from this clinical schedule). in addition, expanding the current schedule to cover more vaccine-preventable infectious diseases and including vaccine formulations incorporating novel immunoengineered delivery systems may be key tools to allow for an accelerated schedule approach ( table ) . as outlined above, with the exception of the hepatitis b virus vaccine and bcg (which in some countries are given at birth), present immunization schedules start mostly after months of age. consequently, the present immunization schedules do not induce protection against the majority of these diseases until the fifth-sixth months of life or later. this creates a period of vulnerability during the first months of life, which is associated with significant mortality and morbidity. therefore, current aspirational efforts often focus on the development of (a) single-dose immunization strategies for newborns that instruct lifelong protection to subsequent challenges or (b) optimizing maternal immunization as means to cover this susceptibility gap. the sixth revolution in pediatric vaccinology: immunoengineering and. . . d soni et al. heterologous prime-boost vaccination strategies (e.g. an attenuated vaccine followed by an inactivated vaccine targeting the same pathogen) may also be advantageous in early life, especially if they incorporate the same antigen but distinct delivery systems. current prime-boost regimens incorporating viral vectors or dna vaccines, followed by a boost with a protein-based vaccine may be able to overcome distinct immune ontogeny in early life while also taking advantage of the relatively lower rates of reactogenicity in the neonatal and early infant periods. furthermore, increased appreciation of immune ontogeny may inform development of rationally designed age-specific vaccine formulations. future studies need to focus not only on safety and efficacy, but also on potential vaccine-vaccine interactions that can lead to interference, and possibly include adjuvants that more effectively enhance immune responses in early life. innate immune memory, i.e. an alteration of reactivity in innate immune cells previously exposed to diverse stimuli, may also confer heterologous immunity that could be leveraged by some adjuvanted vaccine formulations in the future. vaccination holds promise for primary prevention not only for neonates and infants but also for diverse age-and target groups, including adults, elderly, adolescents, pregnant women, preterm infants, fetuses, and people suffering from chronic and immunecompromising diseases. these groups represent heterogeneous populations, separated by genetic background, gender, pregnancy, microbiome, diet, lifestyle, poverty, risk of infectious diseases, and geography (those in the developed and developing world where future vaccines would be deployed) among others. therefore, considering these broad differences, it is critical to understand how novel delivery systems may be utilized effectively to optimize vaccine efficacy. two of foremost the target populations are pregnant women and preterm infants. notably, immunity of preterm infants is distinct from newborns, rendering them particularly susceptible to infection. , every year,~ million newborns are born preterm worldwide, which is~ % of all live births. preterm infants demonstrate impairments in innate and acquired immunity, significantly less maternal-derived ab than term infants, and a higher risk of infection-induced disability and death. early life immunization to close the window of disease vulnerability may be key to preventing these infections, but immune responses to subunit vaccines are impaired among preterm infants. for example, although administered at birth to term infants, hepatitis b virus immunization is often delayed in preterms due to reduced immunogenicity in this population. similarly, preterm infants demonstrate impaired serotype-specific immune responses to pneumococcal conjugate vaccine (pcv)- . novel use of preexisting and clinical licensed vaccine formulations may shed light on areas of optimization. co-administration of bcg with hbv, for example, significantly enhanced anti-hbv igg titers in mouse models of both term and preterm birth, equally, but not in adult mice. accordingly, there is an unmet need for safe and immunogenic vaccines for preterm infants, including those targeting hbv and pneumococcus. maternal vaccination has emerged as a favorable public health approach in the past decade. this approach is highly promising as it may decrease maternal, fetal, and neonatal susceptibility to infections. while implementation may vary by region, currently a number of vaccines are universally recommended/indicated during pregnancy. these include vaccines against tetanus, influenza, and pertussis, along with new vaccines for group b streptococcus and respiratory syncytial virus that are currently being developed to prevent neonatal infections. as the efficacy of maternal vaccines significantly relies on active transport of antibodies at the maternal-fetal interface (through the placenta using an fc receptor and concentrated in the fetus), antigen/ table . potential uses and benefits of "novel delivery systems" to future early life vaccination strategies. • instruct an accelerated, targeted, potent, and durable immune response in humans against pathogens (e.g. overcome pathogen diversity and immune evasion) • allow for dose sparing and reduced vaccine manufacturing costs, thereby increasing the global access to pediatric vaccines • dramatically increase the number of antigens per formulation/immunization • modulation of antigen delivery and persistence (i.e., single bolus vs. slow release formulations) • act as immunomodulators to enhance th (e.g., t helper [th ] cell versus th ) or achieve qualitative alteration of the immune response (cd + versus cd + t cells) • allow cell-mediated t cell vaccination strategies • induction of un-natural immunity (i.e., broadly protective universal vaccines) • instruct heterologous immunity, thereby reducing overall mortality rates the sixth revolution in pediatric vaccinology: immunoengineering and. . . d soni et al. adjuvant delivery systems may become key tools in expanding these efforts to cover a broader range of diseases. in addition, adjuvanted vaccine formulations may be specifically designed to polarize maternal responses to minimize interference with infant responses to subsequent vaccination or infection. lastly, passive immunization (via the use of monoclonal abs) may also be considered a relevant tool in areas where no vaccine currently exists. this includes the mab palivizumab (brand name synagis), produced by recombinant dna technology for the prevention of rsv infections, especially in at-risk preterm infants. modern vaccine design and development strategies endeavor to integrate knowledge of formulation composition and understanding of immunostimulatory mechanisms to generate immune responses otherwise unachievable in relevant target populations. , rational vaccine design approaches, employing novel immunoengineering strategies, and targeted delivery systems may allow for the controlled preparation of vaccine formulations of the desired immunostimulatory properties, particulate size, and antigen load, all of which can greatly improve safety and limit systemic toxicities due to their targeted nature. these potential advances may be key to tailored vaccines capable of matching unique characteristics of the developing immune system during the neonatal period and infancy. plotkin highlighted various candidates for the sixth revolution of vaccinology, including the expanded use of combination vaccine strategies, new adjuvants, vaccines for non-infectious 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propensity score weighted regression analysis designing tomorrow's vaccines vaccines for the st century the authors d.s. and d.j.d. would like to thank dr. ofer levy for his mentorship, and boston children's hospital precision vaccines program for publication support. this work was supported by u.s. national institutes of health grant r ai - a and adjuvant discovery program n c . all figures for this review have been created with biorender. d.j.d. contributed to initial article conceptualization. d.s. and d.j.d. contributed to outline/framework, initial drafting, and editing/finalization for publication. s.b. and s.l. contributed to preparation of initial and advanced drafts. e.a.s. reviewed the manuscript and provided editorial input. all authors gave their final approval of this paper for publication. competing interests: the authors declare no conflict of interest.patient consent: patient consent was not required for this review.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -fgwf wy authors: wang, ben x.; fish, eleanor n. title: the yin and yang of viruses and interferons date: - - journal: trends immunol doi: . /j.it. . . sha: doc_id: cord_uid: fgwf wy interferons (ifns)-α/β are critical effectors of the innate immune response to virus infections. through activation of the ifn-α/β receptor (ifnar), they induce expression of ifn-stimulated genes (isgs) that encode antiviral proteins capable of suppressing viral replication and promoting viral clearance. many highly pathogenic viruses have evolved mechanisms to evade an ifn response and the balance between the robustness of the host immune response and viral antagonistic mechanisms determines whether or not the virus is cleared. here, we discuss ifns as broad-spectrum antivirals for treatment of acute virus infections. in particular, they are useful for treatment of re-emerging virus infections, where direct-acting antivirals (daas) have limited utility due to daa-resistant mutations, and for newly emerging virus strains in which the time to vaccine availability precludes vaccination at the onset of an outbreak. interferons (ifns)-a/b are critical effectors of the innate immune response to virus infections. through activation of the ifn-a/b receptor (ifnar), they induce expression of ifn-stimulated genes (isgs) that encode antiviral proteins capable of suppressing viral replication and promoting viral clearance. many highly pathogenic viruses have evolved mechanisms to evade an ifn response and the balance between the robustness of the host immune response and viral antagonistic mechanisms determines whether or not the virus is cleared. here, we discuss ifns as broad-spectrum antivirals for treatment of acute virus infections. in particular, they are useful for treatment of re-emerging virus infections, where direct-acting antivirals (daas) have limited utility due to daa-resistant mutations, and for newly emerging virus strains in which the time to vaccine availability precludes vaccination at the onset of an outbreak. ifns-a/b: host-derived broad spectrum antivirals virus infections range from mild and benign to highly virulent epidemics and pandemics, and significantly affect global health. daas, which target specific steps of virus replication, and vaccines, are currently the most effective therapeutic intervention strategies used against virus infections. newly emerging or re-emerging viruses that have undergone mutations may, however, be resistant to the effects of daas, whereas vaccines require that the virus strain be identified before vaccine production, precluding their use at the onset of any new virus infection outbreak. broad-spectrum antivirals, capable of modulating the innate immune response regardless of the infecting virus, present as ideal candidates as a first-line treatment for acute virus infections such as respiratory tract or sexually transmitted infections. ifns-a/b are produced by plasmacytoid dendritic cells (pdcs), macrophages, fibroblasts and endothelial cells, and are critical effectors in an innate immune response to virus infections [ ] . ifns-a/b are induced following pattern recognition receptor (prr) activation by viruses (box , as reviewed in [ ] ) and target many different stages of viral replication: for example, viral entry, envelope uncoating, genome replication, protein assembly, and release of viral progeny [ , ] . ifns-a/b also activate different cell types in the immune system to promote viral clearance and induce apoptosis of cells to prevent viral replication [ , ] . as host-derived innate immune response factors, ifns are, therefore, broad-spectrum antivirals, crucial for the primary host response to viral infection. ifns-a/b bind to and activate the ifnar complex, resulting in the rapid induction of transcription and translation of isgs ( figure , as reviewed in [ ] ). notably, ifnars are ubiquitously expressed on all cell lineages; probably an evolutionary consequence of different viruses being able to target and infect different cell types. ifnar expression on any and all cell types ensures that an ifn response to a virus may be induced upon infection. as an ifn response is central to a robust innate immune response, viruses have evolved a variety of mechanisms to interfere with ifn production and signaling, to disrupt innate host antiviral factors. successful viral clearance is determined by the balance between virus-encoded molecules that antagonize the host innate immune response and the robustness of the host innate immune response. here, we discuss how ifn therapy presents as a viable treatment option for a range of acute virus infections that target a variety of tissues, including respiratory tract infections by severe acute respiratory syndrome coronavirus (sars-cov) and influenza a viruses, infections of the liver by hepatitis c virus (hcv) and hepatitis b virus (hbv), and mucosal infections by herpes simplex virus (hsv). this may be particularly important given the paucity of broad-spectrum antivirals for treating newly emerging and re-emerging virus infections, which present a major threat to human health. yin: ifn activates the immune system in addition to the induction of isg expression in all cell types, ifns shape the landscape of the immune system in response to virus infection by promoting neutrophil survival [ , ] and the activation of macrophages [ ] , natural killer cells [ ] , dcs [ , ] , b cells [ ] and cd + t cells [ ] , and t helper (th) polarization of effector cd + t cells [ ] ( figure ). ifn therapy therefore has the advantage over daa treatments in that, in addition to stimulating genes that block viral replication in infected cells, ifns activate other innate and adaptive immune responses to combat the virus. polymorphisms in genes encoding factors involved in different stages of the ifn response can lead to marked differences in susceptibility to virus infection and severity of disease, and can also serve as predictive markers for the outcome of ifn treatment. for example, polymorphisms in host genes encoding proteins associated with regulation of an ifn response such as interferon receptor a-chain (ifnar ) [ ] , the ifn-inducible myxovirus resistance gtpase protein, mx [ ] , the ifn-inducible , -oligoadenylate synthetase (oas) [ ] and the suppressor of cytokine signaling (socs) associated with regulation of an ifn response [ ] , are predictive markers linked with the rate of sustained virological response (svr) to hcv infection following ifn-a treatment. this highlights the importance of an intact ifn response during viral infection and indicates that genetic variations among patients can present as a challenge for optimizing ifn therapy. yang: virulence factors antagonize the ifn response it is not surprising that many pathogenic viruses, including sars-cov, influenza a viruses, hcv and hsv, have developed mechanisms to disrupt and limit the ifn-a/b response (table ) . many viruses are able to evade the innate immune system by directly targeting pathways required for the induction of ifn-a/b production [ ] [ ] [ ] [ ] [ ] . these viruses are also able to inhibit an ifn response, by interfering with effectors in ifn-inducible signaling cascades [ ] [ ] [ ] . understanding the basis of these antagonistic mechanisms is essential for optimizing the timing and dosage of ifn treatments as a viable therapy for acute virus infections. box . ifn-a/b induction by viral pathogen-associated molecular patterns (pamps) prrs, including membrane-associated toll-like receptors (tlrs), and cytosolic rna and dna sensors such as rig-i, are able to detect both extracellular, endosomal or cytosolic viral pamps: viral genomic material. the primary outcome of this nonspecific surveillance system that detects any and all viruses, regardless of the target cell or tissue tropism of the virus, and independent of where the virus is located, is the transcriptional activation of ifn-a/b genes and the rapid production and secretion of these ifns. upon viral pamp recognition, prrs are able to trigger a phosphorylationdependent signaling cascade to activate irf and/or irf . for instance, endosomal rna/dna-sensing tlr and tlr activate irf via myeloid differentiation primary response gene (myd ), whereas endosomal dsrna-binding tlr and rig-i, are able to activate irf and irf through tbk and inhibitor of ikke. irf and irf activation results in their nuclear translocation where they act as transcription factors and up-regulate ifn-a/b gene expression. ifns-a/b bind with high affinity to the ifnar complex, composed of an a-chain, ifnar , which is structurally modified by cell membrane glycosphingolipids, galabiosylceramide (gb ) and globotriaosylceramide (gb ) to promote efficient ifn binding [ , ] , and a b-chain, ifnar . ifn binding to ifnar induces phosphorylation of the receptor-bound tyrosine kinases, tyrosine kinase (tyk ) and jak , leading to the subsequent regulation of: (a) protein synthesis via the activation of pi k and mammalian target of rapamycin (mtor); (b) histone modification, via the activation of p mapk; and (c) gene expression via the phosphorylation of stat proteins and mapk activation. tyk -and jak -mediated activation of insulin receptor substrate (irs) and irs is required for recruitment and activation of pi k. pi k phosphorylates akt, which inactivates inhibitors of mtor, tuberous sclerosis protein (tsc) and tsc [ ] . mtor activates the serine/ threonine kinase p s k, and inactivates eukaryotic translation initiation factor e-binding protein ( e-bp ), to upregulate cap-dependent mrna translation and protein synthesis. p is activated downstream of mapk kinases and (mkk / ) and regulates histone modification and gene expression through mitogen-and stress-activated protein kinase (msk) and msk . in addition, ifn signaling invokes promyelocytic leukemia zinc finger (plzf) protein-mediated histone modification to regulate isg expression [ ] . phosphorylation of stat proteins results in their dimerization and ifn-stimulated gene factor (isgf) formation. stat complexes translocate to the nucleus and bind to specific gene elements in the promoters of isgs, ifn-g activated sequence (gas) and ifn-sensitive response element (isre), to induce expression of antiviral genes. ifn therapy as a first-line treatment against newly emerging or re-emerging virus outbreaks: sars-cov the sars-cov outbreak originated in hong kong in late - and resulted in > cases of disease worldwide, with a . % mortality rate between november and july (http://www.who.int/csr/sars/country/ table _ _ /en/index.html). sars-cov is a singlestranded rna virus that encodes in its genome virulence factors that antagonize the ifn-a/b response. in infected host cells, sars-cov expresses the nonstructural protein (nsp) and nsp . nsp suppresses host gene expression by disrupting mrna translation and by upregulating mrna degradation [ , ] . immunoprecipitation and luciferase reporter studies have shown that nsp directly associates with the s ribosomal subunit to inhibit its translational activity [ ] . in addition, the nsp - s complex is able to modify mature -capped rnas to limit translation and promote degradation [ ] . in the context of an ifn response, these antagonistic mechanisms of nsp on host gene expression and protein synthesis inhibit ifn-a/b expression and production [ ] . in vitro, nsp also inhibits signal transducer and activator of transcription (stat) protein phosphorylation induced by ifn-a treatment [ ] . both nsp and nsp inhibit interferon regulatory factor (irf) and irf activation to downregulate ifn production in response to viral infection [ , ] . specifically, nsp inhibits irf in human bronchial epithelial cells via its papain-like protease (plp) domain, which [ , ] , tripartite motif-containing protein a (trim a) and trim , which are antiviral factors that limit hiv- infection [ ] , and transcription factor jun-d (jund) and claudin (cldn ) [ ] . ifn treatment primes cells for apoptosis by modulating the expression of proteasome subunits, major histocompatibility complex (mhc) class i, and fas receptor (cd ) [ ] [ ] [ ] [ ] . ifns-a/b also contribute to the activation and differentiation of cells involved in the (b) innate and (c) adaptive immune responses to virus infection. ifn-a/b induces production of interleukin (il)- , il- , and il- by dcs, and il- by macrophages to modulate b and t cell differentiation (th polarization) and activation [ ] . ifn-b signaling in pdcs leads to altered cd and sphingosine- -phosphate (s p ) receptor expression, thereby affecting pdc retention in lymph nodes [ ] . ifns-a/b increase mhc class ii, cd and cd expression on antigen presenting cells. ifn-a/b treatment induces macrophage and neutrophil phagocytosis [ , ] . moreover, ifns-a/b promote neutrophil survival by activating cellular inhibitor of apoptosis (ciap ) [ ] . natural killer (nk) cells respond to ifns-a/b with increased fas ligand (fasl) and perforin expression, and ifn-g production [ , ] . in response to ifns-a/b, b cells upregulate l-selectin and igg production [ , ] . trends in immunology april , vol. , no. interacts with irf to inhibit irf phosphorylation and nuclear translocation [ ] . in addition to nsp and nsp , the open reading frame (orf ) and matrix (m) proteins of the sars-cov also inhibit an ifn response [ , ] . orf localizes to the host endoplasmic reticulum (er) and blocks the transcription factor function of phosphorylated stat , by binding to nuclear import factors to prevent its translocation to the nucleus [ ] . the m protein interacts with rna sensor retinoic acid-inducible gene (rig-i), an rna helicase and key intracellular prr associated with induction of irf-dependent ifn production following detection of viral rnas. the m protein also interacts with the signaling effectors serine/threonine-protein kinase (tbk ), inhibitor of nuclear factor-kb kinase subunit e (ikke), and tumor necrosis factor (tnf)-associated factor (traf ), again associated with ifn gene induction [ ] . thus, there are multiple mechanisms by which sars-cov might inhibit the host ifn response. the implications are that an ifn response to sars-cov infection must be dramatically limited for virus replication to proceed, suggesting that the dominant immune response is the ifn response. different recombinant ifn-as and ifn-b are now approved for various clinical indications, including the treatment of chronic hcv infections [ , ] . through a comprehensive analysis of how structural features in the ifn-a/b moleculescrucial clusters of amino acidsaffect the sensitivity of target cells to ifn-induced biological responses, specific epitopes on the exposed surface of the ifn molecule have been identified that are associated with receptor recognition [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . accumulating evidence suggests that the affinity of a particular ifn-a/b subtype for ifnar determines the biopotency of the ifn, specifically in the context of antiviral and antiproliferative responses [ , ] . a direct consequence of this was the design and development of a synthetic ifn-a, ifn alfacon- , that exhibits optimized affinity for ifnar [ ] [ ] [ ] [ ] . initially, treatment for sars-cov infection focused on the use of a daa, ribavirin, in combination with corticosteroid therapy [ , ] . however, in a pilot clinical study, the therapeutic potential of ifn alfacon- was evaluated in individuals infected with sars-cov and hospitalized in toronto, canada [ ] . ifn alfacon- treatment together with corticosteroids is associated with reduced disease-associated impaired oxygen saturation, more rapid resolution of radiographic lung abnormalities and lower levels of disease-associated creatine kinase. in vitro studies to examine the mechanism of action of ifn against the sars-cov have revealed that ifn-inducible janus kinase (jak ), protein kinase c (pkc)-d and p mitogen-activated protein kinase (mapk) activation mediate ifn antiviral protection. target genes downstream of activation of these kinases are differentially expressed in the peripheral blood cells of sars patients treated with ifn alfacon- compared with patients not treated with ifn, and functionally these genes are associated with antimicrobial activity [ ] . treatment of a human bronchial epithelial cell line, calu- , with ifn alfacon- before infection with the sars-cov results in inhibition of virus infection and a reduction in overall virus yield, further supporting the idea that ifn alfacon- demonstrates antiviral activity against the sars-cov [ ] . these data demonstrate that despite the inherent ability of the sars-cov to inhibit ifn production and limit an ifn response, treatment with exogenous ifn-a overrides these inhibitory effects. these results support the further evaluation of ifn alfacon- as a first-line treatment for acute sars-cov infection and approved inhibits the function of pkr and , -oas via its dsrna-binding domain. [ ] review trends in immunology april , vol. , no. randomized clinical trial protocols are in place in the usa and canada should there be outbreaks of sars-cov. seasonal influenza a virus infections are a considerable health burden and vaccine programs are currently implemented in most developed countries. vaccines, however, are not relevant during an outbreak involving an emergent variant. the h n swine-origin influenza a virus is a prime example of how quickly a pandemic can develop given the potential for genetic shift and mutation of influenza a viruses among natural hosts. during the h n pandemic, daas such as the neuraminidase inhibitors oseltamivir and zanamivir were widely used before a vaccine became available [ ] . not surprisingly, however, daa-resistant variants of pandemic h n emerged [ , ] . avian h n influenza virus outbreaks, now affecting populations throughout asia and europe, are associated with mortality rates around % [ ] . notably, a number of h n strains are resistant to oseltamivir [ ] . to date, there have been no reported cases of human-to-human transmission of this lethal h n influenza virus infection, but if a newly emerging strain capable of human-to-human transmission appears, daa resistance will develop and until a vaccine becomes availableprobably - monthspopulations will be at risk in the absence of access to broad-spectrum antivirals. ns is the primary virulence factor encoded by influenza a viruses and it is expressed in host cells during the earliest stages of infection [ ] . in comparison with sars-cov nsp , influenza virus ns has both overlapping functions as well as unique mechanisms to inhibit the ifn response. ns acts both in the nucleus and cytoplasm of an infected cell, and is the primary antagonist of the host innate immune response. remarkably, ns has evolved to inhibit virtually all stages of the ifn response to virus infection, including inhibition of ifn production, interference with ifn signaling events, and inhibiting the function of antiviral factors induced by ifn signaling. ns inhibits the activity of rig-i (box ) where the ns dsrna-binding domain interacts directly with rig-i [ ] . within the nucleus of an infected cell, ns inhibits the processing and synthesis of host mrnas, including ifn-a/b mrnas, by binding to and inhibiting both cleavage and polyadenylation specific factor kda (cpsf ) and poly(a)-binding protein ii (pabpii), via its proteinbinding domain [ ] . the expression of avian h n ns disrupts ifn signaling events by downregulating the surface expression of one of the ifnar subunits, ifnar , and by upregulating socs protein expression, leading to a reduction in ifn-inducible stat phosphorylation and stat homo/heterodimer nuclear translocation [ ] . ns is able to block directly the antiviral activities of ifninducible antiviral proteins such as protein kinase rnaactivated (pkr) and , -oas/rnasel, via its proteinbinding domain and dsrna-binding domain, respectively [ ] . the src homology (sh )-binding domain within the protein-binding region of ns permits interaction with the internal sh domain of p b, the inhibitory subunit of phosphoinositide -kinase (pi k). this leads to activation of the pi k-akt pathway [ ] . activation of pi k, a downstream target of ifn-a/b signaling, by a specific ns promotes cell survival during the early stages of infection, illustrating the complex interplay between virus encoded factors and the ifn-a/b response [ ] . remarkably, distinct highly pathogenic respiratory viruses, namely influenza viruses and the sars-cov, encode nonstructural proteins in their genomes that function as virulence factors that specifically target the host innate ifn response, further emphasizing the importance of ifns as broad-spectrum antivirals. a recently completed randomized controlled trial has examined the safety and efficacy of recombinant ifn-a (rifn-a) treatment, administered in the form of a nasal spray, in military recruits, in the context of protection from respiratory virus infections [ ] . serum igm levels were measured as evidence of virus infection. subjects receiving rifn-a had lower concentrations of serum igm specific for h n influenza a virus, influenza b virus, adenovirus (species b), and parainfluenza virus types , and [ ] . specifically with regard to influenza a virus, only recruits treated with rifn-a had detectable levels of influenza a virus igm compared with recruits in the untreated control group [ ] . no adverse events were reported in the treatment group, the data demonstrating that ifn was well tolerated and was effective in preventing a variety of common viral respiratory infections. thus as for the sars-cov, the implications are that treatment with ifn-a can override the inhibitory effects of ns on an ifn response during influenza a virus infection. ifn therapy for highly pathogenic and oncogenic viral infections: hbv and hcv worldwide, > million people are infected with hcv, resulting in approximately deaths each year (http://www.who.int/mediacentre/factsheets/fs /en/). more than an estimated million people are chronically infected with hbv, resulting in approximately deaths each year (http://www.who.int/mediacentre/factsheets/fs /en/). hcv and hbv target the liver and cause both acute and chronic infections, resulting in liver cirrhosis and eventually, hepatocellular carcinoma [ , ] . the current approved standard-of-care treatment for hcv infection comprises daily ribavirin in combination with weekly pegylated ifn-a (peg-ifn-a) . the covalent linkage of polyethylene glycol to ifn-a increases the halflife of ifn-a in the circulation. common side effects associated with ifn therapy include a range of flu-like symptoms (fatigue, fever, myalgia), that often diminish spontaneously during the first few weeks of therapy. more severe neuropsychiatric disturbances including sleep disturbances and depressive mood changes have their onset within the first months of ifn therapy. hematological disturbances such as neutropenia or anemia may occur and are responsive to ifn dose reduction or treatment [granulocyte colony-stimulating factor (g-csf), erythropoietin (epo), respectively]. this combination ifn/ribavirin therapy has been very successful in patients infected with hcv genotypes or , and - % of patients go on to achieve an svr, characterized by undetectable hcv rna following weeks of treatment [ ] . the rate of svr falls to - % in patients infected with hcv genotypes or , trends in immunology april , vol. , no. following weeks of treatment with peg-ifn-a and ribavirin [ ] . the incomplete response to ifn treatment is partially attributable to virally encoded virulence factors that interfere with an ifn response: ns / a and ns a. ns / a is an hcv serine protease that targets mitochondrial antiviral signaling (mavs) proteins required for rig-i-mediated irf activation and subsequent ifn production [ ] . recently, the secondary structure of the hcv genotype b ns n-terminal region was identified as a predictive marker for the virological response in patients who had received ifn and ribavirin combination therapy for weeks. specifically, polymorphisms in the secondary structure of the ns amino-terminal region segregate hcv genotype b infected individuals into two groups and are predictive of the virological response to peg-ifn plus ribavirin therapy [ ] . ns a associates with intracellular membranes and its expression is vital for hcv genome replication. ns a is able to interact with ifn-inducible pkr to evade an ifn-induced antiviral response. polymorphisms in amino acid residues and in the hcv core and in ns a are also predictive markers of the virological response in patients receiving ifn and ribavirin therapy [ ] . notably, ns a is a target of ifn, because the ifn-activated gene (ifi )/interferon stimulated gene (isg ) encoding a -kda protein, promotes isgylation of ns a to enhance its degradation, thereby inhibiting hcv replication [ ] . in addition to hcv ns a, both the hcv envelope protein e and the internal ribosome entry site (ires) are able to inhibit ifn-inducible pkr activity [ ] . e contains a eukaryotic translation initiation factor (eif- a) phosphorylation homology domain through which it is able to interact with pkr, whereas the hcv ires binds to pkr, precluding dsrna binding, thereby preventing pkr activation [ ] . despite these potent inhibitory effects of hcv-encoded factors on an ifn response, clinical data provide direct evidence that peg-ifn-a treatment in combination with ribavirin is effective at limiting hcv infection and, dependent on the hcv genotype, may invoke a svr. the mechanisms by which hbv evades an innate immune response are less well understood. the hbv polymerase (pol) blocks irf signaling and subsequent ifn production by inhibiting tbk /ikke activity, associated with prr signaling [ ] . this inhibition is mediated by direct protein-protein interactions between pol and the host dead box (d-e-a-d amino acid sequence motif) rna helicase, ddx , that enhances tbk /ikke activity [ ] . peg-ifn-a is an effective treatment for hbv infection, again suggesting that ifn treatment can overcome virus-imposed inhibition of the innate immune response, specifically ifn production. peg-ifn-a is an effective treatment option for hepatitis b e core antigen (hbeag)positive disease, where detection of hbeag in the blood is indicative of viral replication. up to % of hbeag-positive patients treated with peg-ifn-a are able to develop hbeag-specific antibodies (seroconversion) by months after the end of treatment. this percentage rises to % at years after the end of treatment [ ] . in comparison to monotherapy with the daa lamivudine, which can lead to the emergence of mutant lamivudine-resistant hbv strains, peg-ifn-a alone or in combination with lamivudine is up to % more effective for inducing hbeag seroconversion, although more side effects are reported in patients receiving peg-ifn-a [ ] . in contrast to hbeag-positive disease, peg-ifn-a, alone or in combination with ribavirin, has limited effect in patients with late stage hbeag-negative disease, where the hbv mutation has resulted in loss of hbeag expression [ ] . in a randomized clinical trial, the percentage of hbeag-negative patients with hbv dna levels < copies/ml, receiving peg-ifn-a monotherapy, dropped from % to %, from the end of treatment to weeks later [ ] . the stage of viral disease can therefore affect the efficacy of ifn therapy and the timing of treatment contributes to the capacity to resolve an infection. moreover, as for influenza viruses and sars-cov, despite hbv and hcv encoding viral factors that antagonize an ifn response, exogenous ifn therapy has proven to be an effective treatment for establishing an svr. ifn therapy for highly transmissible viral infections: hsv- hsv- is a highly contagious virus, prevalent among sexually transmitted infections. hsv- is able to establish a latent infection in immunocompetent individuals by evading the immune system and is only reactivated when the host immune system is weakened [ , ] . the hsv- genome encodes a number of virulence factors, namely infected cell protein (icp) . , icp and icp , which are associated with immunoevasion and suppression of the innate immune response to virus infection [ , , , ] . specifically, icp . dephosphorylates eif- a to reverse pkr-mediated inactivation of eif- a [ ] , icp localizes to the cytoplasm and inhibits irf activity [ ] , and icp blunts the ifn-inducible jak-stat signaling pathway by inhibiting ifn-inducible stat phosphorylation and nuclear translocation [ ] . furthermore, hsv structural protein us , which has a dsrna-binding domain, disrupts the activation of the ifninducible antiviral proteins , -oas and pkr [ ] . for immunocompromised individuals infected with hsv- , viral pathogenesis can lead to serious life-threatening disease; more so in the context of emergent drug-resistant hsv- strains [ ] [ ] [ ] . different daas have been used to control hsv- infection, including acyclovir, penciclovir and foscarnet, resulting in the emergence of daa-resistant hsv- strains [ ] [ ] [ ] . ifn-g is able to exert antiviral activity by stimulating a t cell response. however, ifn-g alone may have limited efficacy in immunocompromised hsv- -infected individuals lacking a robust adaptive immune response. recent studies have shown that when immunocompromised nude mice are infected with a daa (acyclovir)-resistant hsv- variant and treated with ifn-b in combination with ifn-g, viral infection is reduced [ ] . these preliminary data are in further support of the broadspectrum antiviral activities of ifns-a/b. shifting the balance to favor the host innate immune response: the future of ifn antiviral therapy mechanisms for viral evasion of the host immune response include both the expression of many virulence factors by a review trends in immunology april , vol. , no. single virus to target different stages of the ifn response, or the expression of a single, highly specialized molecule that alone targets multiple facets of an ifn response. a priori, the widespread existence of these virally encoded virulence factors that target an ifn response highlights the critical role of a robust ifn response to limiting virus infection. the ability of ifns-a/b to target multiple types of viruses at different stages of viral replication, and the ubiquitous expression of ifn receptors on cells that are susceptible to different virus infections with different tissue tropisms, as well as the ability of ifns to activate innate immune cells and influence the adaptive immune response, emphasizes the relevance of ifns-a/b as broadspectrum antivirals. understanding the viral strategies for evasion of an ifn will permit the design of strategic ifn treatment regimens to both protect from and clear virus infections. the opportunity to limit virus infections even in the absence of characterizing the specific infecting virus, a reality during an outbreak of unknown etiology, or during a pandemic of a newly emerging or re-emerging virus strain, has profound implications for global health. indeed, early data indicate that ifn therapy may be effective in treating west nile virus [ ] , hemorrhagic yellow fever virus [ ] and ebola virus infections [ ] . moreover, short-term ifn therapy for an acute virus infection may not invoke the debilitating side effects associated with long-term ifn therapy for chronic infections such as hbv and hcv. preliminary data from pilot clinical trials of ifn treatment for sars-cov and for influenza a viruses showed this to be the case [ , ] . cognizance of the yin and yang of viruses and ifns opens the door to the widespread clinical application of these broad-spectrum antiviral ifns. type i interferons in host defense pathogen recognition by the innate immune system mechanisms of type-i-and type-ii-interferonmediated 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immunoproteasomes at the site of infection ifn-a secretion by type predendritic cells upregulates mhc class i in the hiv- -infected thymus involvement of fas-mediated apoptosis in the inhibitory effects of interferon-alpha in chronic myelogenous leukemia dynamic accumulation of plasmacytoid dendritic cells in lymph nodes is regulated by interferon-b effect of interferon-alpha( a) on neutrophil adhesion and phagocytosis in chronic myeloid leukemia and behçet's disease ifnalpha regulates nk cell cytotoxicity through stat pathway induction of interferon-gamma from natural killer cells by immunostimulatory cpg dna is mediated through plasmacytoid-dendritic-cell-produced interferon-alpha and tumour necrosis factor-alpha inhibition of the interferon-inducible protein kinase pkr by hcv e protein key: cord- -v iyur authors: abe, takayuki; marutani, yuki; shoji, ikuo title: cytosolic dna‐sensing immune response and viral infection date: - - journal: microbiol immunol doi: . / - . sha: doc_id: cord_uid: v iyur how host cells recognize many kinds of rna and dna viruses and initiate innate antiviral responses against them has not yet been fully elucidated. over the past decade, investigations into the mechanisms underlying these antiviral responses have focused extensively on immune surveillance sensors that recognize virus‐derived components (such as lipids, sugars and nucleic acids). the findings of these studies have suggested that antiviral responses are mediated by cytosolic or intracellular compartment sensors and their adaptor molecules (e.g., tlr, myeloid differentiation primary response , retinoic acid inducible gene‐i, ifn‐β promoter stimulator‐ , cyclic gmp‐amp synthase and stimulator of ifn genes axis) for the primary sensing of virus‐derived nucleic acids, leading to production of type i ifns, pro‐inflammatory cytokines and chemokines by the host cells. thus, host cells have evolved an elaborate host defense machinery to recognize and eliminate virus infections. in turn, to achieve sustained viral infection and induce pathogenesis, viruses have also evolved several counteracting strategies for achieving immune escape by targeting immune sensors, adaptor molecules, intracellular kinases and transcription factors. in this review, we discuss recent discoveries concerning the role of the cytosolic nucleic acid‐sensing immune response in viral recognition and control of viral infection. in addition, we consider the regulatory machinery of the cytosolic nucleic acid‐sensing immune response because these immune surveillance systems must be tightly regulated to prevent aberrant immune responses to self and non‐self‐nucleic acids. vertebrate cells possess prrs to enable detection of pathogens such as viruses, bacteria, fungi and parasites. immediately after a pathogen invades the cells, the prrs detect different combinations of pathogen-specific molecules, such as lipids, sugars, and nucleic acids, called pathogen-associated molecular patterns, and activate an innate antiviral response to eliminate the pathogens [ ] . among the molecules composing the pathogen-associated molecular patterns, pathogen-derived nucleic acids are the most potent mediators of innate antiviral responses. the primary induction of innate antiviral responses through detection of nucleic acids is also critical for subsequent induction of an acquired immunity response. over the past ten years, the responsible immune surveillance sensors, such as the tlrs, cytosolic rna sensors (primary rig-i/ mda ), cytosolic dna sensors (primary cgas and others), and sensors of the inflammasome pathway (e.g., the primary nlr family, which consists of two major subfamilies, nlrc and nlrp), have been well characterized [ ] [ ] [ ] . these immune surveillance sensors are ubiquitously expressed, enabling detection of pathogens invading at the cell surface and cytoplasmic or nuclear compartment in several cell types and leading to production of type i ifns, proinflammatory cytokines and chemokines by host cells. however, it has also been suggested that these immune surveillance systems must be tightly regulated to prevent aberrant immune responses to especially self-nucleic acids derived from damaged cells, senescent cells, apoptotic cells and fertilization [ , ] . indeed, rig-i, the cytosolic sensor for rna, distinguishes self from non-self rna through its interaction with ′-triphosphate or ′-diphosphate, which are not present in the transcribed rna species in vertebrate cells [ ] . both rig-i and mda are also known to sense the synthetic or viral dsrna; additionally, mda may also detect dsrna with a high molecular weight, in contrast to the preferential detection of short dsrna by rig-i [ ] . studies using genetically engineered mice have revealed that rig-i is crucial for detection of several negative-stranded rna viruses (e.g., vesicular stomatitis virus, ndv and influenza a and b virus), as well as detection of positivestranded rna viruses (e.g., jev and hcv) [ ] . in contrast, mda dominantly detects picornaviruses (e.g., encephalomyocarditis virus). though exceptions to this requirement have been reported, the ability of rig-i and mda to distinguish self from non-self in this way ensures that anomalous immune responses to cellular rna do not occur. responses to self-dna such as those described above are far less clear. cytoplasmic dna-sensing by cgas in collaboration with an adaptor protein, sting, does not distinguish between cellular and pathogen-derived dna [ , ] . of note, it has also been reported that the chronic inflammatory response induced by self-dna via cgas/sting may be responsible for induction of aberrant inflammatory diseases such as systemic lupus erythematosus, ags and polyarthritis [ , ] . while engulfed apoptotic cells represent a possible source of self-dna, endogenous dnases in both the cytoplasmic (e.g., dnase-iii, also referred to as trex ) and lysosomal (e.g., dnase-ii) compartments can degrade these endogenous self-dnas and ensure that inappropriate responses are not initiated [ , ] . in the case of the leaking of mitochondrial dna into the cytoplasm following mitochondrial damage, intracellular caspase activation can control the aberrant immune response [ ] [ ] [ ] . similarly, cgas/sting and its necessary cofactors and cellular dna are compartmentalized such that sensing of self-dna is avoided; the receptor in the cytosol and the ligand (dna) in the nucleus are sequestered [ ] . additionally, recent studies have shown that cell cycle progression during mitosis following a dna-damage response may lead to formation of micronuclei, thereby eliciting a cgas/sting-mediated micronuclear dnasensing immune response [ , ] . then, following dna damage and micronuclei formation, cgas may re-localize to the micronuclei bodies and recognize micronuclear dna, followed by initiation of downstream signal activation. in addition to the dna-damage response discussed above, two independent research groups have also reported that the cgas/sting axis also recognizes an intrinsic dna during cellular senescence and that this activation precedes induction of an inflammatory response (this is defined as the senescence-associated secretory phenotype) [ , ] . subsequently, production of senescence-associated secretory phenotype factors such as inflammatory cytokines and chemokines may reinforce senescence-associated cells via autocrine and paracrine routes. thus, remarkably, the cgas/ sting pathway appears to regulate inflammatory disorders manifested through detection of self-dna during dna damage and cellular senescence. the cgas/sting pathway may not only be important for recognition of dna virus infection, but also be critical for the host defense against rna virus infection. however, the detailed mechanism by which sting controls rna virus infection remains to be determined. of note, viruses have evolved an elaborate mechanism for escaping detection by the cgas/ sting axis or for suppressing activation of that pathway; this escape mechanism includes downstream signal activation of the cgas/sting axis. in this report, we provide an overview of recent discoveries regarding the cytosolic dna-sensing pathway, with a focus on the cgas/sting pathway and its modulation by various host factors. we also discuss the elaborate evasion strategies of certain viruses that target different steps in this signaling pathway, with a focus on recently published work concerning clinically important viruses. in contrast to the more clearly identified mechanisms of the tlr and rig-i-like receptor signaling pathways, there is still no universal agreement regarding the cytosolic dna sensors and their regulatory mechanisms [ ] . following reports that cytosolic dna-sensing tlr-independent ifn production is implicated in the transfected synthetic dsdna in murine fibroblast and immunocompetent cells [ , ] , many studies have attempted to identify the putative dna sensors that may activate the region downstream of the tbk /irf axis. accumulated evidence for involvement of these putative dna sensors is briefly described below. the first of the putative dna sensors was identified by takaoka et al., who reported that a dai (also referred to as dlm- /zbp ), which is one of the ifn-inducible genes, is involved in dsdna recognition for tbk -mediated irf activation [ ] . dai may form a signaling complex with tbk and irf for production of type i ifns in response to synthetic dsdna or dna virus infection (e.g., hsv- ) in l murine fibroblast cells. however, ishii and colleagues reported that ifn production is not impaired in response to synthetic b-form dsdna and hsv- infection in several types of cells that lack dai function; additionally, daideficient mice have a normal response to plasmid-based dna immunization [ ] . these findings indicate that the function of dai may be cell-type specific or redundantly replicate the dna-sensing innate immune response. . | critical role of the cytosolic dna-sensing innate immune response via the cgas/sting axis prior to the consecutive introduction of several dna sensors (described below), barber and colleagues introduced sting (also referred to as mita, mpys or eris), which is encoded by the tmem gene and enables activation of the ifnβ promoter. sting is a amino acid protein consisting of multiple transmembrane regions; it is localized in the er and plays an essential role in cytosolic dna-mediated innate immune responses and responses to dna-based immunization [ ] [ ] [ ] . several cytosolic dna species derived from microbial pathogens can trigger sting-dependent signal activation via either the tbk -mediated irf axis or ikksmediated nf-κb axis. in response to being stimulated by a dna ligand, sting may dynamically translocate from the er to the perinuclear-golgi region, and form a signaling complex with kinase tbk in order to induce irf activation. sting may also lead to protein degradation via a ubiquitin-mediated proteasome pathway to terminate signal activation near the perinuclear-golgi region. it has also been suggested that sting associates with dsdna directly; however, the physiological relevance such an association remains to be clarified [ ] . in a later study, it was found that sting binds directly to cdns, which are known to be bacterially derived second messenger molecules, with high affinity via formation of dimers [ ] . around the same time, it was also found that genetically engineered mice with a single point mutation in the sting gene (t a, referred to as goldenticket) failed to associate with cdns or produce type i ifn in response to bacterially produced cdns [ ] , suggesting that sting is a direct innate immune sensor for production of cdn-mediated type i ifns. in addition, several groups have determined the crystal structure of complexes formed by binding between cdns and sting via the cytoplasmic c-terminal region [ ] . through all these points of enquiry, the discovery of sting should improve our understanding of the molecular mechanisms of cytosolic dna-mediated innate immune responses; however, a cytosolic dna sensor located in the region upstream of sting may be identified in the future in a universally accepted manner. such a cytosolic dna sensor may be implicated in generation of an endogenous catalytic enzyme that enables production of cdns in response to dna pathogens for production of type i ifns. in , chen and colleagues reported a major breakthrough in this line of research. namely, they discovered cgamp, a type of cnd consisting of different phosphodiester linkages, and its catalytic enzyme (cgas), which is encoded by mb d or c orf gene [ , ] . in the cytoplasm, cgas may bind directly to viral dna derived from not only several species of dna viruses, but also from reverse-transcribed dna that is produced by retroviruses through reverse transcription of the rna genome. following binding of dna, cgas may induce conformational rearrangement and catalyze synthesis of cgamp using cellular adenosine triphosphate and guanosine- ′-triphosphate. indeed, it has been demonstrated that cgamp may induce sting-dependent signal activation and that this induction would occur via direct association of cgamp with bacterially-derived cdns [ ] . details of this signaling transduction via the cgas/sting axis are summarized in figure . of note, the cgas/sting-mediated signal transduction does not require protein-protein interactions between sensor and adaptor that characterizes pathways of other prrs (e.g., the tlr, rig-i-like receptor and inflammasome pathways); rather, it may be mediated by cgamp or cdns. another interesting point is that cgamp may be transferred from virus-infected cells to neighboring uninfected cells via gap-junction channels, thereby promoting sting activation independently of type i ifn signaling mediated by the ifn-receptor/jak-stat axis [ ] . this may be considered a novel strategy for rapid conveyance of anti-viral signals in a horizontal manner. importantly, it has been confirmed that cytosolic dnamediated innate immune responses and responses to dnabased immunization play non-redundant roles in mice, including several cgas-lacking mouse cell types. the cgas/sting pathway not only plays crucial roles in host defense against dna pathogens, but also in induction of autoimmune and inflammatory diseases through sensing of self-dna. thus, the cgas/sting pathway must be properly regulated for maintenance of cellular homeostasis and immune responses. however, details of these subjects are beyond the scope of this review; they have been reported extensively elsewhere. another dna sensor candidate gene, ifi , which is a member of the pyhin family of proteins, has also been shown to play a role in recognition of synthetic dsdna and viral dna derived from viruses that are replicated in the nucleus (e.g., hsv- , kshv, human cytomegalovirus and epstein-barr virus) [ ] . although ifi is predominantly expressed in the nucleus in the steady state, it may shuttle between the nucleus and cytosol for sensing of viral dna [ ] . in addition, ifi may reportedly be involved in both dna-sensing inflammasome activation and dna damage responses in apoptotic cells [ ] . similar to ifi , a cytosolic dna sensor aim , which may induce il- β and il- rather than type-i ifns production through the inflammasome pathway after dna sensing in the cytosol has been proposed [ ] [ ] [ ] . recently, aim -like receptors, of which there are members, all of which possess a pyrinsignaling domain and a dna-binding hin domain (e.g., p , which is seen as a mouse ortholog of human ifi ), have been shown not to contribute to dna sensing for type-i ifn production or induction of self-dna-mediated autoimmune diseases such as ags [ ] . however, most recently, the establishment of human ifi -deficient cells on the basis of macrophages and keratinocytes using crispr/cas or a talen approach indicates that production of type-i ifns the cgas/sting pathway. upon dna virus infection, the cytosolic dna sensor cgas directly recognizes viral dna and catalyzes cgamp, which utilizes cellular gtp/adenosine triphosphate, thereby triggering activation of the signal adaptor sting via direct interaction with cgamp. cgas may also recognize the transfected dsdna or viral dna that is produced by hiv- through reverse-transcription of viral rna. following the binding of cgamp, sting is translocated from the er to perinuclear-golgi and may form a signaling complex with kinase tbk (phosphorylation of sting at serine[s]- occurs here after translocation), thus inducing production of irf -mediated type i ifns. sting may also activate production of nf-κb(p )-mediated pro-inflammatory cytokines. cgas may also recognize self-dnas, such as the released nucleosome and micronuclear dna in the cytoplasm during dna damage or cellular senescence, promoting sting-dependent signal activation. missense mutation of a number of cellular dnases may induce aberrant inflammatory responses via the cgas/sting axis as a result of failure of self-dna digestion in necrotic or inappropriately apoptotic cells. p; phosphorylation and isgs in response to synthetic dsdna and hsv- infection is impaired [ , ] . interestingly, these studies also showed that ifi is a prerequisite for full-activation of cgas/sting function; however, in vivo evaluation is needed to fully clarify the physiological relevance of ifi . subsequent studies using sub-sirna libraries targeted to the members of dexd/h helicase have identified helicase ddx as a putative dna sensor and also shown that ddx is involved in dna recognition and regulation of dna virus infection in immunocompetent rather than epithelial cells [ ] . surprisingly, ddx has also been shown to be involved in recognition of bacterial-derived second messenger molecules (such as cdns, which include cyclic di-amp and cyclic di-gmp) for type-i ifn production [ ] . crystal structure analyses have revealed that the binding regions for dsdna and cdns overlap, suggesting that ddx has the potential to recognize different ligands via the dead domain [ ] . of note, it has been proposed that ifi and ddx , but not dai, function as upstream molecules of sting through their physical interactions. it has been suggested that ddx plays a role in cytosolic dna-mediated immune response in collaboration with the adaptor sting; however, a detailed investigation using genetically engineered mice is needed to clarify the physiological relevance of sting for ddx . there may be a correlation between the dna damage response and innate immune responses mediated by virus infection; however, stetson et al. have reported conflicting findings [ ] . most recently, it has been suggested that several dna damage-inducible host factors, such as the catalytic subunit of dna-pkcs, and its binding co-factors ku / , or mre , are involved in the cytosolic dnasensing immune response through direct interaction with dna ligands [ , ] . upon simulation with synthetic dna ligands or infection with dna virus, only partial redundancy of isg expression was observed in dna-pkcsdeficient murine cells, suggesting a high potential for a cytosolic dna-sensing innate immune response. however, it is interesting to note that mre may be specifically involved in the response to synthetic dsdna, but not involved in the response to dna virus infection or to treatment with dmxaa, which is known as a sting specific agonist. additionally, these observations indicate that type-i ifns are produced in response to stimulation with dna damage-inducing chemical agents (e.g., etoposide or cisplatin) [ ] . recent studies using dna damage-inducing agents such as , -dimethylbenz-α-anthracene have suggested that the following pathway underlies the dna damage-induced immune response via the cytosolic dnasensing pathway: , -dimethylbenz-α-anthracene-induced dna damage results in nucleosome leakage into the cytosol and then elicits cgas/sting-dependent signal activation via self-dna recognition [ ] . it has been proposed that, in addition to direct sensing of dna, the indirect machinery of the cytosolic dna-sensing innate immune response is also involved. the leucine-rich repeat flightless-interacting protein has been shown to function as an amplifier of cytosolic nucleic acid-sensing immune responses for production of type-i ifns via the transcriptional co-activator β-catenin but not via irf activation [ ] . thus, accumulating evidence indicates that various genes function in cytosolic nucleic acid-sensing immune responses in a ligand-specific or cell type-specific manner. taken together, these results show that, although the functional relevance of some of the dna sensor candidates (e.g., ddx , ifi , dna-pkcs, mre ) still needs to be fully investigated, there is consensus that cgas and sting are bona fide cytosolic dna-mediated regulators. recent investigations have demonstrated that intracellular ptm systems, particularly phosphorylation and ubiquitination, participate in the cgas/sting pathways that positively or negatively modulate enzymatic activity, subcellular distribution, protein stabilization and degradation, conformational rearrangement and signal transduction. here, we provide an update on the role of ptms in regulation of the cgas/sting pathway (table ). it has been shown that, upon dna ligand stimulation, sting may be phosphorylated at amino acid position serine- (s ) as a primary acceptor site of phosphorylation through several intracellular kinases [ , ] . the kinase tbk was the first to be proposed to be involved in positive regulation of sting phosphorylation to promote irf activation. a subsequent study showed that the autophagy-related serine/ threonine protein kinases ulk and ulk are involved in the process of sting phosphorylation that enables termination of signal activation. these results indicate that these distinct kinases, tbk and ulk / , may possess opposite functions via the same residue s for sting phosphorylation. further studies using genetically engineered mice are needed to resolve these conflicting observations and determine the physiological relevance of ulk / . the presence of an additional residue at position s of human sting also has the potential to affect sting phosphorylation; however, the impact of this phosphorylation remains to be clarified [ ] . most recently, it was shown that ribosomal protein s k may also participate in the positive regulation of stingdependent signal activation; however, the kinase activity of s k has been found to be dispensable [ ] . in contrast to the observation of sting-mediated phosphorylation, only one intracellular kinase that is involved in modulation of cgas function has been reported. a dna virus (like hsv- )inducible akt (also referred to as protein kinase b) may be involved in phosphorylation of cgas at s (at s in murine cgas) within the enzymatic domain of cgas, thereby suppressing the enzymatic activity of cgas [ ] . this may be one of the strategies by which hsv- escapes the dna-sensing immune response and achieves sustained infection. further studies are needed to determine whether there is an intracellular phosphorylation kinase that can positively regulate cgas function. attempts to determine the detailed mechanism of cgas/ sting function have indicated that sting may also accept an intracellular ubiquitination process at multiple lysine (k) residues through recruitment of distinct ubiquitin e ligases. first, it has been proposed that several members of the trim family of ring e ligases, such as trim and trim , play a role in sting ubiquitination via conjugation of k linked poly-ubiquitination in a manner that positively impacts sting function [ , ] . conversely, in addition to trim and trim α, e ubiquitin ligase rnf may induce degradation of sting via conjugation of k -linked polyubiquitination for termination of signal activation [ ] [ ] [ ] . additionally, each of two distinct molecules-the amfrinsulin-induced gene (gp /insig ) e ubiquitin ligase complex and mitochondrial e ubiquitin protein ligase have also been shown to be involved in sting ubiquitination via conjugation of k -and k -linked poly-ubiquitination, respectively, to facilitate sting-dependent signal activation [ , ] . most recently, it has been reported that rnf may catalyze sting ubiquitination via conjugation of k linked poly-ubiquitination at the same conjugating site of rnf . this may be considered to contribute positively to sting function through competition with rnf -mediated this table summarizes the various kinases, phosphatase, ubiquitin ligases and de-ubiquitin ligases that modulate cgas/sting-dependent signal activation. these enzymes may be catalyzed or remove specific amino acid residues or specific linkage types of ubiquitin to the target substrate. irhom may recruit eif s , which acts as a deubiquitin ligase to remove k -linked poly-ubiquitin from sting. poly-ubiquitination of sting [ ] . in contrast to the accumulated evidence regarding sting ubiquitination, little is known about cgas function. the e ubiquitin ligase rnf may be involved in cgas ubiquitination via conjugation of k -linked poly-ubiquitination for promotion of an enzymatic activity of cgas [ ] . moreover, it has been shown that trim may also play a role in cgas-mediated mono-ubiquitination for facilitation of cgamp production [ ] . thus far, however, an intracellular e ubiquitin ligase that negatively regulates cgas function has not yet been identified. given the involvement of multiple e ubiquitin ligases in modulation of the cgas/sting pathway, further investigations are needed to clarify the elaborate interactions. the ptms system must be reversible in order to alternatively regulate prr-mediated signal transduction via de-ubiquitination or de-phosphorylation. for instance, ppm a has been shown to be involved in de-phosphorylation of s of sting to prevent sting-dependent signal activation [ ] , whereas the kinase that is responsible for dephosphorylation of s has not yet been identified. recently, it was reported that irhom may facilitate deconjugation of rnf -mediated k -linked poly-ubiquitination of sting through recruitment of a de-ubiquitination enzyme, eif s [ ] . another possible ptm-independent function of irhom is regulation of sting translocation through recruitment of the er translocon-associated protein trapβ [ ] . on the other hand, it has also been shown that some types of usp are involved in modulation of sting function. usp and usp may facilitate de-conjugation of rnf -mediated k -linked poly-ubiquitination of sting in addition to k -linked poly-ubiquitination [ ] . usp has also been shown to act as a de-conjugated enzyme to stingmediated k -linked and k -linked poly-ubiquitination [ ] . although the impact of k -linked poly-ubiquitination of sting has not yet been determined, distinct usps may manipulate sting function via de-conjugation at multiple lysine residues. while the ptms that control cgas/sting function are yet to be fully defined (e.g., the physiological role of noncanonical polyubiquitin linkage types such as k and k on sting ubiquitination), elucidation of the mechanism underlying this signal modification could help to establish a potent therapeutic approach against auto-inflammatory diseases that are mediated by the cgas/sting axis. upon dna virus infection, host cells may initiate induction of various effector anti-viral genes, such as type-i ifns, isgs and pro-inflammatory cytokines or chemokines, through the cgas/sting pathway. however, it has been widely reported that a number of viruses are equipped with mechanisms for counteracting the cgas/sting pathway during both acute and persistent viral infection and that these activities may be the means by which these viruses escape the host immune surveillance system. thus far, many different immune evasion strategies employed by various viruses have been identified, including: (i) interference with the functions of the host innate immune response via physical interactions with viral antagonistic proteins targeted to sensors, adaptors, related intracellular kinases and transcription factors; (ii) inducing degradation or specific cleavage at the protein level; and (iii) sequestration of signal transduction molecules targeting the ptm systems. here, we summarize these strategies with a focus on recently published studies ( figure ). chronic hbv infection is a major cause of chronic liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma. a recent study has shown that the hbv polymerase (pol) gene prevents sting-dependent signal activation by blocking sting-mediated k -linked polyubiquitination, leading to chronic persistent infection of hepatocytes by hbv [ ] . additionally, packaging of relaxed-circular hbv dna into the viral capsid protein has been shown to block direct recognition by the cgasmediated sensing process; this was recently reported to be an alternative evasion strategy of hbv [ ] . it has also been suggested that hepatocytes do not produce type-i ifns in response to synthetic dsdna or hbv infection because hepatocytes lack the cgas/sting pathway [ , ] . this may also be considered a reasonable explanation for hbv's ability to specifically adapt in hepatocyte cells and may contribute to these cells' weak capacity to eliminate hbv infection. further investigation of the interaction between hbv infection and sting-dependent signal activation in hepatocytes is necessary. members of the herpesvirus family are widely used as ligands for induction of dna-mediated immune responses both in vitro and in vivo and there is accumulated evidence that these viruses have antagonistic effects on the cgas/ sting pathway. the hsv- -encoded tegument protein ul has been shown to induce cgas degradation via the proteasomal pathway; additionally, hsv- immediate early protein icp may affect the stability and function of sting in certain cell types [ , ] . other tegument proteins such as ul reportedly interfere with the function of cgampmediated sting by physically interacting with sting and its downstream kinase tbk [ ] . hsv- icp , an immediate early protein that is conserved in all herpesviruses, may also interact with sting and tbk , thus interfering with sting-dependent signal activation [ ] . hsv- vp , which is encoded by the ul gene, may also possess an antagonistic function against the cytosolic dna-sensing immune response through modulation of the enzymatic activity of cgas [ ] . deamidation impairs the ability of cgas to catalyze cgamp synthesis; additionally, it has been shown that hsv- ul tegument protein may promote cgas deamidation, thus attenuating cgamp-mediated anti-viral activity [ ] . recent studies have demonstrated that hsv- may target transcription factors that are located downstream of the sting/tbk axis as part of its immune evasion strategy. in addition to the viral serine protease of hsv- , vp , some viral tegument proteins, such as ul and ul , have evolved certain strategies to target irf and nf-κb by negatively regulating them [ ] [ ] [ ] . interestingly, it has been demonstrated that the γ . gene of hsv- encodes a virulence factor for hsv- mediated pathogenesis that may also act as an antagonistic factor against the cgas/sting pathway [ ] , which is consistent with the finding that an hsv- mutant in which the γ . gene has been deleted no longer exhibits an antagonistic function in infected cells, thereby facilitating ifn production in a sting-dependent manner [ ] . on the other hand, an oncogenic herpesvirus, khsv, which encodes viral interferon regulatory factor gene, has been shown to prevent an association between sting and tbk , thereby inhibiting initiation of irf -mediated signal activation [ ] . the authors of this paper also identified five other kshv-encoded proteins that can suppress sting-dependent signal activation. the lana of kshv has been shown to play a pivotal role in viral replication. zhang and colleagues have also reported that n-terminal truncated cytoplasmic isoforms of lana may associate with cgas directly, thus interfering with cgas-dependent signal activation [ ] . moreover, mhv encoding dub enzyme orf (also referred to as a kshv orf homolog) has also been shown to suppress sting-dependent signal activation [ ] . the mechanism responsible for this involves a dna-sensing process in a dub activity-dependent manner. additionally, a kshv-encoded tegument protein, orf , has also been shown to antagonize cgas function through its direct association with both cgas and viral dna [ ] . furthermore, the authors of this paper showed that, in addition to mhv , the homologs of orf genes derived from epstein-barr virus and rhesus monkey rhadinovirus exhibit similar inhibitory functions targeting cgas, suggesting that the antagonistic function of gamma herpesviruses is evolutionally conserved. overall, these results indicated that herpesviruses may modulate the cytosolic dna-mediated immune response via an elaborate mechanism that involves viral encoding of several antagonistic genes. moreover, a viral oncoprotein containing the lxcxe motif, which is also conserved among a small number of dna tumor viruses, was recently shown to suppress the cytosolic dna-sensing immune response. for instance, viral encoding oncoproteins such as e and e a derived from human papillomavirus and human adenovirus type- suppress the cytosolic dna-sensing immune response through direct association with sting [ ] . interestingly, it has been shown that lxcxe-containing tumor proteins are also expressed in many types of immortalized cells and permanently impair the cytosolic dna-sensing immune response via cgas/sting. this may be a component of the cytotoxic mechanism of the viral oncoprotein that exhibits lxcxe-mediated antagonism of the sting pathway. although the role of cgas/sting in recognition and counteraction of dna viruses has been well described, as detailed above, recent studies have also reported on a number of rna viruses, particularly positive-stranded rna viruses, that are targeted by this pathway. the non-structural protein b (ns b) of yfv, which belongs to the flaviviridae family, was first reported as a viral protein that interacts with sting. analysis of the sequence alignment revealed that sting possesses a highly structural homology domain with ns b of denv and hcv in addition to yfv [ ] . subsequent studies have found that hcv ns b suppresses sting-dependent signal activation via its direct interaction with sting near the viral replication complex on the er [ ] [ ] [ ] . although details of the mechanism of the ns b-mediated counteraction of sting are not fully understood, it is necessary to obtain functional evidence of the cgas/sting pathway in hepatocytes, as described above for hbv infection. another elegant viral immune evasion strategy is disruption of dsrna-mediated innate immune responses via proteolysis of mitochondrial adaptor ips- (also known as mavs, visa and cardif) by hcv ns / a serine protease [ ] . some rna viruses that encode viral proteases (e.g., hepatitis a virus c protease, enterovirus a protease, rhinovirus a and c protease and coxsackievirus ( ) . (b) enveloped rna viruses such as iav, ndv and sendai virus activate ifn production through a viral envelope-mediated fusion process in a sting-dependent but cgasindependent manner; however, the molecular mechanism of signal transduction is yet to be clarified ( ) . as shown in figure , the hemagglutinin fusion peptide of iav may also associate with sting via its dimerization interphase domain, thereby inhibiting stingdependent signal activation. abbreviations: fp, fusion peptide b c protease) can directly cleave ips- by means of this strategy [ ] . with regard to the cgas/sting pathway, ns b/ns protease of denv was initially found to have a similar inhibitory evasion mechanism via proteolysis of human, but not murine, sting [ , ] , which is consistent with the observation that denv replication is restricted in mice because denv protease cannot cleave murine sting. most recently, this conserved strategy for cleaving sting was also observed in other flavivirus ns b/ns proteases derived from zika virus, west nile virus and jev, but not yfv [ ] . accordingly, there may be a non-proteolytic mechanism for counteracting sting function by yfvencoded viral protease, as described above. it has also been proposed that the zika virus may possess an alternative strategy for immune escape. zika virus-encoded ns protein has been shown to promote proteolysis of cgas rather than sting through recruitment of caspase- activation, suggesting that zika virus infection manipulates the interplay between the inflammasome and cgas/sting pathways [ ] . these results indicate that the flaviviridae family has evolved the mechanism of targeting distinct signal adaptor molecules of cytosolic nucleic acid sensors to escape from innate immune responses in humans. some rna viruses have also evolved a mechanism for evading innate immune responses that targets the ptm systems involved in the cgas/sting pathway. virusencoded dub enzymes, such as papain-like proteases derived from human coronavirus-nl , severe acute respiratory syndrome cov and porcine epidemic diarrhea virus, have also been shown to suppress sting-dependent signal activation in a manner similar to that of the hbv polymerase gene [ ] [ ] [ ] . similarly, the tax protein of human t lymphotropic virus type- , which plays a critical role in promoting viral replication and t cell transformation, has also been shown to interfere with sting-mediated k -linked poly-ubiquitination via physical interaction [ ] . as described in the section above, conjugation of k -linked polyubiquitination has been shown to be involved in positive regulation of prr-mediated signal activation. thus, one strategy for viral-mediated immune evasion could be to counteract sting-mediated k -linked poly-ubiquitination. counteraction of the cgas/sting pathway by rna viruses classified as positive-sense rna viruses has been reasonably well elucidated; however, much less is known about the actions of negative-sense rna viruses. iav belongs to the orthomyxoviridae family, the members of which possess negative-sense rna. it was recently reported that iav elicits ifn production through a viral envelopemediated fusion process in a sting-dependent, but cgasindependent, manner [ ] . similarly, other enveloped rna viruses, such as ndv and sendai virus also play the same roles in inducing innate immune response; however, the molecular mechanism of signal transduction is yet to be clarified ( figure ) . interestingly, it has been shown that the hemagglutinin fusion peptide of iav may associate with sting via its dimerization interphase domain, thereby inhibiting the sting-mediated dimer formation that initiates sting-dependent signal activation. thus, this is the first evidence for a negative-sense rna virus exerting an agonistic effect on sting. in contrast to the well-studied viral escape strategies that directly target the functioning of sting, less is known about the function of cgas during rna virus infection. it has recently been reported that the viral capsid proteins of hiv- and hiv- may suppress the cgas-mediated dna-sensing process by recruiting host factors for hiv replication, such as cofactors cleavage and polyadenylation specificity factor subunit and cyclophilin-a, respectively [ , ] . more specifically, mutated hiv capsids in which cyclophilin-a association is impaired can be stimulated by cgas-mediated immune activation, suggesting that an intact hiv capsid is a determinant factor for the immune evasion strategy of hiv- . despite the fact that the counterpart to sting functions via proteolysis by denv protease, it has been shown that release of mitochondrial dna via induction of damage to denv-infected cells may have the potential to stimulate a cgas/sting-mediated immune response ( figure ) . to avoid such signal activation, an ns b protease co-factor derived from denv may directly target cgas, thus leading to proteasomal degradation [ ] . it remains unclear whether species-specific effects are involved in this ns b protease co-factor-dependent immune evasion strategy, for example, proteolysis of human, but not murine, sting in a proteasedependent manner. the past decade has seen a rapid advance in our understanding of the cytosolic dna-sensing pathways, especially the cgas/sting axis, including their regulatory mechanisms and development of diseases associated with them, such as systemic lupus erythematosus, ags and polyarthritis. it remains to be determined whether other types of inflammatory diseases may also be associated with defects in cgas/ sting function (e.g., mediated by gain-or loss-of-function mutations). thus, an improved understanding of the mechanisms underlying overactivity of inflammation mediated via the cgas/sting pathway could lead to design of potent therapeutic agents and strategies for overcoming undesirable inflammatory diseases and certain types of cancer. in addition, although the cgas/sting pathway may contribute to detection and elimination of infection by both dna and rna viruses, over time these viruses have also evolved the ability to escape or manipulate this signal activation through several evasion strategies, as described above. indeed, such manipulation is considered critical to the ability of viruses to establish both lytic and persistent infection and elicit characteristic pathogenesis. further information on the non-canonical mechanism of cgas-independent sting activation and the evasion strategies of rna viruses will strongly facilitate understanding of the host-pathogen interaction. the cgas/sting pathway may function in a variety of cell types; however, details of the mechanisms by which hepatotropic viruses achieve persistent infection of hepatocytes have yet to be elucidated. similarly, an understanding of virus-mediated immune evasion strategies could provide novel insights into viral evolution and the potential design of novel anti-viral agents. finally, along with the accumulating insights regarding the cgas/sting pathway and its involvement in virus-host interactions, it is also important to better understand the loss of cgas/sting function in some types of cancer or transformed cells by viral encoding oncoprotein. this loss may involve signal manipulation via epigenetic silencing and insertion of missense mutations into the cgas/ sting locus. further elucidation of this point may provide important information for the treatment of several viral oncogeneses. we apologize to the authors whose research is relevant to this topic but not cited because of space limitations. we thank the members our laboratory at kobe university for their helpful input. we are also grateful to dr. glen n. barber (university of miami, miami, fl, usa) and dr. sagi d. shapira (columbia university, new york, ny, usa) for providing assistance in association with their related research projects. this work was supported by grants to the daiichi sankyo foundation of life science (t.a.) the authors declare that they have no conflicts of interest regarding this work. innate immune sensing and signaling of cytosolic nucleic acids pathogen recognition by the innate immune system newly described pattern recognition receptors team up against intracellular pathogens multi-receptor detection of individual bacterial products by the innate immune system cgas conducts micronuclei dna surveillance germ-cell-specific inflammsome component nlrp negatively regulates cytosolic nucleic acid sensing to promote fertilization length-dependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiation-associated gene differential roles of mda and rig-i helicases in the recognition of rna viruses sting: infection, inflammation and cancer intracellular nucleic acid detection in autoimmunity apoptotic caspases suppress mtdna-induced sting-mediated type i ifn production apoptotic caspases prevent the induction of type i interferons by mitochondrial dna bak/bax macropores facilitate mitochondrial herniation and mtdna efflux during apoptosis cgas surveillance of micronuclei links genome instability to innate immunity mitotic progression following dna damage enables pattern recognition within micronuclei cgas is essential for cellular senescence innate immune sensing of cytosolic chromatin fragments through cgas promotes senescence the interferon response to intracellular dna: why so many receptors? a toll-like receptor-independent antiviral response induced by double-stranded b-form dna recognition of cytosolic dna activates an irf -dependent innate immune response dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response tank-binding kinase- delineates innate and adaptive immune responses to dna vaccines sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity sting-dependent cytosolic dna sensing pathways sting recognition of cytoplasmic dna instigates cellular defense sting is a direct innate immune sensor of cyclic di-gmp the n-ethyl-nnitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides cyclic dinucleotides and the innate immune response cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp ifi is an innate immune sensor for intracellular dna a member of the pyrin family, ifi , is a novel brca -associated protein involved in the p -mediated apoptosis pathway aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc aim activates the inflammasome and cell death in response to cytoplasmic dna an orthogonal proteomic-genomic screen identifies aim as a cytoplasmic dna sensor for the inflammasome the aim -like receptors are dispensable for the interferon response to intracellular dna ifi is required for dna sensing in human macrophages by promoting production and function of cgamp ifi and cgas cooperate in the activation of sting during dna sensing in human keratinocytes the helicase ddx senses intracellular dna mediated by the adaptor sting in dendritic cells the helicase ddx recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response structural and functional analysis of ddx : a bispecific immune receptor for dna and cyclic dinucleotide dna-pk is a dna sensor for irf- -dependent innate immunity dna damage sensor mre recognizes cytosolic double-stranded dna and induces type i interferon by regulating sting trafficking the dna damage response induces ifn the cytosolic nucleic acid sensor lrrfip mediates the production of type i interferon via a betacatenin-dependent pathway sting specifies irf phosphorylation by tbk in the cytosolic dna signaling pathway cyclic dinucleotides trigger ulk (atg ) phosphorylation of sting to prevent sustained innate immune signaling the adaptor protein mita links virus-sensing receptors to irf transcription factor activation s k-sting interaction regulates cytosolic dna-mediated activation of the transcription factor i rf akt kinase-mediated checkpoint of cgas dna sensing pathway the ubiquitin ligase trim regulates innate immune responses to intracellular doublestranded dna trim protein modulates type i interferon induction and cellular antiviral response by targeting mita/sting protein for k -linked ubiquitination the ubiquitin ligase rnf regulates antiviral responses by mediating degradation of the adaptor protein mita trim alpha is a negativefeedback regulator of the intracellular dna and dna virustriggered response by targeting sting trim promotes dna virus infections by inhibiting innate immune response the e ubiquitin ligase amfr and insig bridge the activation of tbk kinase by modifying the adaptor sting ubiquitination of sting at lysine controls irf activation rnf temporally regulates virus-triggered type i interferon induction by two distinct mechanisms the e ubiquitin ligase rnf facilitates the cgas-mediated innate immune response trim -mediated monoubiquitination of cgas for cytosolic dna sensing ppm a regulates antiviral signaling by antagonizing tbk -mediated sting phosphorylation and aggregation irhom is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting usp recruits usp to promote innate antiviral response through deubiquitinating sting/mita usp negatively regulates antiviral responses by deubiquitinating sting hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dna-sensing pathways hepatitis b virus evasion from cyclic guanosine monophosphate-adenosine monophosphate synthase sensing in human hepatocytes lack of immunological dna sensing in hepatocytes facilitates hepatitis b virus infection hbv bypasses the innate immune response and does not protect hcv from antiviral activity of interferon herpes simplex virus abrogates the cgas/ sting-mediated cytosolic dna-sensing pathway via its virion host shutoff protein, ul hsv- degrades, stabilizes, requires, or is stung by sting depending on icp , the us protein kinase, and cell derivation evasion of the sting dnasensing pathway by vp / of herpes simplex virus- hsv- icp targets the tbk -activated sting signalsome to inhibit virusinduced type i ifn expression herpes simplex virus tegument protein vp abrogates cgas/sting-mediated antiviral innate immunity species-specific deamidation of cgas by herpes simplex virus ul protein facilitates viral replication herpes simplex virus serine protease vp blocks the dna-sensing signal pathway by abrogating activation of interferon regulatory factor herpes simplex virus ul abrogates the dna sensing signal pathway by inhibiting nf-kappab activation herpes simplex virus ubiquitinspecific protease ul abrogates nf-kappab activation in dna sensing signal pathway herpes simplex virus g . protein inhibits sting activation that restricts viral replication modulation of the cgas-sting dna sensing pathway by gammaherpesviruses cytoplasmic isoforms of kaposi sarcoma herpesvirus lana recruit and antagonize the innate immune dna sensor cgas evasion of innate cytosolic dna sensing by a gammaherpesvirus facilitates establishment of latent infection inhibition of cgas dna sensing by a herpesvirus virion protein dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway hepatitis c virus ns b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis c virus ns b blocks the interaction of sting and tbk to evade host innate immunity viral evasion of intracellular dna and rna sensing denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity species-specific disruption of sting-dependent antiviral cellular defenses by the zika virus ns b protease zika virus elicits inflammation to evade antiviral response by cleaving cgas via ns -caspase- axis coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex htlv- tax impairs k -linked ubiquitination of sting to evade host innate immunity influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses hiv- evades innate immune recognition through specific cofactor recruitment the capsids of hiv- and hiv- determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells dengue virus ns b protein targets cgas for degradation and prevents mitochondrial dna sensing during infection cytosolic dna-sensing immune response and viral infection key: cord- -ddau fu authors: ciottone, gregory r. title: introduction to disaster medicine date: - - journal: ciottone's disaster medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ddau fu nan c h a p t e r introduction to disaster medicine what exactly is disaster medicine? if you have decided to purchase and read this book, it is likely a question you have wrestled with. disaster itself is not an easily defined entity, thus the new medical specialty evolving around it is continuously undergoing metamorphosis. because a disaster is a local event, throughout history, the local medical responders have cared for the victims of disaster. the same medical personnel who provide health care on a daily basis also assume the responsibility of providing care to patients with illness or injury resulting from a disaster. unlike other areas of medicine, however, the care of casualties from a disaster requires the health care provider to integrate into the larger, predominantly nonmedical multidisciplinary response. this demands a knowledge base far greater than medicine alone. to operate safely as part of a coordinated disaster response, either in a hospital or in the field, an understanding of the basic principles of emergency management is necessary. now we begin to see the evolution of the specialty of disaster medicine. to respond properly and efficiently to disasters, all health care personnel should have a fundamental understanding of the principles of disaster medicine (which incorporates emergency management in its practice) and what their particular role would be in the response to the many different types of disasters. in the mid- s, disaster medicine began to evolve from the union of disaster management (now called emergency management) and emergency medicine. although disaster medicine is not yet an accredited medical subspecialty, those who practice it have been involved in some of the most catastrophic events in human history. practitioners of presentday disaster medicine have responded to the aftermaths of the tsunami in southeast asia, hurricane andrew, the haiti earthquake, the madrid train bombings, and the world trade center attacks, to name a few. during the past several decades, we have seen the first applications of basic disaster medicine principles in real-time events, and as demonstrated by the devastation caused by hurricane sandy in and the devastating ebola outbreak of - , there is sure to be continued need for such applications. the impetus for this text grew from a realization that as the specialty of emergency medicine grows, emergency physicians must take ownership of this new field of disaster medicine and ensure that it meets the rigorous demands put upon it by the very nature of human disaster. if we are to call ourselves disaster medicine specialists and are to be entrusted by society to respond to the most catastrophic human events, it is imperative that we pursue the highest level of scholarly knowledge and moral conduct in this very dynamic area. until there is oversight from a certifying board, it is our responsibility to the public to maintain this high level of excellence. as in medical ethics, where patients rely on the virtue of their physicians to compel them to abide by moral standards, so must we exercise virtue in how we conduct the medical response to disaster. because disasters strike without warning, in areas often unprepared for such events, it is essential for all emergency services personnel to have a foundation in the practical aspects of disaster preparedness and response. the first step is to understand that disaster can strike here at home. i can assure you the people of haiti minutes before the earthquake of and the people of japan minutes before the earthquake and tsunami of all were going about their normal daily routine, not expecting disaster to strike. then it did. as is discussed in other chapters throughout this text, emergency responders have an integrated role in disaster management. all disasters follow a cyclical pattern known as the disaster cycle ( figure - ) , which describes four reactionary stages: preparedness, response, recovery, and mitigation or prevention. emergency medicine specialists have a role in each part of this cycle. as active members of their community, emergency specialists should take part in mitigation and preparedness on the hospital, local, and regional levels. once disaster strikes, their role continues in the response and recovery phases. by participating in the varied areas of disaster preparation and response, including hazard vulnerability analyses, resource allocation, and creation of disaster legislation, the emergency medicine specialist integrates into the disaster cycle as an active participant. possessing a thorough understanding of the disaster medicine needs of the community allows one to contribute to the overall preparedness and response mission. over the course of recorded history, natural disasters have predominated in frequency and magnitude over human-made ones. some of the earliest disasters have caused enormous numbers of casualties, with resultant disruption of the underlying community infrastructure. yersinia pestis caused the death of countless millions in several epidemics over hundreds of years. the etiologic agent of bubonic plague, y. pestis, devastated europe by killing large numbers of people and leaving societal ruin in its wake. during the writing of this chapter, an ebola outbreak raged in west africa, along with concern that a worldwide pandemic might ensue. the and ebola and middle east respiratory syndrome (mers) outbreaks have proven that, despite the passage of time and the great advances in medicine, the world continues to be affected by disease outbreaks. in addition, diseases that have been eradicated have the potential of being reintroduced into society, either accidentally from the few remaining sources in existence around the world, as in the measles outbreak in the united states, or by intentional release. such events have the potential of devastating results, as the baseline intrinsic immunity the world population developed during the natural presence of the disease has faded over time, putting much larger numbers of people at risk. finally, with the advent of air travel allowing people to be on the opposite side of the world in a matter of hours, the bloom effect of an outbreak is much harder to predict and control. disease outbreaks that were previously controlled by natural borders, such as oceans, no longer have those barriers, making the likelihood of worldwide outbreak much greater now than it was hundreds of years ago. we saw evidence of this in , with ebola-infected patients arriving in spain and the united states from west africa. during that outbreak, naysayers to intrusive actions such as quarantine and travel restrictions cited following the "science" learned since the disease emerged in central africa in the s. the problem with such logic was that ebola had never before been seen in urban settings such as nigeria, new york city, and dallas, texas. transmission parameters in such settings were truly uncharted waters for the medical community. in addition to epidemics, with each passing year, natural disasters in the form of earthquakes, floods, and deadly storms batter populations. one need only to remember the destruction in terms of both human life and community resources caused by the indian ocean earthquake and tsunami of , the haiti earthquake in , or the earthquake, tsunami, and radiation disaster in japan in to understand the need for preparedness and response to such natural events. considering that the earthquakes that caused these tsunamis occurred hours before the devastation, it is difficult to understand how today's advanced society, able to travel far into space among other great achievements, was unable to mitigate against some of the most deadly natural events in recent history. the realization that disaster can strike without warning and inflict casualties on the order of the , , and earthquakes and tsunamis, despite our many technological advances, serves as a warning that mitigation, preparedness, response, and recovery to natural disaster must continue to be studied and practiced vigorously. today, the possibility of terrorist attack threatens populations across the globe. both industrialized and developing countries have witnessed some of the most callous and senseless taking of life, for reasons not easily fathomed by civilized people. it is unusual to read an internet news article or watch a television newscast without learning of a terrorist attack in some part of the world. with the advent of more organized groups such as the islamic state of iraq and syria (isis), boko haram, the revolutionary armed forces of colombia (farc), and the epanastatikos agonas (ea), these attacks are more frequent and deadly, often using horrifying means of execution. the commonplace nature of a terrorist attack in modern society ensures it is unquestionably something that will continue long into the future, and will very likely escalate in scale and frequency. the multilayered foundation on which ideological belief evolves into violent attack is beyond the scope of analysis that this book ventures to undertake. these ongoing events do demonstrate, however, that the principles studied in the field of disaster medicine must include those that are designed to prepare for and respond to a terrorist attack. because there are very intelligent minds at work designing systems to bring disaster on others, equally there must be as robust an effort to prepare for and respond to those disasters. such response involves the deployment of law enforcement, evidence collection, and military personnel and equipment, which are typically not seen in the response to a natural disaster. the integration of these unique assets into the overall response is essential for the success of the mission. the disaster medicine specialist must have a thorough understanding of the role of each. a thorough discussion of disaster preparedness and response must be predicated on a clear definition of what, in fact, constitutes a disaster. used commonly to describe many different events, the word disaster is not easily defined. the indian ocean tsunami in and the haiti earthquake in , each killing significantly more than , people, would certainly meet the criteria for disaster. however, the flood in peru that killed people and tropical storm in madagascar that killed have also been called disasters. likewise, the explosion of the space shuttle columbia on reentry into earth's atmosphere that killed the crew of seven astronauts onboard has often been referred to as the columbia disaster in the lay press. how can an event resulting in the loss of seven people be placed in the same category as one that kills hundreds of thousands? herein lies the paradox of disaster. what is it? who defines it, and by what criteria? it is difficult to dispute that an event causing thousands of casualties should be considered a disaster, but let us analyze why that is the case. what is it about the sheer number of dead and injured that allows the event to be called a disaster? in terms of medical needs, it is simply because there is no health care system on earth that can handle that number of casualties. therefore an event of such magnitude is a disaster because it has overwhelmed the infrastructure of the community in which it occurred. following this logic, we can then also make the statement that any event that overwhelms existing societal systems is a disaster. this definition is close to the definition of disaster given by the united nations international strategy for disaster reduction (unisdr) : a serious disruption of the functioning of a community or a society involving widespread human, material, economic, or environmental losses and impacts, which exceeds the ability of the affected community or society to cope using its own resources. a similar definition is used by the world health organization (who). by applying these definitions, one can understand how an event in a rural area with to casualties may also be considered a disaster because the limited resources in that area may prevent an adequate response without outside assistance. the widely accepted unisdr and who definitions of disaster justify describing both the haiti earthquake and the flood in peru as disasters. however, what about the destruction of the space shuttle columbia on reentry? clearly, this definition does not allow one to justify the use of disaster in describing that horrific accident, which brings to light a discrepancy in how disaster specialists and the public term events. the columbia accident, as an example, does not meet any accepted criteria of disaster. it was, however, an exceedingly tragic event, seen by millions on television, as it was unfolding. it was tragic by the word's very definition in the cambridge dictionary: "a very sad event, especially one involving death or suffering." public perception of such events may cause this misnomer, with a tragic incident being termed a disaster. much like disaster, tragedy can also have a profound and lasting effect on society, especially a tragedy that is widely viewed through modern media outlets. this text, however, will follow the unisdr and who definitions when discussing disaster. disaster medicine is a discipline resulting from the marriage of emergency medicine and disaster management. the role of medicine and emergency medical services in disaster response has abundant historical precedence. responsibility for the care of the injured from a disaster has been borne by the emergency specialist or its equivalent throughout history. therefore disaster medical response, in its many forms, has been around for thousands of years. whenever a disaster has struck, there has been some degree of a medical response to care for the casualties. in the united states, much of the disaster medical response has followed a military model, with lessons learned through battlefield scenarios during the last two centuries. the military experience has demonstrated how to orchestrate efficient care to mass casualties in austere environments. however, it does not translate directly into civilian practice. for instance, scenarios encountered on the battlefield with young, fit soldiers injured by trauma are vastly different from those encountered in a rural setting, where an earthquake may inflict casualties on a population with baseline malnutrition or advanced age. with this realization came the need to create disaster medicine as an evolution from the military practice. this recent organization of the medical role in disasters into a more formalized specialty of disaster medicine has enabled practitioners to define further their role in the overall disaster preparedness and response system. disaster medicine is truly a systems-oriented specialty, and disaster specialists are required to be familiar and interact with multiple responding agencies. the reality is there is no "disaster clinic." no practitioners leave home in the morning intent on seeing disaster patients. disaster medical care is often thrust upon the practitioner and is not necessarily something that is sought out. the exception to this is the medical specialist who becomes part of an organized (usually federal) disaster team, such as a disaster medical assistance team (dmat). in this case, one may be transported to a disaster site with the intention of treating the victims of a catastrophic event. in all other circumstances, however, the disaster falls on an unsuspecting emergency responder who is forced to abandon his or her normal duties and adopt a role in the overall disaster response. unlike the organized disaster team member, if an emergency provider treats casualties from a disaster, it will most likely be because of an event that has occurred in his or her immediate area. because of the random nature of disaster, it is not possible to predict who will be put into that role next. therefore it is imperative for all who practice in emergency health services to have a working knowledge of the basics of disaster medicine and disaster management. in addition, especially with natural disease outbreaks and the escalation in perceived and real terrorist threats of - , there are several possible natural or attack scenarios that may involve dangerous chemical, biological, or nuclear agents and modalities. a response to these events may also require a robust public health system and knowledgeable health care practitioners spanning all specialties. most clinicians will have a very limited knowledge of many of these agents, so it is therefore important to educate our potential disaster responders on their specifics. the field of disaster medicine involves the study of subject matter from multiple medical disciplines. disasters may result in varying injury and disease patterns, depending on the type of event that has occurred. earthquakes can cause entrapment and resultant crush syndrome; tornados may cause penetrating trauma from flying debris; and infectious disease outbreak, either natural or intentional, can result from many different bacteria, viruses, and fungi. because of the potential variability in casualty scenarios, the disaster medicine specialist must have training in the many injury and illness patterns seen in disaster victims. even though the expanse of knowledge required is vast, the focus on areas specifically related to disaster medicine allows the science to be manageable. the study of disaster medicine should not be undertaken without prerequisite medical training. a disaster medicine specialist is always a practicing clinician from another field of medicine first and a disaster specialist second. by integrating these many disciplines, one is better prepared for the variety of injury and illness patterns that may be faced. finally, disaster medicine presents unique ethical situations not seen in other areas of medicine. disaster medicine is predicated on the principle of providing care to the most victims possible, as dictated by the resources available and by patient condition and likelihood of survival. this amounts to a balance of needs versus assets, an equation that can change over time as more resources are pulled into the response. thus the triage of patients in disasters is fluid and should be repeated regularly. disaster triage involves assigning patients into treatment categories based on their predicted survivability. this triage process may dictate that the most severely injured patient is not given medical care but rather, it is given to a less critically injured patient. to the best of his or her ability, the triage officer must make a determination as to whether, in the environment of the specific disaster and the availability of resources, a given patient has a significant probability of survival or does not. if it is the latter, disaster triage principles mandate that care be given to the patient with a higher likelihood of survival. this basic disaster triage principle can have a profound psychological effect on the care provider. as a physician, one is trained to render care to the sick and not to leave the side of a needy patient. to deny care to a critically ill or injured patient can be one of the most emotionally stressful tasks a disaster medicine specialist performs. the unique and ever-changing circumstances under which disaster medicine specialists operate mandate the continued evolution and vigorous pursuit of academic excellence in this new specialty. a comprehensive approach that unifies medical principles with a sound understanding of disaster management procedures will yield a wellrounded and better-prepared disaster responder. if emergency medicine providers around the world can develop a basic understanding of the fundamental principles of this specialty, great advances in the systems included in the disaster cycle will surely follow. the more widely dispersed this knowledge becomes, the better prepared we are as a society to respond to the next catastrophic event. tsunami in thailanddisaster management in a district hospital a comparison of patient needs following two hurricanes analysis of the international and us response to the haiti earthquake: recommendations for change casualties treated at the closest hospital in the madrid the world trade center attack: lessons for disaster management identifying sources of human exposure to plague the ebola crisis: perspectives from european public health united nations international strategy for disaster reduction worldwide disaster medical response: an historical perspective key: cord- -l yshyc authors: alekseeva, ekaterina; sominskaya, irina; skrastina, dace; egorova, irina; starodubova, elizaveta; kushners, eriks; mihailova, marija; petrakova, natalia; bruvere, ruta; kozlovskaya, tatyana; isaguliants, maria; pumpens, paul title: enhancement of the expression of hcv core gene does not enhance core-specific immune response in dna immunization: advantages of the heterologous dna prime, protein boost immunization regimen date: - - journal: genet vaccines ther doi: . / - - - sha: doc_id: cord_uid: l yshyc background: hepatitis c core protein is an attractive target for hcv vaccine aimed to exterminate hcv infected cells. however, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. we aimed to design an hcv vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed. methods: plasmids encoding core with no translation initiation signal (pcmvcore); with kozak sequence (pcmvcorekozak); and with hcv ires (pcmvcoreires) were designed and expressed in a variety of eukaryotic cells. polyproteins corresponding to hcv b amino acids (aa) – and – were expressed in e. coli. c bl/ mice were immunized with four -μg doses of pcmvcorekozak, or pcmv (i). balb/c mice were immunized with μg of either pcmvcore, or pcmvcorekozak, or pcmvcoreires, or empty pcmv (ii). lastly, balb/c mice were immunized with μg of core aa – in prime and boost, or with μg of pcmvcorekozak in prime and μg of core aa – in boost (iii). antibody response, [( )h]-t-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization. results: plasmids differed in core-expression capacity: mouse fibroblasts transfected with pcmvcore, pcmvcoreires and pcmvcorekozak expressed . ± . , . ± . , and ± ng core per cell, respectively. single immunization with highly expressing pcmvcorekozak induced specific ifn-γ and il- , and weak antibody response. single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong t-cell proliferation (pcmvcoreires), and antibodies in titer ( )(pcmvcore). boosting with pcmvcorekozak induced low antibody response, core-specific t-cell proliferation and ifn-γ secretion that subsided after the rd plasmid injection. the latter also led to a decrease in specific il- secretion. the best was the heterologous pcmvcorekozak prime/protein boost regimen that generated mixed th /th -cellular response with core-specific antibodies in titer ≥ × ( ). conclusion: thus, administration of highly expressed hcv core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. instead, the latter is induced by a heterologous dna prime/protein boost regimen that circumvents the negative effects of intracellular core expression. (pcmvcoreires), and antibodies in titer (pcmvcore). boosting with pcmvcorekozak induced low antibody response, core-specific t-cell proliferation and ifn- secretion that subsided after the rd plasmid injection. the latter also led to a decrease in specific il- secretion. the best was the heterologous pcmvcorekozak prime/protein boost regimen that generated mixed th /th cellular response with core-specific antibodies in titer  × . thus, administration of highly expressed hcv core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. instead, the latter is induced by a heterologous dna prime/protein boost regimen that circumvents the negative effects of intracellular core expression. globally, an estimated million people are chronically infected with hepatitis c virus (hcv), and to million persons are newly infected each year [ , ] . the human immune system has difficulties in clearing the virus in either the acute, or chronic phase of the infection with up to % of patients progressing to cirrhosis and liver failure [ ] [ ] [ ] [ ] . extensive studies have unraveled important reliable correlates of viral clearance [ ] [ ] [ ] [ ] [ ] . this, together with the growing need to diminish the magnitude of hcv associated liver disease served as a basis for intensive hcv vaccine research. a series of hcv vaccine candidates have moved into clinical trials [ ] . one such is the peptide vaccine ic consisting of a panel of mhc class i and class ii restricted epitopes adjuvanted by poly-l-arginine administered to healthy volunteers [ ] and to chronic hcv patients including non-responders to the standard therapy [ , ] . another therapeutic vaccine employed peptides chosen individually for their ability to induce the strongest in vitro cellular response [ ] . in a further vaccine trial, chronic hepatitis c patients received the recombinant hcv envelope protein e [ ] . the first clinical trial of an hcv dna vaccine consisting of a codon-optimized ns / a gene administered to chronic hepatitis c patients is currently ongoing (chronvac-c ® ; http:// www.clinicaltrials.gov/ct /results?term=nct ; http://www.bion.no/moter/vaccine/ matti_s%e llberg.pdf). so far, none of the peptide or protein vaccines were able to induce a significant improvement in the health conditions of chronic hcv patients, or a significant decrease of hcv rna load, specifically if compared to the conventional ifn-based therapy [ , , ] . the vaccine trials have, however, demonstrated that when achieved, hcv rna decline in the vaccine recipients correlates with induction of strong ifn-gamma t-cell response [ ] . such a response can best be recruited by dna vaccines, either alone or with the aid of heterologous boosts [ , ] . indeed, vaccination of chimpanzees showed the ability to elicit effective immunity against heterologous hcv strains using t-cell oriented hcv genetic vaccines that stimulated only the cellular arm of the immune system [ , ]. an attractive target for hcv vaccine is the nucleocapsid (core) protein [ ] [ ] [ ] . it is highly conserved among various hcv genotypes with amino acid homology exceeding % [ , ] . core binds and packages the viral genomic rna, regulates its translation [ ] [ ] [ ] [ ] and drives the production of infectious viruses [ ] [ ] [ ] . core contributes to hcv persistence also indirectly by interfering with host cell transcription, apoptosis, lipid metabolism, and the development of immune response [ ] [ ] [ ] [ ] . extermination of core expressing cells and inhibition of the activity of extracellular core (non-enveloped particles containing hcv rna [ ]) could be highly beneficial. ideally, hcv core could be eliminated by a specific vaccine-induced immune response. it is a strong immunogen with anti-core immune response evolving very early in infection [ , ] . early and broad peripheral and intrahepatic cd + t-cell and antibody response to core/core epitopes is registered in chimpanzees controlling hcv infection hcv, but not in chimpanzees that become chronically infected [ ] [ ] [ ] . in mice, potent experimentally induced anti-core immune response conferred partial protection against challenge with core expressing recombinant vaccinia virus [ ] . however, despite high immunogenicity in the natural infection, core does not perform well as an immunogen, specifically if introduced as naked dna [ , [ ] [ ] [ ] . attempts to enhance core immunogenicity by targeting hcv core protein to specific cellular compartments [ ] , co-immunization with cytokine expressing plasmids [ , ] , adjuvants as cpg [ ] , or truncated core gene versions [ ] had limited or no success. prime-boost strategies have been used to increase immune responses to a number of dna vaccines. immunization regimens comprised of a dna prime and a viral vector boost for instance for vaccinia virus [ , ] , adenovirus [ ] , fowlpox [ , ] , and retrovirus [ ] . priming with dna and boosting with protein is another promising approach. this regimen has been studied for hiv [ , ] , hepatitis c virus [ , ] , anthrax [ ] , mycobacteria [ , ] , streptococcus pneumoniae [ ] and bvdv [ ] . dna vaccines and recombinant protein vaccines utilize different mechanisms to elicit antigen-specific responses. due to the production of antigen in transfected cells of the host, a dna vaccine induces robust t-cell responses, which are critical for the development of t-cell-dependent antibody responses [ ] . dna immunization is also highly effective in priming antigen-specific memory b cells. in contrast, a recombinant protein vaccine is generally more effective at eliciting antibody responses than cell-mediated immune responses and may directly stimulate antigen-specific memory b cells to differentiate into antibody-secreting cells, resulting in production of high titer antigen-specific antibodies [ ] . therefore, a dna prime plus protein boost is a complementary approach that overcomes each of their respective shortcomings. certain improvement of the immune response was reached after co-delivery of hcv core dna and recombinant core [ , , ] . in this study, we have shown that in dna immunization, poor core-specific immune response can be a consequence of high levels of intracellular core expression, and that such a response can be improved by using low-expressing core genes, or single core gene primes in combination with recombinant core protein boosts. region encoding aa - of hcv core was reverse-transcribed and amplified from hcv b isolate ru (genebank accession #af ) [ ] using oligonucleotide primers: sense gatccaagcttatgagcac-gaatcc and antisense gatccctcgagtcaagcggaagctgg containing recognition sites of hindiii and xhoi restriction endonucleases. the amplified dna was cleaved with hindiii/xhoi and inserted into pcdna (invitrogen, usa) cleaved with hindiii/xhoi resulting in pcmvcore. region encoding aa - of hcv core was also reverse-transcribed and amplified from hcv isolate ru using another set of primers that carried kozak consensus sequence sense agctgctagcgccgccaccatgagcacgaatcct and antisense gatcgttaactaagcggaagctggatgg primers containing recognition sites of restriction endonucleases nhei and xhoi, respectively. the amplified dna was cleaved with nhei/kspai and inserted into the plasmid pcmve /p - [ ] cleaved with nhei/xhoi, resulting in pcmvcorekozak. the region corresponding to hcv 'utr, and coding sequences for aa - was reverse-transcribed and amplified from hcv b isolate ad p (genebank accession #aj ) [ ] , kindly provided by prof. m. roggendorf (essen, germany) using sense-gacccaagcttcgtagaccgtgcaccat and antisense catgctcgagttaggcgtatgctcg primers. the amplified dna was cleaved with hindiii/xhoi and inserted into pcdna cleaved with hindiii/xhoi resulting in pcmvcoreires. hcv ru core differed from hcv ad p core in positions (h versus r), (t versus a), and (v versus t), respectively. growth of pcdna , pcmvcore, pcmvcorekozak, and pcmvcoreires was accomplished in the e. coli strain dh alpha. plasmid dna was extracted and purified by endo free plasmid maxi kit (qiagen gmbh, germany). the purified plasmids were dissolved in the phosphate buffered saline (pbs) and used for in vitro expression assays and for dna immunization. bhk- , cos- , and nih t cells were seeded into plates ( × cells/well) and transfected by plasmid dna ( g) using lipofectamine (gibco-brl, scotland) or exgen (fermentas, lithuania) as described by the manufacturers. hcv core expression was analyzed , and h post transfection. cells were lysed for min at °c in the buffer containing mm tris-hcl, ph . , mm edta, mm pmsf and % np- . lysates were cleared by min centrifugation at g, resolved by % sds-paag, and transferred to pvdf membranes (amersham pharmacia biotech, ireland). hcv core expression was detected by immunostaining with polyclonal rabbit anti-core antibodies [ ] , and secondary horseradish peroxidase (hrp)-conjugated anti-rabbit immunoglobulins (amersham pharmacia biotech, ireland) followed by ecl™ detection (ecl plus, amersham pharmacia biotech, ireland). nih t cells were transfected with either pcdna , pcm-vcore, pcmvcorekozak, pcmvcoreires, or pegfp-n (clontech, ca, usa). the percent of transfection was evaluated by counting the number of gfp expressing cells per transfected nih t cells using a fluorescence leica dm microscope (leica camera ag, germany). cells were harvested h post-transfection, counted, and cells were lysed in × sds sample buffer. lysates and samples containing to ng of recombinant core aa - (corresponding to p ) were run simultaneously on % sds-paag and transferred onto pvdf membrane for calibration. blots were blocked overnight in pbs-t with % non-fat dry milk, stained with polyclonal anti-core antibodies # - ( : ) followed by the secondary antirabbit hrp-conjugated antibodies (dakopatts ab, denmark). signals were detected using the ecl™ system (amersham pharmacia biotech, ireland). x-ray films were scanned, and processed using image j software http:// rsb.info.nih.gov/ij. the data are presented as the mean grey values (mgv). the core content was quantified by plotting the mgv of each sample onto a calibration curve prepared using recombinant core aa - . after core detection, blots were striped according to the ecl protocol and re-stained with monoclonal anti-tubulin antibod-ies (sigma, usa) and secondary anti-mouse hrpconjugated antibodies (dakopatts ab, denmark). core content per transfected cell was evaluated after accounting for the percent of transfection and normalization to the tubulin content per well. immunofluorescence staining bhk- cells were seeded on the chamber slides (nunc international, denmark) and transfected as above. h post transfection, the slides were dried, fixed with acetic acid and ethanol ( : ) for min and rinsed thoroughly in distilled water. fixed cells were re-hydrated in pbs, and incubated for h at °c with anti-hcv core rabbit polyclonal antibodies ( : ) in the blocking buffer (pbs with . mm edta and % bsa). secondary antibodies were goat anti-rabbit immunoglobulins labeled with tritc ( : ; dako, denmark). slides were then mounted with permafluor aqueous mounting medium (immunon, pa., usa) and read using a fluorescence microscope. peptides covering core amino acids - , - , - , - , - and a control peptide ttavpwnas from gp of hiv- were purchased from thermo electron gmbh (germany). core proteins representing aa - of hcv ru and aa - , and - of ad p were expressed in e. coli and purified by chromatography as was described earlier [ , ] . purified proteins were dissolved in pbs. the following immunizations were performed: scheme i groups of female -week old c bl/ mice (stolbovaya, moscow region, russia) were immunized with a total of g of pcmvcorekozak, or empty vector, split into four i.m. injections done with - week intervals. control mice were mock-immunized with pbs. scheme ii female - week old balb/c mice (animal breeding centre of the institute of microbiology and virology, riga) had injected into their tibialis anterior (ta), l of . mm cardiotoxin (latoxan, france) in sterile . % nacl five days prior to immunization. groups of to mice were immunized with a single g dose of either pcm-vcoreires, or pcmvcore, or pcmvcorekozak, or empty vector, all dissolved in l pbs, applied intramuscularly (i.m.) into the cardiotoxin-treated ta. control mice were left untreated. groups of to female - week old balb/c mice pretreated with cardiotoxin, were injected i.m. with g of pcmvcorekozak and boosted three weeks later with g of core aa - in pbs, or primed and boosted subcutaneously with g of core aa - in pbs. control animals were left untreated. mice were bled from retro-orbital sinus prior to, and to weeks after each immunization, or weeks post a single gene immunization (in scheme ii). peptides corresponding to core aa - , - or - were coated onto -well maxisorp plates (nunc, denmark), and recombinant core aa - , - , or - , on the -well polysorp plates (nunc, denmark). coating was done overnight at °c in mm carbonate buffer, ph . at antigen concentration of g/ml. after blocking with pbs containing % bsa for h at °c, serial dilutions of mouse sera were applied on the plates and incubated for an additional hour at °c. incubation was followed by three washings with pbs containing . % tween- . afterwards, plates were incubated with the horseradish peroxidase-conjugated anti-mouse antibody (sigma, usa) for h at °c, washed, and substrate opd (sigma, usa) added for color development. plates were read on an automatic reader (multiscan, sweden) at nm. elisa performed on plates coated with core aa - , - , or - showed similar results (data not shown). immune serum was considered positive for anti-core antibodies whenever a specific od value exceeded, by at least two-fold, the signals generated by: pre-immune serum reacting with core-derived antigen, and by immune serum reacting with bsa-coated plate, the assays performed simultaneously. for t-cell proliferation tests, mice were sacrificed and spleens were obtained two weeks after each immunization in scheme i; and three and five weeks after the last immunization in schemes ii and iii. murine splenocytes were harvested using red blood cell lysing buffer (sigma, usa), single cell-suspensions were prepared in rpmi supplemented with mm l-glutamine and % fetal calf serum (gibco brl, scotland) at × cells/ml. cell were cultured in u-bottomed microculture plates at °c in a humidified % co chamber (gibco, germany). cell stimulation was performed with peptides representing core aa - , - , - and recombinant core aa - , - , and aa - at dilutions to . , . , . , . , . , and g/ml, all in duplicate. concanavalin a (cona) was used as a positive control at g/ml. cells were grown for h, after which [ h]-thymidine ( ci per well; amersham pharmacia biotech, ireland) was added. after an additional h, cells were harvested onto cellulose filters and the radioactivity was measured on a beta counter (beckman, usa). the results were presented as stimulation indexes (si), which were calculated as a ratio of mean cpm obtained in the presence and absence of a stimulator (protein or peptide). emptyvector immunized and control mice showed si values of . ± . . si values  . were considered as indicators of specific t-cell stimulation. for detection of cytokines, cell culture fluids from t-cell proliferation tests were collected, for il- - h, and for il- and ifn- - h post the on-start of t-cell stimulation. detection of cytokines in the cell supernatants was performed using commercial elisa kits (pharmingen, bd biosciences, ca, usa) according to the manufacturers' instructions. plasmids were constructed encoding core of hcv b isolate ru without translation initiation signals (pcmvcore); and with kozak translation initiation signal (pcmvcorekozak). core with viral translation initiation signal ires taken in the natural context was derived from hcv b isolate ad p [ ] . viral cores had a minimal sequence difference in positions , , and , all three cases representing homologous substitutions. expression from these plasmids was tested both in vitro and in cell cultures. plasmids pcmvcore and pcmvcore-kozak were used as the templates for the t -driven mrna transcription; mrna was translated in vitro in the rabbit reticulocyte lysate system. both mrnas generated a translation product of approximately kda corresponding to the molecular mass of unprocessed hcv core (p ; data not shown). next, core-expressing vectors were used to transfect a series of mammalian cell lines. western blotting of bhk- and cos- cells transfected with pcmvcore, pcmvcorekozak and pcmvcoreires using corespecific antibodies demonstrated an accumulation of proteins with the expected molecular mass of kda that corresponds to core aa - cleaved from the full-length core by cellular proteases [ , ] (fig. ) . minimal amounts of p were also detected, specifically after transfections of bhk- with pcmvcore and pcmvcoreires (fig. ) . the overall level of hcv core synthesis in bhk- cells was somewhat higher than in cos- cells (fig. ) . in both cell lines, the highest level of core expression was achieved with pcmvcorekozak ( fig. , ) . all cells expressing core and immunostained with core-specific antibodies demonstrated cytoplasmic granular staining characteristic of the processed p form of hcv core [ ] [ ] [ ] (fig. ) . the expression capacity of the vectors was quantified in murine fibroblasts to reproduce dna immunization that was to be done in mice. core expression was assessed on western blots of sds-paag resolving lysates of nih t transfected with core expressing and control plasmids ( fig. a and b) . images of western blots were processed using the imagej software and individual bands were represented in arbitrary units (mean grey values, mgv). their correspondence to core quantity was established using calibration curves built with the use of recombinant core aa - (see additional file ) after normalization to the percent of transfection and protein content of the samples. plasmid pcmvcore with no translation initiation signals provided the lowest level of core expression (fig. b) . ires promoted a two-fold increase, and the kozak sequence, a -fold increase of core expression with > ng of protein produced per expressing cell (fig. b) . immunization of mice with hcv core dna all plasmids were purified by standard protocols in accordance with a glp practice for preparation of dna vaccines, and used in a series of mouse immunization experiments. plasmid directing the highest level of core expression was selected and a pilot experiment defining the strategy of dna immunization was performed. c bl/ mice were immunized four times with g of pcmvcorekozak, and core-specific antibody and cellular responses were evaluated. no specific response was registered after the st injection (data not shown). the immune response generated after the following three boosts is illustrated by fig. . three injections of g led to no increase of core-specific igg response over the initial levels achieved after the first two plasmid injections (fig. a) . three plasmid injections generated a better t-cell proliferative response to core and core-derived peptides than two. however, the response could not be boosted further (fig. b) . ifn- and il- response to core was also boostable. however again, no boosting was seen after the initial two pcmvcorekozak injections (fig. c) . furthermore, the repeated injections led to a significant decrease of il- secretion in response to splenocyte stimulation by recombinant core and peptides representing core n-terminus (p < . ; fig. b , and data not shown). core-specific il- secretion was not detected. thus, the development of core-specific immune responses occurred within six weeks after the on-start of immunization; repeated boosts with hcv core gene did not lead to a significant enhancement of core-specific immunity. in the next series of experiments, we selected balb/c mice as a strain that is expected to support a better th -type response with stronger antibody production [ ] . plasmid pcmvcore kozak was given as a single g injection with the effect of repeated intramuscular dna boosts substituted by pre-treatment of the injection sites by cardiotoxin [ ] . t-cell proliferative response, antibody production and cytokine secretion were monitored two and five weeks after immunization. significant responses in the form of core-specific ifn- and il- secretion exceeding the background levels in empty-vector-immunized mice were detected five weeks after a single administration of hcv core gene (fig. ) . immunization generated no core-specific t-cell response and a low titer of core-specific igg. antibody response against hcv core has already been shown to develop slowly [ ] , mirroring the development of anti-core antibody response in hcv infected individuals [ ] . here as well, a slow increase in the level of anti-core antibodies expression of hcv core proteins plasmids used for nih t transfection core expression (units) was observed days after a single gene injection as compared to levels detected at day (data not shown). there was no difference between balb/c and c bl/ mice with respect to core-specific ifn- secretion (fig. c versus fig. c ), or core-specific igg production (p > . mann whitney u-test; fig. a versus fig. a and additional file ). the magnitude of anti-core response suggested that the increase of hcv core gene dose either by one-time large dose injection, or by repeated injections of smaller doses, did not significantly enhance core-specific immunity. to delineate if that could be influenced by core expression level, balb/c mice were immunized with a single dose of low-expressing core genes with no translation initiation signals (pcmvcore), or with ires (pcmvcoreires). the results were compared to immunization with core gene regulated by the kozak sequence (pcmvcoreires) (fig. ) . the t-cell proliferative response to core-and corederived peptides was stronger in mice immunized with pcmvcoreires (fig. ) . the highest anti-core igg response was raised in mice immunized with pcmvcore that directed the lowest level of hcv core expression ( fig. ; fig. a ). it was significantly higher than the antibody response induced by pcmvcorekozak (p < . ); the immune response in pcmvcoreires-immunized mice was intermediate (fig. a) . the t-cell proliferative response to core-and core-derived peptides was stronger in mice immunized with pcmvcoreires ( fig. b ; p < . ). while il- secretion was somewhat higher in mice immunized with highly expressing pcmvcorekozak, both dna immunogens provided a similar level of core-specific ifn- secretion (fig. c) . we aimed to see if core-specific immune response could be enhanced without increasing core gene doses, but instead by using the heterologous prime-boost immunization regimens. hcv core protein aa - and pcmv-corekozak were used to immunize balb/c mice either separately, or in the dna prime-protein boost regimen. a high titer of core-specific antibodies was achieved only after the heterologous boost (fig. a) . the heterologous regimen effectively induced a proliferative response, both in si values (p levels . , mann whitney u-test) and in the number of positive t-cell proliferation tests (p level . ; fig. b ); and potent core-specific ifn- and il- secretion (fig. c) . core-specific il- secretion was, in all cases, very low. heterologous regimen induced significant anti-core antibody production (fig. a) . sera of mice primed with pcmvcorekozak and boosted with core aa - were analysed for the presence of anti-core antibodies of igg, igg , igg a, igg b and igm subclasses, and the results were compared to seroreactvivity in mice immunized with single injection of core or core expressing plasmids (fig. ) . mice primed with pcmvcorekozak and boosted with core protein had significantly higher levels of anti-core igg than mice immunized with pcmvcorekozak (p = . , mann-whitney u-test) or pcmvcore (p = . ) (immunization with pcmvcore gave higher level of igg than immunization with pcmvcorekozak, p < . ). group with heterologous prime/boost regimen had also an increased levels of anti-core igg , although the difference with the control group did not reach the level of significance (p < . ). antibodies of igg a or igg b subclasses were not found. low specific anti-core igm were observed only in mice immunized with recombinant core aa - (p < . ; fig. ) . it was higher than in mice primed with core dna and boosted with core protein (p level . ). at the same time, core-immunized mice had no anti-core igg or igg (fig. ) . thus, the heterologous core dna prime/core protein boost regimen preferentially induced anti-core igg, while protein immunization triggered mostly low-level anti-core igm. the immune response in dna immunization depends on the amount of antigen produced from the immunogen in vivo as predetermined by the gene dose and by the gene capacity to direct an efficient antigen expression [ , ] . normally, the response increases with the increase of the dose and efficacy of gene expression (for examples, see [ ] [ ] [ ] [ ] ). however, the dna immunogen used here encodes not just a structural component of the virus, but also a pathogenic factor. hcv core protein interacts with spectrum of core-specific immune response figure spectrum of core-specific immune response. , , , ] . of importance for hcv vaccine design was to find to what extent the immune response to hcv core in dna immunization is influenced (positively, or negatively) by the level of core expression as determined by gene dose (i), and gene expression efficacy (ii). the first issue was addressed in a series of immunizations in which the same dose of hcv core was given as a single or split into multiple injections. we and others have earlier observed that repeated hcv core gene boosts do not lead to an enhancement of core-specific immune response [ , , ] . on the contrary, both core-specific ifn- and il- production [ ] and anti-core antibody response [ , , ] appear to be down-regulated. here as well, the overall comparison between immunizations carried out by single and multiple core gene injections in different mouse strains demonstrated that the outcomes of immunization with one g versus two to four g core gene doses were quite similar ( fig. ; see also the summary in additional file ). furthermore, antibody response was not boosted; t-cell proliferative response and core-specific ifn- secretion could not be boosted beyond the levels reached after the initial two injections, and core-specific il- secretion even appeared to be suppressed. thus, core-specific immune response can be achieved after single dna immunization, while repeated core gene administration may actually suppress core-specific immunity. the issue of translation efficacy was assessed in singledose immunizations with plasmids directing different levels of hcv core expression. there are different ways to increase the level of gene expression efficacy such as the use of strong promoters, optimal species-specific codons, and manipulations with rna folding [ , , ] . an important factor is the efficacy of translation initiation. in the cap-dependent translation of mammalian genes it is determined by sequences flanking the aug initiator codon. high levels of translation are achieved with the kozak sequence, a guanine at position + and an adenine at - from aug [ , ] . the alternative mechanisms of initiation site selection on eukaryotic cellular and viral mrnas, also of hcv, include the translation initiation from iress (internal ribosome entry sites/segments) [ ] . located in the '-utr region of viral genome, hcv ires is optimized to hijack the ribosomes and translation factors from the host for the translation of hcv polyprotein [ ] . core is tightly involved in the ires-mediated regulation of hcv translation with several regulatory signals localized in both core protein and core coding sequence [ , , ] . thus, the '-end of hcv genome incorporating '-utr and core coding sequence were harmonized during evolution to provide for the levels of core expression essential for the virus. both cap and ires translation initiation options were employed in the design of core dna immunogens. eukaryotic expression vectors were constructed encoding core of hcv b without translation initiation signal (pcmvcore), core preceded by the '-utr of hcv b isolate ad (pcmvcoreires), and core preceded by the consensus kozak sequence (pcmvcorekozak). the latter directed the expression of -fold more core than the gene devoid of the translation initiation signals, and -fold more core than the gene regulated by ires. however, despite a considerable difference in the core expression capacity, kozak-and ires-regulated dna immunogens induced similar levels of core-specific ifn- secretion (fig. c ). more so, while il- secretion was somewhat higher in mice immunized with highly expressing pcmvcorekozak, a t-cell proliferative response to core-and corederived peptides was stronger in mice immunized with pcmvcoreires ( fig. b) . thus, high core expression levels did not promote a better core specific cellular response. dna-based immunization can induce potent antibody response including virus neutralizing antibodies [ ] [ ] [ ] [ ] [ ] . however, no significant antibody response has ever been induced in core gene immunization unless it was followed by the protein boost [ , ] . anti-core antibody titers obtained here after immunization with both cap-and ires-regulated core genes were also low. interestingly, however, significantly higher titers of anti-core antibodies were obtained in mice that received the least expressed core gene devoid of any translation regulation signals (pcmv core; figs. a, ). thus, the use of highly expressing hcv core dna did not promote an effective core-specific antibody response. altogether, this points to possible adverse effects of the high-level as well as of the prolonged hcv core gene expression. we have additional data in support of this concept from immunization of c bl/ mice with a synthetic truncated hcv core gene devoid of hcv core nucleotide-sequence dependent regulatory signals. the latter expressed hcv b core at five to six-fold lower levels than the viral full-length core gene [ ] , but nevertheless, was capable of inducing potent core-specific cellular and antibody response http://www.meetingsmanagement.com/ dna_ /index.htm [ ] . dna immunization with antigens co-expressed in natural virus infection can result in inhibition of both protein expression and specific immune response [ ] . more so, pathological effects were reported of the repeated immunization with certain microbial genes, for example the hsp gene of mycobacterium that causes necrotizing bronchointerstitial pneumonia and bronchiolitis in healthy mouse recipients, and multifocal regions of cellular necrosis in lungs when applied therapeutically [ , ] . hcv core is the factor of hcv pathogenicity. it activates cellular and viral promoters [ ] , induces er-and mitochondrial stress [ , ] , regulates apoptosis [ , ] , tumorigenesis [ , ] , and induces abnormal lipid metabolism [ ] . in experimental systems, core expression leads to the development of diverse pathological effects including cd + t-cell depletion, liver steatosis, insulin resistance, and hepatocellular carcinoma [ , ] . one of the notable although controversial features is the capacity of hcv core to suppresses host immunity [ , , , ] . these features of hcv core may explain why here a better immune response was achieved after single immunization with vectors providing for comparatively low hcv core expression. altogether, this points to the necessity to devise alternative immunization regimens that would help to circumvent possible adverse effects of hcv core. many approaches can be pursued, with dna vaccination combined with heterologous protein or recombinant viral boosts considered as the most promising [ ] . the principle of this strategy is to prime t-cells to be antigen-specific and then, upon repeated exposure to a specific antigen, induce a rapid t-cell expansion. in heterologous boosts, the encoded antigen is delivered in a different form/different vehicle [ ] . dna plasmids are perfectly fit for priming since they are internalized by antigen presenting cells and can induce antigen presentation via mhc class i or class ii. such heterologous regimens can be effective when infection occurs with both viral particles and virusinfected cells, and neither cellular, nor antibody response is sufficient for sterilizing protection or viral clearance, if acting alone. this approach may help to circumvent the negative effects of intracellular core expression. indeed, here, the heterologous dna-prime/protein boost strategy was shown to be advantageous to both immunizations with core dna and with the recombinant core protein (figs. , ) . protein alone performed even worse than single dna injections (figs. , ) . only the heterologous dna-prime-protein boost regimen induced a significant core-specific antibody production and potent t-cell response of mainly th -profile. this may be beneficial since most correlates of spontaneous hcv clearance are th -oriented [ , , , ] . this data suggests that the administration of highly expressed hcv core gene, as well as repeated core gene injections may hamper core-specific immune response. the boosting effect of repeated core gene injections is transient as it disappears with subsequent injections. one possible way to enhance core-specific response is to deliver limited intracellular amounts of core, either by giving lower plasmid doses, or by giving vectors with low expression efficacy. an additional option is the use of 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syndrome coronavirus (sars-cov) s protein results in the production of high levels of sars-cov-neutralizing antibodies field trials of a very potent rabies dna vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions gene immunization may induce secondary antibodies reacting with dna bacterialized" gene for the c-terminus truncated version of human hepatitis c virus core induces potent th -type immune response in mice interactions of single and combined human immunodeficiency virus type (hiv- ) dna vaccines pulmonary necrosis resulting from dna vaccination against tuberculosis lack of protection in mice and necrotizing bronchointerstitial pneumonia with bronchiolitis in guinea pigs immunized with vaccines directed against the hsp molecule of mycobacterium tuberculosis transcriptional regulation of cellular and viral promoters by the hepatitis c virus core protein cellular response to conditional expression of hepatitis c virus core protein in huh cultured human hepatoma cells oxidative stress in the absence of inflammation in a mouse model for hepatitis c virus-associated hepatocarcinogenesis the hcv core protein acts as a positive regulator of fas-mediated apoptosis in a human lymphoblastoid t cell line suppression of apoptotic cell death by hepatitis c virus core protein the core protein of hepatitis c virus induces hepatocellular carcinoma in transgenic mice hepatitis c virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype hepatitis c virus core protein modulates fatty acid metabolism and thereby causes lipid accumulation in the liver hepatitis c virus core protein induces hepatic steatosis in transgenic mice the prime-boost strategy: exciting prospects for improved vaccination we thank prof. eva stankevica and her group for the oligonucleotide synthesis and automatic sequencing and ms. natalija gabrusheva and ms. irena timofeeva for technical assistance. this work was supported by grants from the latvian council of science . , eraf vpd /eraf/cfla/ / apk/ . . ./ / , eu # - - , the european social fund (esf), the new visby program of the swedish institute, compuvac grant #lshb-ct- - and the russian foundation for basic research # - - -a. the authors declare that they have no competing interests. is and ek constructed plasmids and screened their immunogenicity; ea did experiments on expression and wrote a draft of the manuscript; es and ei carried out quantifications of core expression; ds and np did immunological experiments; rb conducted the immunocytochemistry; mi was involved with the immunological experiments, statistical evaluations and worked with the manuscript; tk and pp give useful scientific advice and revised the manuscript. all authors read and approved the final manuscript. establishment of calibration curves for quantification of core expression in vitro. recombinant core aa - in serial dilutions in the range of to ng (a), to ng (b), or . to ng (c) was loaded on % sds-paag and resolved by gel electrophoresis together with the study samples. proteins were transferred to pvdf membrane and subjected to western blotting with core-specific rabbit antibodies, and secondary anti-rabbit hrp-conjugated antibodies (dakopatts). signals were registered using x-ray films and ecl detection system, images were saved, scanned, and signal of individual band corresponding to core was quantified by image j http://rsb.info.nih.gov, and calibration curves were built (d key: cord- -dmpoy b authors: rowe, john c.; attia, zayed; kim, eunsoo; cormet-boyaka, estelle; boyaka, prosper n. title: a novel supplementation approach to enhance host response to sublingual vaccination date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: dmpoy b sublingual immunization is emerging as an alternative to nasal immunization and induction of mucosal iga responses. using bacillus anthracis edema toxin (edtx) as an adjuvant, we previously showed that innate responses triggered after sublingual immunization could limit generation of iga responses. we tested whether co-administration of a neutrophil elastase inhibitor (nei) could rescue the ability of edtx to induce broad antibody responses, including mucosal iga. nei supplementation of sublingual vaccines containing edtx promoted antigen-specific serum iga responses but also enhanced serum igg , and igg b responses. this enhancing effect of nei did not extend to all antibody isotypes and igg sublclasses, since nei reduced serum ige responses and did not affect igg a/c and igg responses. nei supplementation also promoted anti-bacillus anthracis protective antigen (pa) neutralizing antibodies and enhanced high affinity igg and iga antibodies. in addition to serum iga, nei supplementation stimulated antigen-specific mucosal iga responses in the gi tract, and enhanced antigen-specific igg responses in vaginal washes. analysis of cd (+) t helper cell responses revealed that co-administration of nei broadened the profile of cytokine responses, by stimulating th , th , th , and tfh cytokines. we also noted that nei had a higher stimulatory effect on il- , il- , il- responses. needle-free vaccines delivered via mucosal surface have the potential of being better-accepted by the most vulnerable and commonly vaccinated population of children. they also present higher likelihood to generate the necessary b and t cell responses for optimal protection at the portal of entry of infectious agents, in addition to promoting the levels of systemic immunity generally achieved by conventional injected vaccines . secretory iga (siga) represents the hallmark of immune responses at mucosal surfaces. the high resistance of these polymeric immunoglobulins to degradation in the harsh environment of mucosal surfaces, including the lumen of the gastrointestinal tract, allow them to provide frontline protection at the portal of entry of most infectious microbes . while mucosally delivered subunit-vaccines have the potential of stimulating broad mucosal and systemic immune responses, their ability to trigger mucosal iga relies on the addition of effective vaccine adjuvants. stimulation of inductive site immune responses in different mucosal sites (i.e., gastrointestinal tract, respiratory tract, rectal) imprints the expression of discrete mucosal homing receptors and adressins which allow effector b and t cells to home in distinct mucosal tissues. for example, intranasal delivery of vaccines containing appropriate mucosal adjuvants can promote specific immune responses in the airways. however, safety issues were reported following intranasal application of a ganglioside-binding toxin as adjuvant. sublingual immunization is now being considered as an alternative to the intranasal route of vaccination. nonetheless, a major challenge for the development of sublingual vaccines is the identification of appropriate antigen-adjuvant formulations , . we have previously shown that bacillus anthracis edema toxin (edtx) is an effective adjuvant capable of promoting both systemic immunity and mucosal siga responses against nasally co-administered vaccine antigens , . however, when edtx was tested as adjuvant for sublingual vaccination, it promoted antigen-specific igg responses in the bloodstream but failed to elicit iga responses in the serum or mucosal secretions . this lack of iga responses was not due to the route of immunization itself, since sublingual immunization could induce these responses when vaccines were administered with a range of adjuvants including bacterial enterotoxins, toll-like receptor ligands, and sting ligands [ ] [ ] [ ] [ ] [ ] . interestingly, the lack of iga response correlated with the recruitment of neutrophils after sublingual administration of edtx, and partial depletion of neutrophils before sublingual immunization restored the adjuvant activity of edtx for iga responses of myeloid cells in the bloodstream and characterize the initial response to inflammatory events through their own degranulation and cytokine production . unfortunately, depletion of neutrophils prior to immunization is not a feasible approach and thus, new strategies are needed to improve the efficacy of edtx-based, and possibly other, sublingual vaccines. neutrophils are recruited by inflammatory cytokines, including il- , il- β, and tnfα, and were more recently recognized to contribute to the chemotaxis of other myeloid cells through the products released after neutrophil degranulation , . neutrophil elastase inhibitors (nei) are a class of serine protease inhibitors that target the neutrophil granule protein elastase, commonly implicated in chronic lung inflammation [ ] [ ] [ ] . co-administration of a nei enhances serum igg and igg b responses to a sublingual vaccine. we first asked whether supplementation with a nei could affects igg responses induced by a model sublingual vaccine containing edtx as adjuvant. vaccines targeting two or more pathogens can reduce the schedule of vaccination. we choose to use ovalbumin (ova) plus bacillus anthracis protective antigen (pa) as a combinatorial antigen to test the ability of nei to regulate the immune response to two different antigens. ova is a well-studied model antigen which allow a more in-depth analysis of immune responses to vaccination thanks to the large number of reagents available to study ova-specific b and t cell responses. on the other hand, the use of pa as antigen allowed us to address the biological significance of the antibody responses through the assessment of anti-pa antibodies (abs) ability to neutralize anthrax lethal toxin (letx). analysis of ova-specific igg responses revealed that nei increased the magnitude of responses induced by edtx, and this effect was evident as early as day after the first immunization (fig. a) . we also found that the nei used in these studies had an intrinsic adjuvant activity and increased igg responses when co-administered with antigen in the absence of edtx (fig. a) . analysis of other igg subclasses showed no evidence that the nei enhanced igg a/c, or igg when co-administered with antigen in the absence of edtx (fig. b,c) . however, the nei significantly increased serum igg b responses induced by edtx as adjuvant (fig. b ,c). since the presence of nei differentially regulated igg subclass responses, we next addressed its potential effects on other immunoglobulin isotypes. sublingual immunization with edtx as adjuvant induced ova-specific ige responses which peaked weeks after the first immunization (day ) (fig. a) . co-administration of nei significantly reduced the magnitude of ige responses to levels barely at the detection threshold ( fig. a) . furthermore, no ige response was detected following sublingual application of nei and antigen alone ( fig. a ). as previously reported , sublingual immunization with edtx as adjuvant failed to induce ova-specific serum iga responses (fig. b) . mice co-administrated antigen and nei showed detectable but minimum levels of serum iga responses at day . minimum serum iga levels were also induced and could be measured on day in mice immunized with antigen (ova + pa) plus ef. interestingly, only mice immunized with antigen and ef in the presence of the nei developed significant serum iga responses (fig. b ). nei supplementation enhances the amount of high affinity abs and toxin neutralizing responses in mice immunized with edtx as adjuvant. results summarized above show that nei supplementation improves the kinetic of igg responses and allows mice immunized with edtx to reach maximum igg titers more quickly ( fig. ). they also show that nei broadens the antibody responses by enhancing igg b and promoting iga (figs and ). to determine the biological significance of the broader antibody responses measured in the blood of mice administered vaccines supplemented with nei, we first assess anti-pa neutralizing antibodies. serum of mice immunized with antigen alone (ova plus pa), antigen plus nei, or antigen plus nei contained low and comparable levels of anti-pa neutralizing antibodies (fig. a ). on the other hand, anti-pa neutralizing antibody titers were significantly increased in the group of mice that received edtx and nei (fig. a) . we also addressed whether the enhanced neutralization of letx toxicity was due to the increase in anti-pa antibody titers or whether it also resulted from an increase in high affinity antibodies. as with anti-ova responses above ( fig. ) , nei supplementation enhanced the titers of pa-specific igg and iga responses in mice with edtx as adjuvant (fig. b ). most importantly, the igg and iga promoted by nei supplementation were high affinity antibodies (fig. c ). our previous studies have shown that a major limitation of edtx as adjuvant for sublingual vaccines is its inability to promote iga responses in the bloodstream and in mucosal secretions . sublingual application of antigen alone or antigen plus nei did not stimulate antigen-specific iga responses in the gut. addition of nei to a vaccine formulation containing antigen and edtx resulted in the induction of fecal iga responses, which were detectable weeks after the first immunization (day ) and were further increased one week later (day ) (fig. a ). to determine if nei supplementation promoted generalized iga responses in other mucosal secretion, we analyzed these responses in vaginal washes. unlike mucosal secretions of the gastrointestinal tract, the vaginal washes of mice immunized with edtx and nei showed only non-significant levels of antigen-specific iga responses (fig. b ). however, nei supplementation was able to enhance the vaginal igg responses induced by edtx as adjuvant (fig. c) . these data suggest that nei might promote expression of gut homing receptors by iga producing cells, while the elevated igg antibodies found in the secretions of the genitourinary tract might be coming from diffusion of igg from the blood. it is important to indicate that anti-pa ab responses measured in mucosal we performed flow cytometry analysis of cytokines produced by cd + t cells upon in vitro restimulation with ova to elucidate the profile of t helper cytokines that supported the antibody responses induced by supplementation with nei. spleen cells from mice immunized with antigens plus nei exhibited the same frequencies of th cells (ifnγ + cd + t cells and tnfα + cd + t cells) as spleen cells from mice that received antigen plus edtx (fig. a ). the frequencies of th cells producing il- were also similar between these two groups, but mice immunized with antigens plus nei exhibited lower frequencies of il- + cd + t cells (fig. b ). these analyses also show that immunization with antigen plus nei promoted higher frequencies of il- + cd + t cells and il- + cd + t cells than immunization with antigen plus edtx (fig. c,d) . furthermore, nei and edtx promoted the same frequencies of antigen-specific il- + cd + t cells (fig. d) . consistent with the broad increase in antibody responses, co-administration of edtx and nei significantly increased all cd + t helper cytokine responses, including th , th , th and tfh responses (fig. ). the sublingual route is currently used for drug delivery and allergen specific immunotherapy (asit). sublingual vaccination is theoretically very simple as it involves application of liquid or rapidly dissolving tablets to the floor of the oral cavity. like vaccines delivered at other mucosal sites, sublingual vaccines have the potential of producing mucosal siga in addition to igg responses in the general bloodstream. because sublingually administered vaccines are less likely to reach central nervous system tissues, they are potentially safer than nasal vaccines. despite these advantages, identification of appropriate adjuvant formulations remains a requirement for the development of sublingual vaccines. here we show that supplementation of the edtx adjuvant with a chemical inhibitor of a neutrophil function broadens b and t cell responses induced after sublingual immunization and promotes antigen-specific siga abs. we have shown that the breath of antibody responses induced by edtx as a sublingual adjuvant is limited by the recruitment of neutrophils in mucosal inductive sites . while this work was the first to establish an inverse relationship between the presence of neutrophils and production of iga, the mechanisms underlying the suppressive effect of neutrophils on iga production remained elusive. it was previously shown that neutrophil peptide defensins enhance igg, but not iga responses against nasally co-administered vaccine antigen . the primary (or azurophilic) granules of neutrophils contain defensins, myeloperoxidase, lysozymes, and three serine proteases: neutrophil elastase, cathepsin g and protease , . we now show that inhibition of a single function of neutrophils can rescue the adjuvant activity of edtx and promote iga responses both in the blood stream and in mucosal tissues. this finding is significant because the use of a serine protease inhibitor might be a more realistic alternative to neutrophil depletion as a strategy for promoting iga responses. it also raises the question of the mechanism behind neutrophil elastase limiting iga production. neutrophil elastase and the related serine proteases cathepsin g and proteinase , were shown to regulate cytokine responses through activation or degradation of cytokines, cytokine receptors, or toll-like receptor , . our separate studies have shown that nei induce the expression of il- mrna but also aid and baff responses by spleen cells in vitro . because il- facilitates ig class switching for production of iga and regulate the expression of aid for induction of ig csr , this may be a mechanism for enhanced iga responses in mice sublingually immunized with edtx in the presence of nei. sublingual immunization of mice with sting ligands as adjuvant induces iga responses in mucosal tissues of the airways , and the genitourinary tract . however, neither cpg nor the sting ligand ′ -cgamp as sublingual adjuvant induced siga in the secretions of the gi tract (i.e., in fecal extracts) . we now show that when added to an edtx-based sublingual vaccine, nei primarily promoted siga responses in mucosal secretions of the gi tract. no significant levels of iga were measured in other mucosal secretions although igg responses were significantly increased in vaginal washes. the best-described factors that regulate homing of immune cells into the gut are ccr and gut homing receptors α β , . these factors are induced through signals provided by retinoid acid, a product of myeloid cells in mucosal tissues . to our knowledge, no study has examined whether neutrophils regulate expression of gut homing receptors. but it is worth noting that in conditions of normal homeostasis, neutrophils are not present in mucosal tissues where the concentration of iga is the highest in the body. the observation that nei primarily promotes siga in the gut suggests that it either directly stimulated retinoic acid production or stimulated cytokines such as il- , il- and il- , which promote or enhance expression of gut homing receptors , . iga was not the only immunoglobulin isotype affected by supplementation of the edtx-based sublingual vaccine with nei. in fact, nei also enhanced igg and igg b responses. this is consistent with the previous report that elastase inhibits the maturation and function of dendritic cells, including expression of the costimulatory molecules cd , cd and cd . thus, the presence of nei may have promoted the maturation of dendritic cells and their expression of baff and april, and perhaps costimulatory molecules (i.e., cd ), for enhanced antibody production. another important point revealed in the present study is that the presence of nei improved the quality of antibody responses. indeed, nei increased the titers of high affinity igg and iga antibodies, an effect that translated into higher titers of anti-pa neutralizing antibodies. our findings are in line with the recent report that neutrophils regulate germinal center formation in secondary lymphoid organs and production of autoreactive plasma cells in lupus . analysis of antigen-specific t helper cytokine responses provided some clues on how nei improved the breath, the magnitude and the quality of antibody responses in mice immunized via the sublingual route with edtx as adjuvant. we found that addition of nei resulted in higher frequencies of th cells producing ifn-γ, th cells producing il- and il- , th cells producing il- , and tfh cells producing il- . this broad profile of th cell cytokine responses has most likely contributed to increase b cell survival via signals from il- and il- [ ] [ ] [ ] [ ] [ ] . it is also consistent with the observation by others that neutrophils preferentially accumulate in proximity of t cells in secondary lymphoid organs during the early stage of lupus and that neutrophil depletion accelerate germinal center formation . in our study, the nei may have played the same role as neutrophil depletion and enhanced development of germinal centers and production of high affinity antibodies by facilitating differentiation of tfh producing il- . altogether, our results show that supplementation of sublingual vaccine by co-administration of a nei can improve the kinetic, breath and quality of antibody responses both in the bloodstream and in mucosal tissues. these findings raise the question of can nei display similar adjuvant activity for vaccines given through other mucosal routes and, possibly non-mucosal vaccines? our separate studies indicate that this is in fact the case and that co-administration of nei modulates immune responses induced by intranasal and injected vaccines (attia z and boyaka pn, manuscript in preparation). it is also worth noting that nei alone exhibited some adjuvant activity and enhanced igg and iga responses against the sublingually co-administered vaccine antigens. this effect does not appear to extend to other immunoglobulin isotypes/subclasses. previous studies have established that after oral administration, the plasma concentration of the nei alvelestat peaked by - . hrs and that it was eliminated thereafter . thus, the nei is believed to act quickly after sublingual immunization and to be eliminated within hrs. future studies will investigate the underlying mechanisms and whether nei can be used as a universal strategy for improving immune responses to sublingual vaccines regardless of the adjuvant or delivery system. ethics statement. all experiments were performed according to the guidelines for the care and use of laboratory animals adopted by the national institutes of health. the institutional animal care and use committee (iacuc) at the ohio state university approved all protocols. all efforts were made to avoid unnecessary pain and distress and to minimize animal suffering during the course of these studies. animals. female c bl/ j mice were obtained from the jackson laboratory (bar harbor, me). mice were maintained in a specific pathogen-free environment and used at ± wks of age. all the experiments were approved by and performed in accordance with the guidelines of nih and the ohio state university iacuc. sublingual immunization and samples collection. sublingual immunization was performed as previously described to address the effect of nei supplementation, groups of mice were given antigens plus μm of alvelestat (azd , c h f n o s) (selleckchem, houston, tx), or antigen plus ef plus alvelestat. mice that received the nei showed no change in their vitality, food consumption or body weight. all groups were immunized at weekly intervals for consecutive weeks (days , , and ). serum, fecal pellet and vaginal wash samples were collected weekly, and nasal washes were collected at day . to determine ova-specific and pa-specific antibody titers, elisa were performed with antigen-coated plates as described previously [ ] [ ] [ ] , briefly, microtiter plates were coated with ova ( mg/ml) or pa ( μg/ml). for detection of ova-or pa-specific igg and iga abs, serum or fecal material extracts were serially diluted in pbs % bsa, added to the plates and the binding antibodies were detected with hrp-conjugated anti-mouse γor α-heavy chain-specific antisera (southern biotech associates inc., birmingham, al). biotin-conjugated rat anti-mouse igg , igg a/c, igg b or igg monoclonal abs and hrp-conjugated streptavidin (bd bioscience, san jose, ca) were used to measure igg subclass responses. the reactions were revealed by addition of the water-soluble hrp substrate abts ( , ′-azinobis [ -ethylbenzothiazoline- -sulfonic acid]-diammonium salt, sigma-aldrich) and the ab titers were determined as the last dilutions of samples with an absorbance of > . above that of control samples from naïve mice. for assessment of iga levels in the intestinal secretions, freshly emitted fecal pellets were normalized by homogenization in pbs ( ml per . g feces). after centrifugation, dilutions of supernatants were used for evaluation of antigen-specific iga levels as described above. quantification of high affinity antibody responses. high affinity antibody responses were measured by elisa as described above with a minor modification. briefly, plates were coated with pa and incubated with dilutions of the samples. urea ( mm) was then added and the plates incubated for min at room temperature to remove antibodies that bound to the antigen with low affinity . after washing, detection antibodies were added and the remaining steps of the elisa conducted as described above. assessment of toxin neutralizing antibodies. toxin neutralization assay was performed as previously described [ ] [ ] [ ] . briefly, sample dilutions were added to j macrophages cultured in cultured in rpmi supplemented with % fetal calf serum. bacillus anthracis lethal toxin (letx) [i.e., pa plus bacillus anthracis lethal factor (lf, list biological, campbell, ca)] was then added to the plates. after overnight incubation, mtt ( -( , -dimethylthiazol- -yl)− , -diphenyl tetrazolium bromide; sigma-aldrich) was added to assess the viability of macrophages as a function of redox potential. toxin neutralizing antibody titers were determined as the lowest concentrations of sera that protect macrophages from the cytotoxicity of letx. mucosal immunity and vaccines inducing mucosal iga: a challenge for vaccine adjuvants and delivery systems topical immunization strategies contributions of edema factor and protective antigen to the induction of protective immunity by bacillus anthracis edema toxin as an intranasal adjuvant bacillus anthracis edema toxin acts as an adjuvant for mucosal immune responses to nasally administered vaccine antigens neutrophils negatively regulate induction of mucosal iga responses after sublingual immunization mucosal administration of cycle-di-nucleotide-adjuvanted virosomes efficiently induces protection against influenza h n in mice sublingual vaccination with sonicated salmonella proteins and mucosal adjuvant induces mucosal and systemic immunity and protects mice from lethal enteritis buccal and sublingual vaccine delivery sublingual targeting of sting with ′ ′-cgamp promotes systemic and mucosal immunity against anthrax toxins sublingual vaccination with influenza virus protects mice against lethal viral infection neutrophil function: from mechanisms to disease mechanisms underlying neutrophil-mediated monocyte recruitment the role of neutrophil elastase in chronic inflammation the potential of neutrophil elastase inhibitors as anti-inflammatory therapies azd : pharmacological characterization of a novel oral inhibitor of neutrophil elastase mechanisms for induction of acquired host immunity by neutrophil peptide defensins neutrophil granules: a library of innate immunity proteins neutrophil serine proteases: mediators of innate immune responses inhibitors of elastase stimulate murine b lymphocyte differentiation into igg-and iga-producing cells new concepts in the generation and functions of iga il- regulates aicda expression through mir- retinoic acid induces homing of protective t and b cells to the gut after subcutaneous immunization in mice differentiation and homing of iga-secreting cells interleukin (il)− promotes intestinal iga response to microbiota generation of gut-homing iga-secreting b cells by intestinal dendritic cells inflammatory lung secretions inhibit dendritic cell maturation and function via neutrophil elastase neutrophils slow disease progression in murine lupus via modulation of autoreactive germinal centers cyclooxygenase- orchestrates germinal center formation and antibody class-switch via regulation of il- plasticity of th cells in peyer's patches is responsible for the induction of t cell-dependent iga responses interleukin- regulates genes involved in b-cell terminal maturation il- induces igg isotype switch recombination in mouse cd -activated sigdpositive b lymphocytes transforming growth factor beta induces iga production and acts additively with interleukin for iga production pharmacokinetics and safety of azd , an oral neutrophil elastase inhibitor, in healthy volunteers and patients with copd ikkbeta in intestinal epithelial cells regulates allergen-specific iga and allergic inflammation at distant mucosal sites routes of allergic sensitization and myeloid cell ikkbeta differentially regulate antibody responses and allergic airway inflammation in male and female mice use of antibody avidity assays for diagnosis of severe acute respiratory syndrome coronavirus infection analysis of antigen-specific t helper cell cytokines. antigen-specific t helper cell cytokine responses were analyzed by flow cytometry after in vitro restimulation and intracellular staining with cytokine-specific fluorescent antibodies. briefly, splenocytes and cervical lymph nodes were collected on day after the first immunization and restimulated with antigen (i.e., ova) in vitro as previously described - . after days culture, cells were subjected to extracellular staining with lineage-specific antibodies [i.e., anti-cd , and anti-cd (biolegend, san diego, ca)] and, after fixation, to intracellular staining with th , th , th , and tfh cytokine-specific antibodies (biolegend). labeled cells were then analyzed with an attune nxt flow cytometer (thermo fisher scientific, waltham, ma). results are expressed as mean ±one standard deviation. statistical significance was determined by one-way anova, followed by tukey post-hoc test. all statistical analyses were performed with the statase . software (statacorp llc, college station, tx) and prism software (graphpad software, la jolla, ca). this work was supported by national institutes of health grants dk and ai . competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -ry o xj authors: ciotti, john robert; valtcheva, manouela v.; cross, anne haney title: effects of ms disease-modifying therapies on responses to vaccinations: a review. date: - - journal: mult scler relat disord doi: . /j.msard. . sha: doc_id: cord_uid: ry o xj background: : development of long-term immunologic memory relies upon humoral and cellular immune responses. vaccinations aim to stimulate these responses against pathogens. several studies have evaluated the impact of multiple sclerosis disease-modifying therapies on immune response to vaccines. findings from these studies have important implications for people with multiple sclerosis who require vaccination and are using disease-modifying therapies. methods: : searches using pubmed and other engines were conducted in may to collect studies evaluating the impact of various disease-modifying therapies on immune responses to vaccination. results: : several studies demonstrated preserved immune responses in people treated with beta-interferons to multiple vaccine types. limited data suggest vaccine responses to be preserved with dimethyl fumarate treatment, as well. vaccine responses were reduced to varying degrees in those treated with glatiramer acetate, teriflunomide, sphingosine- -phosphate receptor modulators, and natalizumab. the timing of vaccination played an important role in those treated with alemtuzumab. humoral vaccine responses were significantly impaired by b cell depleting anti-cd monoclonal antibody therapies, particularly to a neoantigen. data are lacking on vaccine responses in patients with multiple sclerosis taking cladribine and high-dose corticosteroids. notably, the majority of these studies have focused on humoral responses, with few examining cellular immune responses to vaccination. conclusions: : prior investigations into the effects of individual disease-modifying therapies on immune responses to existing vaccines can serve as a guide to expected responses to a sars-cov- vaccine. responses to any vaccination depend on the vaccine type, the type of response (recall versus response to a novel antigen), and the impact of the individual disease-modifying therapy on humoral and cellular immunity in response to that vaccine type. when considering a given therapy, clinicians should weigh its efficacy against ms for the individual patient versus potential impact on responses to vaccinations that may be needed in the future. multiple sclerosis (ms) is an immune-mediated demyelinating central nervous system (cns) condition characterized by attacks of neurologic symptoms disseminated in space and time that often leads to disability. ms affects over , people in the united states with enormous costs to society. ms disease-modifying therapies (dmts) act on the immune system, by modulation or suppression. this review assesses the current evidence regarding the impact of ms dmts on immune responses to existing vaccinations, highlighting implications for response to a potential vaccine against sars-cov- . an effective immune response that provides long-term immunologic memory is driven primarily by the adaptive immune system, consisting of b cells (responsible for humoral, or antibody-mediated, immunity) and t cells (responsible for cell-mediated immunity). when stimulated in the presence of their target antigen, b and t cells clonally expand, with some transforming into memory cells, able to rapidly proliferate and become effector cells upon re-exposure to their target antigen. upon activation, b cells can also differentiate into plasma cells that generate initially igm and then igg antibodies specific to the antigen. table summarizes vaccine types and how the immune responses they generate differ. humoral responses to vaccines are generally measured using titers of igg antibodies against the particular antigen, though use of the hemagglutination inhibition (hi) assay is an exception. the hi assay reports the inverse of the dilution (the titer) at which a patient's antibody-containing serum is no longer able to inhibit the viral hemagglutination property. for inactivated influenza vaccine, an hi titer of ≥ is considered protective. cellular immune responses to vaccines are less well-studied, and measurement methods are highly variable. irrespective of vaccine type, immune responses to vaccination are generally more robust in women, in whom ms has a predilection. vaccine safety in ms was a subject of debate throughout the s and s, as seasonal influenza, measles/mumps/rubella (mmr), hepatitis b (hbv), h n influenza, and human papillomavirus (hpv) ciotti jr, valtcheva mv, cross ah. effects of ms disease-modifying therapies on responses to vaccinations: a review. vaccines were all implicated and subsequently refuted as being linked to ms development or worsening. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] vaccine efficacy in ms has been less controversial, as studies of untreated ms patients have not shown differences in responses compared to healthy controls (hc). regulatory bodies now recommend vaccinating people with ms on a normal schedule, with some caveats regarding live attenuated vaccines. [ ] [ ] [ ] the various immunomodulatory and immunosuppressive effects of different dmts add complexity regarding vaccinations. live vaccines are generally contraindicated in ms patients on immunosuppressive treatments. mechanistically, dmts that impact the adaptive immune system may decrease the efficacy of vaccines by impairing the development of long-term memory. this review evaluates the current evidence regarding the impact of dmts for ms on vaccine responses in humans. a pubmed search was performed on may , for english language articles that were published between january , and may , using the mesh terms multiple sclerosis and vaccine with each individual dmt. articles not focusing on vaccine response in the setting of dmt use, such as basic pathophysiologic reviews, author commentaries, reports of vaccines used as ms therapy, and animal studies were excluded. additional references were obtained from a google search of each individual dmt and immunization and vaccination (may - , ), secondary review of the articles discovered in these searches, searches of clinicaltrials.gov (may , ) and cdc.gov (may , ), and review of manufacturer prescribing information for each dmt. bias was qualitatively assessed for each study and funding sources are noted in table . levels of evidence for each study are assigned based on the oxford centre for evidence-based medicine levels of evidence. ciotti jr, valtcheva mv, cross ah. effects of ms disease-modifying therapies on responses to vaccinations: a review. table provides a summary of all published studies of vaccine responses in people using fda-approved dmts for ms. a prospective, non-randomized, open label study compared responses to an inactivated influenza vaccine in relapsing ms patients taking interferon beta- a mcg three times weekly and untreated relapsing ms patients. there was no difference in the proportion of patients in each group with seroprotective hi titers ( . % beta-interferon group vs. . % untreated group), or the proportions mounting -fold ( . % vs. . %) and -fold ( . % vs. . %) increase in hi titers. this study offers level evidence that ms patients taking high-dose, high-frequency beta-interferon mount an appropriate immune response to the influenza vaccine. another study compared immune responses after seasonal influenza vaccination in teriflunomidetreated relapsing ms patients to beta-interferon-treated relapsing ms patients. for all influenza strains used, > % of those in the beta-interferon group had protective hi titers days postvaccination. ratios of post-vaccination to pre-vaccination geometric mean titers (gmt) were all ≥ . , indicating an effective immune response. this study was limited by lack of an untreated ms control group. level evidence. a prospective observational study evaluated the effects of the inactivated influenza vaccine in patients taking a variety of beta-interferon preparations, comparing anti-influenza igm and igg titers to those in hc at multiple time-points post-vaccination. no significant difference between groups was found in the degree or duration of these humoral immune responses, with the exception of a significantly higher anti-influenza b igg titer at days and in the beta-interferon group. cellular immune responses were also compared by measuring the frequency of t cells secreting gammainterferon in response to influenza antigen, with no differences between groups. level evidence. the same investigator group performed another observational study of ms patients taking betainterferons and compared influenza vaccine responses at multiple post-vaccination intervals to hc. no differences in the proportion reaching a protective hi titer were observed between the two groups at any time, including at the peak antibody response time of months ( . % in the beta-interferon group vs. . % in hc). level evidence. another observational study evaluating vaccine responses in patients taking dimethyl fumarate included an arm of relapsing-remitting ms (rrms) patients taking beta-interferons. igg titers were assessed pre-and post-vaccination with vaccines to assess different types of immune responses: tetanus-diphtheria toxoid vaccine to assess t-cell dependent anamnestic humoral response, -valent pneumococcal polysaccharide vaccine (ppsv ) to assess t-cell independent humoral response, and quadrivalent meningococcal conjugate vaccine (mcv ) to assess neoantigen responses. those with a ≥ -fold rise in igg levels after vaccination were considered responders. for anti-tetanus/diphtheria, there was no difference in the responder proportion (dimethyl fumarate group % vs. beta-interferon group %). pneumococcal vaccination responses were not significantly different between the two groups, though there was considerable variability in gmt ratios across serotypes. neoantigen responses ciotti jr, valtcheva mv, cross ah. effects of ms disease-modifying therapies on responses to vaccinations: a review. to mcv were not different, with % of each group demonstrating a -fold rise in igg. post-to prevaccination gmt ratios were similar in the dimethyl fumarate and beta-interferon groups ( . vs . , respectively). level evidence. a prospective, multicenter, non-randomized study evaluated influenza vaccine responses in patients treated with a variety of dmts. patients taking beta-interferons showed a significantly greater proportional vaccine response as measured by hi titer than other dmt groups taking glatiramer acetate, fingolimod, and natalizumab. beta-interferon-treated patients reached seroprotective rates of > % for each strain, and reached protective hi titers to all strains ( . % of patients) more frequently than those treated with glatiramer acetate ( . % of patients), fingolimod ( . % of patients), and natalizumab ( . % of patients). this study was limited by lack of an untreated control group and low numbers, especially in the fingolimod and natalizumab groups. level evidence. together, these studies convincingly demonstrate adequate immune responses to a variety of vaccine mechanisms in ms patients treated with beta-interferons. although immune responses to influenza vaccines were observed in glatiramer acetate-treated patients in these studies, the results suggest that responses were reduced compared to hc and to those treated with beta-interferons. these studies regarding inactivated vaccination responses may not be generalizable to other vaccine types (such as live attenuated, nucleic acid, recombinant vector, or subunit vaccines), for which immune responses have not been reported in people on glatiramer acetate. a study already mentioned in the beta-interferon section investigated the effect of teriflunomide on influenza vaccination responses in ms patients. this non-blinded, nonrandomized, multicenter, multinational, parallel-group study included patients in groups: teriflunomide mg (n= ), teriflunomide mg daily (n= ), and beta-interferons (n= , the reference population). more than % of all patients in all groups achieved seroprotection (hi titer ≥ ) for the h n and influenza b antigens. seroprotection was lower in the h n teriflunomide mg group ( . %), compared to % ciotti jr, valtcheva mv, cross ah. effects of ms disease-modifying therapies on responses to vaccinations: a review. in the mg per day teriflunomide and beta-interferon groups. gmt ratios were reduced in the teriflunomide groups ( . - . ) compared to the beta-interferon group ( . - . ) . a limitation of this study is that it was not powered for comparisons of immune responses in the teriflunomide and betainterferon groups. level evidence. a prospective, randomized, double-blind, parallel-group, placebo-controlled study compared antibody responses to rabies vaccine (neoantigen) and delayed type hypersensitivity (recall) to candida albicans, trichophyton, and tuberculin in healthy people assigned to mg/day teriflunomide with healthy individuals assigned to placebo. gmts for rabies antibodies were lower with teriflunomide than with placebo, but all subjects assigned to teriflunomide achieved seroprotective antibody levels. teriflunomide had no adverse impact on the cellular memory response to recall antigens. level evidence. overall, these studies indicate modest negative effects of teriflunomide mg/day on immune response to influenza and rabies vaccinations. . . . dimethyl fumarate. a single open-label, multicenter, non-randomized study evaluated the effects of dimethyl fumarate treatment on vaccination responses. patients on dimethyl fumarate mg twice daily were compared to patients treated with beta-interferon after vaccination with vaccines to assess different types of immune responses. this study is discussed in detail in the section on betainterferons above and provided level evidence that dimethyl fumarate treatment did not reduce t-cell dependent and humoral immune responses. igm and igg, and the frequency of gamma-interferon secreting cells after immunization, were not significantly altered by fingolimod treatment compared to hc. however, the two groups were not well matched, with hc being younger (mean age , range - ) than the ms patients (mean age , range - ), and hc were % female compared with % female in the ms group. level evidence. a blinded, randomized, multicenter, placebo-controlled study of response to seasonal influenza vaccine and tetanus toxoid (tt) booster was performed in relapsing ms patients on either fingolimod . mg/day (n= ) or placebo (n= ). at weeks post-vaccination, responder rates (proportion achieving seroprotective hi titers or a -fold increase in antibody titers against at least one influenza strain) for fingolimod vs. placebo, respectively, were % vs. %. at weeks, responder rates were % vs. %. for tt, responder rates were % vs. % at weeks and % vs. % at weeks. although many fingolimod-treated ms patients were able to mount protective immune responses, this study provided level evidence that response rates were reduced in patients on fingolimod compared with placebotreated patients. fifteen patients on fingolimod were among the ms patients and hc included in a prospective study to measure antibody responses to the / influenza a h n and h n vaccine viruses. the fingolimod group developed reduced rates of seroprotection to h n compared with controls or ms patients on beta-interferons and glatiramer acetate. at months, months, and months, seroprotection rates were . %, . %, and . % in the fingolimod group vs. . %, %, and . % in hc. the response to h n was even poorer in those on fingolimod, with . % protected at months, . % protected at months, and % at months post-vaccination compared with . %, %, and . % for hc. level evidence. ciotti jr, valtcheva mv, cross ah. effects of ms disease-modifying therapies on responses to vaccinations: a review. a non-randomized, prospective, non-controlled study of ms patients who underwent seasonal influenza vaccination discussed in earlier sections of this review included people on fingolimod. a lower proportion of fingolimod-treated patients achieved protection to h n and influenza b compared to those on beta-interferons or glatiramer acetate. interpretation of these results is limited by the very small size of the fingolimod subgroup. level evidence. taken together, these studies indicate that concurrent fingolimod reduces immune response to influenza vaccinations. . . . siponimod. responses to seasonal influenza and ppsv vaccines were assessed in healthy persons treated with siponimod mg/day or placebo. the randomized, prospective study enrolled people per group into siponimod treatment groups and a placebo group. treatment groups were "preceding siponimod" (stopping days prior to immunization), "concomitant" (non-interrupted siponimod), and "interrupted siponimod" (treatment interrupted days prior to and for days after immunization). the durations of stopping or interrupting siponimod were based on the known time of - days for circulating lymphocytes to return after drug discontinuation. each person received seasonal influenza and ppsv vaccines, with blood samples obtained at baseline and multiple times after immunization. seroprotection rate > %, gmt increase of > . vs. baseline, and igg response rate of > % were examined. at days, each group exceeded the % response threshold and a gmt increase > . -fold for both influenza a antigens compared with baseline. for one of the two influenza b viruses, the seroprotection response threshold of > % was not met for the interrupted and concomitant siponimod groups. over % in each group responded to ppsv with > -fold increase in igg on day vs. baseline. compared to the placebo group, the proportions of people with titer increased > -fold at day were decreased in the concomitant and interrupted siponimod groups for h n , h n , and one of the influenza b viruses. gmts over time were lower for the concomitant siponimod group for both influenza a strains and one of the influenza b strains compared to the other ciotti jr, valtcheva mv, cross ah. effects of ms disease-modifying therapies on responses to vaccinations: a review. groups. this study provides level evidence of a lower response to influenza vaccines in those on siponimod at time of vaccination. stopping siponimod at least days prior to administration of a vaccine and resuming siponimod (after up-titration) or more weeks later is a potential strategy to improve vaccine response. . . . ozanimod. no relevant studies were found. no relevant studies have been reported in ms patients on oral cladribine. a vaccine study is being planned by the manufacturer. an early study of natalizumab-treated ms patients ( female) and hc ( female) examined antibody response to seasonal influenza vaccination. mean antibody titers to influenza a and b were not different between the two groups, with a non-significant trend towards lower titers to influenza a for the natalizumab group. this study was likely underpowered, and the study groups were not well matched. level evidence. a randomized, controlled, open-label study of people with relapsing ms was done to study the response to a recall antigen (tt) and the neoantigen keyhole limpet hemocyanin (klh). patients were randomized : to control or natalizumab groups. the control group received immunizations shortly after randomization and delayed starting natalizumab until after day , whereas those randomized to natalizumab were treated with natalizumab beginning months prior to immunizations. a lower proportion of those in the natalizumab group responded to tt and to klh at day . although the differences were not statistically significant, the study may have been underpowered. level evidence. ciotti jr, valtcheva mv, cross ah. effects of ms disease-modifying therapies on responses to vaccinations: a review. a previously mentioned real-world study of ms patients and hc examined response to the h n pandemic "swine flu" vaccine. seventeen of the ms patients in that study were on natalizumab. only of the ( . %) achieved seroprotective hi titers after immunization, compared to of controls ( . %) and of ( . %) of those on beta-interferon. level evidence. the same group of investigators performed a prospective study of responses to the seasonal influenza vaccination in / in ms patients on four different immunomodulatory therapies and hc at baseline and , , and months post-immunization. the proportion of those few patients on natalizumab (n= at months, n= at months, and n= at months) that had adequate response to the immunization was consistently % or more lower than hc and ms patients on beta-interferons. in the previously-discussed non-randomized, prospective, study of ms patients who underwent seasonal influenza vaccination, were on natalizumab. for h n and the influenza b antigen, only . % and . %, respectively, of those on natalizumab achieved sufficient response, compared to . % and . % for the people taking beta-interferon. level evidence. overall, these studies provide evidence that an inadequate response to some immunizations occurs in a sizeable proportion of people being treated with natalizumab. response to the seasonal influenza vaccine tested response to an inactivated vaccine, and immunization with klh tested the humoral response to a previously unknown antigen. the ocrelizumab group had a poorer humoral response to vaccinations. . % of the ocrelizumab group vs. . % of the control group had responded ( -fold increase in antigen-specific igg from baseline or development of protective antibody levels) to tt booster at weeks post-vaccination. positive response to ≥ serotypes in ppsv at weeks was . % in the ocrelizumab and % in the control group. the pcv booster did not enhance the response to serotypes in common with ppsv in the ocrelizumab group, whereas it did for the control group. the humoral response to klh was greatly decreased in the ocrelizumab group vs. the control group. after immunization with klh, the gmts for igm and igg for the control group were almost , and , , respectively, but were less than for igm and igg in those treated with ocrelizumab. seroprotective titers at weeks against five influenza strains (season / and / ) ranged from . % to . % in the ocrelizumab group, compared to . % to . % in the control group. level evidence. . . . rituximab. responses to vaccination were studied in non-ms populations treated with the b cell depleting chimeric monoclonal antibody, rituximab. in one study of rheumatoid arthritis patients, patients were randomized : to take rituximab mg iv twice two weeks apart in addition to methotrexate ( - mg po weekly) vs. methotrexate alone. patients in each treatment group were examined for response to tt, ppsv , and klh, and for dth to candida albicans. these two studies indicate that responses to neoantigens and t cell-independent antigens are greatly reduced by b cell depletion with anti-cd monoclonal antibody treatments. recall responses to the t cell-dependent tt antigen and dth responses were less affected by b cell depletion, with some differences noted in response to tt between the two studies which used different b-cell depleting agents. both studies were done in the first year after b cell depletion; responses might change after longer treatment duration. twenty-four people with ms taking alemtuzumab for median months (range . to months) took part in an investigation of the effects of alemtuzumab on vaccination responses. to test t-celldependent antigen recall responses, igg levels were measured before and weeks after vaccinations with diphtheria and tetanus in ms patients taking alemtuzumab, and in patients taking alemtuzumab before and weeks after inactivated polio , , and . pre-vaccination, all had protection to diphtheria and tt that was maintained after alemtuzumab. protection improved from % to % for polio and from % to % for polio after vaccination. at the time of vaccination, the median cd t-cell count was low and median cd t-cell and cd b-cell counts were normal. ppsv was used to test responses to t-cell-independent antigens. of the ms patients who were immunized, the proportion achieving seroconversion for serotypes and exceeded that of literature controls. similarly, the proportion protected against haemophilus influenzae type b and meningococcal group c (neoantigen) increased from % and %, respectively, to % and % post-vaccination, equivalent to published seroconversion rates for controls. the investigators noted that vaccination within months of treatment resulted in a smaller proportion of responders. this study provides level ciotti jr, valtcheva mv, cross ah. effects of ms disease-modifying therapies on responses to vaccinations: a review. evidence that response to prior vaccinations is maintained following alemtuzumab treatment, but suggests that vaccinations be delayed until at least at months after alemtuzumab treatment. several studies in non-ms patient populations (e.g. asthma, rheumatoid arthritis, systemic lupus erythematosus) have provided level evidence of minimal impact of chronic oral corticosteroids on vaccine responses. [ ] [ ] [ ] [ ] however, the doses of corticosteroids in these studies were all lower than those typically used for ms relapses. in their guidelines, the infectious diseases society of america recognized the lack of data on vaccine efficacy in people treated with high doses of corticosteroids (≥ mg prednisone equivalents for ≥ days). it is generally recommended to avoid administering live vaccines during treatment with and until at least weeks after discontinuing high-dose corticosteroids. , this research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. ciotti jr, valtcheva mv, cross ah. effects of ms disease-modifying therapies on responses to vaccinations: a review. responses to inactivated and toxoid vaccines were diminished in those taking fingolimod at the time of vaccination. responses to the inactivated influenza vaccine were diminished in those taking siponimod at the time of vaccination. the prevalence of ms in the united states: a population-based estimate using health claims data fundamentals of vaccine immunology measuring vaccine responses in the multiplex era standardization of hemagglutination inhibition assay for influenza serology allows for high reproducibility between laboratories sex and gender differences in the outcomes of vaccination over the life course vaccines and multiple sclerosis: a systematic review adverse effects of vaccines: evidence and causality influenza vaccination in ms: absence of t-cell response against white matter proteins effects of ms disease-modifying therapies on responses to vaccinations: a review a multicenter, randomized, double-blind, placebocontrolled trial of influenza immunization in multiple sclerosis quadrivalent hpv vaccination and risk of multiple sclerosis and other demyelinating diseases of the central nervous system seasonal and h n v influenza vaccines in ms: safety and compliance vaccinations and the risk of relapse in multiple sclerosis. vaccines in multiple sclerosis study group vaccines and the risk of multiple sclerosis and other central nervous system demyelinating diseases acip altered immunocompetence guidelines for immunizations | recommendations | cdc immunization and multiple sclerosis: recommendations from the french multiple sclerosis society infectious complications of multiple sclerosis therapies: implications for screening, prophylaxis, and management. open forum infect dis effects of ms disease-modifying therapies on responses to vaccinations: a review vaccination against infection in patients with multiple sclerosis immune response to influenza vaccine is maintained in patients with multiple sclerosis receiving interferon beta- a preserved antigen-specific immune response in patients with multiple sclerosis responding to ifnβ-therapy immunotherapies influence the influenza vaccination response in multiple sclerosis patients: an explorative study antibody response to seasonal influenza vaccination in patients with multiple sclerosis receiving immunomodulatory therapy immune response to vaccines is maintained in patients treated with dimethyl fumarate effects of ms disease-modifying therapies on responses to vaccinations: a review immunization responses in rheumatoid arthritis patients treated with rituximab: results from a controlled clinical trial immune competence after alemtuzumab treatment of multiple sclerosis influenza vaccination in kidney transplant recipients: cellular and humoral immune responses antibody levels and response to pneumococcal vaccine in steroid-dependent asthma immunogenicity and safety of pneumococcal vaccination in patients with rheumatoid arthritis or systemic lupus erythematosus safety and efficiency of influenza vaccination in systemic lupus erythematosus patients idsa clinical practice guideline for vaccination of the immunocompromised host effects of ms disease-modifying therapies on responses to vaccinations: a review ciotti jr, valtcheva mv, cross ah. effects of ms disease-modifying therapies on responses to vaccinations: a review. key: cord- -msuluuuu authors: ballesteros-briones, maría cristina; silva-pilipich, noelia; herrador-cañete, guillermo; vanrell, lucia; smerdou, cristian title: a new generation of vaccines based on alphavirus self-amplifying rna date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: msuluuuu dna or mrna vaccines have potential advantages over conventional vaccines since they are easier to manufacture and have higher safety profiles. in particular, self-amplifying rna (sarna) derived from alphavirus expression vectors has shown to be very efficient to induce humoral and cellular responses against many antigens in preclinical models, being superior to non-replicating mrna and dna. this is mainly due to the fact that sarna can provide very high expression levels and simultaneously induces strong innate responses, potentiating immunity. sarna can be administered as viral particles or dna, but direct delivery as rna represents a safer and more simple approach. although sarna can be delivered as naked rna, in vivo transfection can be enhanced by electroporation or by complexing it with cationic lipids or polymers. alphavirus sarna could have broad application to vaccinate against human pathogens, including emerging ones like sars-cov- , for which clinical trials have been recently initiated. a new generation of vaccines based on alphavirus self-amplifying rna maría cristina ballesteros-briones , , noelia silva-pilipich , , guillermo herrador-cañ ete , lucia vanrell and cristian smerdou dna or mrna vaccines have potential advantages over conventional vaccines since they are easier to manufacture and have higher safety profiles. in particular, self-amplifying rna (sarna) derived from alphavirus expression vectors has shown to be very efficient to induce humoral and cellular responses against many antigens in preclinical models, being superior to non-replicating mrna and dna. this is mainly due to the fact that sarna can provide very high expression levels and simultaneously induces strong innate responses, potentiating immunity. sarna can be administered as viral particles or dna, but direct delivery as rna represents a safer and more simple approach. although sarna can be delivered as naked rna, in vivo transfection can be enhanced by electroporation or by complexing it with cationic lipids or polymers. alphavirus sarna could have broad application to vaccinate against human pathogens, including emerging ones like sars-cov- , for which clinical trials have been recently initiated. the use of nucleic acids to induce immune responses against encoded antigens represents a very attractive type of vaccines, compared to more classical approaches based on peptides, proteins, attenuated and inactivated viruses, or viral vectors expressing antigens. the first nucleic acid vaccines to be tested in animals were based on plasmid dna, resulting in induction of both humoral and cellular immune responses against a great variety of antigens [ ] . soon it became clear that to obtain optimal results, dna had to be delivered in vivo by complexing it with different molecules, like cationic lipids, polymers or peptides, that could compact it and facilitate entry through the cytoplasmic membrane [ ] . physical methods, like electroporation or gene gun, were also effective at enhancing dna delivery. however, a dna vaccine presents some drawbacks, for example, it needs to reach the cell nucleus to be functional, a process that is usually not very efficient. at the same time, its presence in the nucleus gives rise to the possibility of genomic integration and induction of oncogenic processes. besides good results in preclinical models, dna vaccination has been relatively modest in inducing immune responses in humans, and no dna vaccine has been approved for clinical use so far [ ] . an alternative to dna is the use of messenger rna (mrna). this molecule presents the same problems for delivery as dna, but it does not need to reach the nucleus to be expressed, facilitating transfection. in addition, mrna cannot integrate, increasing the safety profile of this approach. however, mrna can be easily degraded by extracellular ribonucleases present in skin and blood. this problem can be avoided by complexing mrna with compounds able to protect it against degradation and facilitate cellular uptake, as will be discussed in this review. besides these advantages, mrna vaccination has not demonstrated efficacy until recently, thanks to the development of new technologies to synthesize more stable rna molecules and new methodologies for efficient in vivo delivery [ ] . a type of mrna that has shown extraordinary properties to induce immune responses is the so-called self-amplifying rna (sarna). sarna is derived from the genome of certain viruses like alphaviruses and flaviviruses and has the capacity of selfamplification due to the fact that it expresses a viral replicase (rep), while the genes coding for the viral structural proteins have been substituted by the transgene of interest [ ] . most sarnas used in vaccination studies derive from alphaviruses, including venezuelan equine encephalitis virus (veev), semliki forest virus (sfv), and sindbis virus. as depicted in figure , when sarna enters the cell cytoplasm, it will translate rep, which will copy this long mrna into a complementary negative strand rna that will be used by rep to make more sarna. simultaneously, rep recognizes a subgenomic promoter in the negative strand from which it will make a smaller mrna (subgenomic rna). this mrna will be produced at levels ten-fold higher than those of genomic rna, leading to high production of antigen in vivo. this, together with the intrinsic properties of sarna in inducing innate immune signals, like activation of several toll-like receptors (tlrs) within cells, contributes to the generation of very strong immune responses. in addition, duration of expression from sarna delivered encapsulated in lipid nanoparticles is longer than the one obtained with mrna, being able to last almost two months in vivo when expressing a reporter gene [ ] . although sarna can also be delivered by packaging it into viral particles (vps) or by launching its expression in cells from a plasmid [ ] , this review will focus on direct delivery of sarna for vaccination. naked sarna is the simplest strategy to deliver rna into cells. the first evidence that naked sarna could be used for vaccination was reported in by zhou et al. [ ] , when they showed that intramuscular (im) injection of mg of sfv rna carrying influenza virus nucleoprotein (sfv-np) induced specific humoral responses in mice, although antibody titers were lower than those obtained with sfv-np vps. a few years later, ying et al. [ ] demonstrated that with only . mg of sfv expressing b-galactosidase (b-gal) rna given im it was possible to elicit antibody and cd t cell responses. this type of immunization was able to protect mice from a challenge with colon tumor cells expressing b-gal, used as a surrogate tumor antigen. other groups have shown that naked sarna can induce immune responses in mice against human pathogens like rabies virus [ ] , influenza preventive and therapeutic vaccines current opinion in virology sarna vectors based on alphavirus. the sarna vector is a positive strand rna containing the genes coding for the viral replicase (rep) and the gene of interest (goi) downstream of a subgenomic promoter (sgpr). upon entry of sarna into cells (i) rep can be translated, being able to use sarna as template to make a complementary negative sarna (-sarna) strand (ii). rep can also use this negative rna as template to make more sarna (+sarna), allowing its self-amplification (iii). in addition, rep can recognize the sgpr in the negative strand from which a subgenomic mrna (+sgrna) of positive polarity is synthesized (iv). sgrna can be translated to produce the desired antigen at very high levels, which will be secreted if having an appropriate signal peptide (v). both +sarna and +sgrna contain a cap at the end and are polyadenilated (not shown). virus [ ] or human immunodeficiency virus (hiv) [ , ] . in particular, moyo et al. cloned highly conserved regions of hiv- gag and pol proteins into a sfv vector to generate mosaic sarna vaccines, which were administered im, generating specific t cell responses [ ] . interestingly, these responses had different time-courses compared to virus-based immunization, mediating a gradual induction of t cells during five weeks, with sustained persistence. it was suggested that this delay in the immune response could be due to a lack of immunogens in the first stage of treatment, until sarna could be translated. despite these encouraging results, several groups have shown that the potency of naked sarna vaccines can be greatly improved by electroporation [ ] , or by complexing them with lipid or polymer formulations [ , ] , as it will be discussed (table and figure ). in vivo electroporation (ep) can enhance the potency of sarna vaccines, by increasing transfection efficiency as shown for the first time by piggott et al. [ ] . in this sense, the skin is a very attractive tissue for immunization, as it is highly immunocompetent and easily accessible, making intradermal (id) ep a non-invasive procedure. the enhancement of immune responses by sarna ep was confirmed by johansson et al. [ ] using sfv vectors expressing b-gal or luciferase (luc) fused to a cd t cell ovalbumin epitope, respectively. this study showed that conventional mrna-immunized mice failed to develop any detectable immune responses, even in combination with ep, indicating that rna replication plays a major role in the induction of immunity. the ability of ep to induce immune responses against human pathogens, like hiv, has been shown using a chimeric veev sarna engineered to contain the terminal untranslated sequences of sindbis virus (cveev), which was injected im in mice [ ] . again, the authors showed that ep mediated higher protein expression and enhanced antigen-specific cellular and humoral immune responses than injection of naked sarna without ep. to get a deeper insight into the mechanism by which ep enhances sarna immune responses, huysmans et al. [ ] have recently characterized the expression kinetics and innate immune responses induced by id delivery of a veev-luc sarna, using as control conventional mrna and a plasmid expressing the same protein (pdna). expression from both sarna and pdna was increased self-amplifying rna vaccines ballesteros-briones et al. table relevant recent sarna-based vaccination strategies with ep, although this effect was significantly more pronounced for sarna. in this study, they also observed striking differences in the expression time-course between ep and lipid nanoparticles (lnps) used for sarna delivery. while sarna ep resulted in a plateau expression between days À , expression after lnp delivery peaked at hour followed by a sharp drop. this could be explained by the fact that sarna delivered by ep generated lower innate immune responses compared to lnp delivery. most preclinical studies using mrna have been performed in mice, which may not mimic humans. in the case of pdna-based vaccines, it is well-known that their efficacy is much lower in larger animals and humans than in mice [ ] . a first attempt has been made in pigs using sarna delivered id in combination with ep [ ] . in this study, the expression obtained with a veev-luc sarna was longer compared to pdna and non-replicating mrnas. while sarna maintained expression during at least days, pdna showed a maximum of expression at day one followed by a steep drop at day two, possibly due to epigenetic silencing [ ] . the fact that porcine and human skin have similarities, make these results more translatable to humans. although the skin is a promising tissue for immunization, it has some shortcomings that should be considered. on one hand, it is a very large and heterologous organ, hence caution should be taken when choosing the immunization site. in mice, ep of a veev-luc sarna at the tail base resulted in a significantly higher and longer luc expression compared with the same administration at the flank [ ] . this observation emphasizes the fact that location of the id immunization should be chosen with thoughtfulness in human clinical trials. on the other hand, the skin contains high levels of rnases that act as a natural protective mechanism against pathogens, which might preventive and therapeutic vaccines be a drawback for rna-based vaccines. preclinical studies delivering sarna id in combination with ep had shown a high variability in expression levels, which could be due to rna degradation. the addition of placental rnase inhibitor to sarna before id injection was shown to increase efficacy and reproducibility of expression in mice [ ] . lnps have demonstrated to be a powerful tool for sarna delivery, generating several vaccine platforms against infectious diseases. this success has been possible thanks to critical advances in lnp formulations focused at improving i) stability ii) infectivity, iii) cytosolic delivery, iv) low immunogenicity, v) capacity to induce humoral and cellular immune responses, and vi) low reactogenicity. the first report on the use of lnps to deliver sarna was described by geall et al. [ ] using a technology previously developed for sirna delivery, based on the use of the ionizable cationic lipid , -dilinoleyloxy- -dimethylaminopropane (dlindma) as a main lnp component. this type of lnp-delivered sarna vaccines, named sam (for self-amplifying mrna) platform, have shown great potential to generate immune responses against influenza virus [ ] [ ] [ ] and toxoplasma gondii [ ] . the group of dr. geall (novartis vaccines, cambridge, ma) has also described an alternative lnp system based on a cationic nanoemulsion (cne), able to bind sarna, enhancing its delivery and increasing the potency of the vaccine [ ] . cne is composed of cationic lipid dotap ( , -dioleoylsn-glycero- -phosphocholine) emulsified with mf , a novartis's proprietary adjuvant based on squalene with a good clinical safety profile. an advantage of cne formulation is that it can be stockpiled separated from sarna, which can be admixed before administration. despite the fact that rna is exposed on the outside of these particles, it is protected from degradation by rnases [ ] . this protection has also been observed for lnps based on cationic lipids formulated with sarna adsorbed to their surface [ ] . the cne platform has shown to be very efficient at inducing immune responses against human pathogens like respiratory syncytial virus [ ] , human cytomegalovirus [ ] , influenza virus [ , ] , hiv [ , ] , and streptococci [ ] , using different animal models. in particular, evidence has been provided that hiv vaccination with a relatively low dose of sarna ( mg) was both safe and immunogenic in nonhuman primates [ ] . the cne system has even been employed to deliver a live-attenuated veev vaccine using the full-length rna genome of veev tc- attenuated strain [ ] . this vaccine induced immune responses similar to those of tc- vps, providing % protection against veev challenge in mice. the advantage of this strategy is that it can eliminate the need for live-attenuated vaccine production, although it does not prevent the possibility of reversion. to make the vaccine safer, the authors generated a second version in which they completely deleted the tc- capsid gene. despite being less immunogenic, this second formulation resulted in significant protection against veev challenge [ ] . regarding the mechanism by which sarna vaccination can induce potent immune responses, it has been proposed that upon im vaccination antigen is expressed in muscle cells and then transferred to antigen presenting cells (apcs), suggesting a cross-priming mechanism able to prime cd t-cells [ ] . following this rationale, manara et al. investigated the possibility to enhance sarna-induced immune responses by increasing recruitment of apcs at the injection site [ ] . for that purpose, they combined a sarna expressing influenza virus nucleoprotein (np) with a second sarna expressing granulocyte-macrophage colony-stimulating factor (gm-csf), a chemoattractant for apcs. vaccination of mice with this sarna combination formulated in cne significantly improved np-specific cellular responses and provided increased protection against influenza virus challenge. a different strategy to increase apcs sarna uptake was described by goswami et al. [ ] , based on the inclusion of mannose-cholesterol amine conjugates in lnps. since apcs express significant amounts of mannose receptors on their surface, this strategy increased humoral and cellular responses against influenza hemagglutinin (ha) expressed from a sarna. a similar approach has been used by perche et al. [ ] using neutral lipopolyplexes (lpps), a tripartite formulation with sarna, a cationic polymer, and anionic liposomes, including a mannosylated lipid to enhance transfection of dendritic cells (dcs). a great effort has been performed in optimizing lnp formulations in order to obtain more stable particles and higher in vivo expression. of note, erasmus et al. [ ] generated a highly stable nanostructured lipid carrier (nlc) based on a mixture of a solid lipid (glyceryl trimyristate-dynasan ) and liquid oil (squalene) able to form a semi-crystalline core upon emulsification. as in the case of cne, this nlc could be stored separated from sarna and admixed at the time of use, greatly facilitating its production. in addition, they showed that combining it with only ng of a veev sarna expressing zika virus antigens could completely protect mice against a lethal viral challenge. using a different approach to enhance complexation and delivery of sarnas, he et al. [ ] developed cationic nanolipoprotein particles, based on a discoidal lipid bilayer stabilized by highdensity lipoprotein. interestingly, these complexes required less amount of cationic lipids compared to other lnp platforms and were efficient for in vivo delivery of sarna. regarding strategies to increase in vivo expression, blakney et al. [ ] observed that by including cephalin (a zwitterionic lipid) in lnp formulations containing veev sarna, luc expression in human skin explants was increased by sevenfold. interestingly, most transfected cells were immune cells, which highlights the potential of this approach for id vaccination. the same group tried to optimize immune responses by using cationic adjuvant formulations combined with tlr / agonists, using sarna expressing the major outer membrane protein (momp) of chlamydia trachomatis [ ] . despite obtaining good momp-specific cellular and humoral responses, immunogenicity was unaffected by tlr-agonists incorporation, and self-adjuvanting effects of sarna seemed to dominate the immune response. an interesting approach to generate broadly neutralizing antibodies against hiv was based on the use of lnps to deliver a veev sarna expressing a gp domain fused to lumazine synthase, a bacterial protein which self-assembles into -mer protein nanoparticles [ ] . this strategy elicited high titers of gp -specific antibodies in mice, and increased levels of antigen-specific germinal center b cells compared to protein immunization, representing a vaccine with potential interest for hiv. vaccines based on sarna conjugated to polyethylenimine (pei) besides lnps, mrnas can also be efficiently delivered if compacted into small particles using cationic polymers like pei, which contain primary amines that facilitate rna condensation, protect rna and enhance cellular uptake. dé moulins et al. [ ] showed for the first time that sarna could be complexed with linear pei to be efficiently delivered to dcs, inducing both humoral and cellular immune responses in vivo against influenza virus ha and np proteins. in a follow-up publication, they fine-tuned pei complexes in order to improve sarna delivery [ ] . thus, they found that three parameters were of paramount importance: (i) pei molecular weight; (ii) sarna:pei (weight:weight) ratio; and (iii) inclusion of cell penetrating peptides. using optimized pei complexes co-administered with a sting-agonist (c-di-amp) they were able to induce immune responses in pigs against influenza virus proteins expressed from a sarna. although in these two studies the authors used sarna derived from the pestivirus classical swine fever virus, these strategies could be equally valid to deliver alphavirus-based sarna. indeed, a medium-length peibased formulation has been used to efficiently deliver an alphavirus sarna expressing influenza virus ha, being able to induce protection in mice against h n influenza challenge using a single dose of . mg of rna [ ] . pei was also used to complex an sfv-based sarna vaccine expressing hiv antigens, resulting in specific plurifunctional cd and cd t cell responses that were higher than those obtained with naked sarna when injected im in mice. furthermore, these responses were sustained during weeks following a single vaccine administration [ ] . a new type of polymer for sarna delivery, called pabol, has been recently developed by the group of robin shattock and molly stevens at imperial college london (london, uk) [ ] . pabol is based on a bioreducible, linear, cationic poly(cba-co- -amino- -butanol) polymer that enhances protein expression and cellular uptake in vivo compared to commercially available pei. interestingly, increasing the molecular weight of pabol enhances delivery efficiency for sarna. besides technological improvements to deliver sarna, reviewed in the previous sections, some recent studies have addressed the possibility to optimize the vector itself. in particular, beissert et al. [ ] have developed a trans-amplifying rna (ta-rna) vaccine based on a bipartite sfv system in which the gene of interest is expressed from a sarna devoid of replicase, providing this one in trans by a non-replicating mrna (mrna-rep) (figure ). despite the fact that the ta-rna system was able to induce good immune responses in vivo against influenza virus ha, it did not outperform vaccination with a single sarna molecule expressing the same antigen. however, this novel ta-rna system might have advantages for vaccination over the single vector system in terms of versatility and ease of manufacturing, since mrna-rep could be produced and stored in advance. one limitation on the efficacy of sarna-based vaccines is the fact that they induce strong innate host immune responses, which could limit the intensity and duration of transgene expression [ ] . minimizing ifn responses could be a useful strategy to increase vaccine potency. this could be achieved by co-administration of compounds able to block ifn responses, like for example vaccinia virus immune evasion proteins [ ] . a different approach to boost sarna vaccines has been based on in vitro evolution of rna replicons in ifn-competent cells [ ] . this strategy led to the identification of six mutations in veev nonstructural proteins (nsps) that promoted subgenomic rna expression. sarna containing an optimal combination of mutations and expressing interleukin- were tested in a murine melanoma murine tumor model, providing enhanced therapeutic activity. the recent covid- pandemic produced by sars-cov- has prompted an unprecedented rapid development of many vaccine formulations, including three prototypes based on sarna. one of them was developed at imperial college london and is based on veev sarna expressing a pre-fusion stabilized sars-cov- spike protein encapsulated in lnps [ ] . this vector induced highly-specific neutralizing antibodies, as well as cellular responses, in mice and is currently being tested in a phase i clinical trial performed with volunteers in the uk. pfizer inc. (new york, ny) in partnership with biontech (germany) has also developed a sarna prototype vaccine, although the details of this vector/formulation have not been disclosed yet. finally, a third candidate based on veev sarna expressing sars-cov- spike encapsulated with lipid inorganic nanoparticles (lion) has been developed by the university of washington in partnership with hdt bio corp. (seattle, wa) [ ] . this vaccine was able to induce neutralizing antibodies in old mice, as well as in nonhuman primates that lasted for at least days. the possibility of using mrna for vaccination has recently become a reality thanks to the development of highly efficient delivery methods, as the ones described in this review. in particular, combination of these new methodologies with sarna has demonstrated to be a very efficient way to induce both humoral and cellular immune responses in both rodents and large animal models, being superior to non-replicating mrna. the reason for this superiority is that transfection with sarna mimics in many ways a viral infection, inducing a plethora of adjuvant signals that enhance immune responses, without apparent toxicity. one important advantage of sarna is its versatility, since new vaccines could be generated quickly by changing the sequence coding for the antigen of interest, something that would not affect its production. in fact, sarna could be efficiently produced at gmp level, using the same technology used to manufacture mrna. this simplicity and fastness in production could allow generating in a very quick way enough vaccine doses to potentially control emerging viruses that are causing global concern, such as zika virus, ebola virus, or the recently appeared sars-cov- . nothing declared. self-amplifying rna vaccines ballesteros-briones et al. papers of particular interest, published within the period of review, have been highlighted as of special interest of outstanding interest dna vaccines: ready for prime time? dna vaccines-how far from clinical use? weissman d: mrna vaccines -a new era in vaccinology liljeströ m p: non-viral amplification systems for gene transfer: vectors based on alphaviruses nonviral delivery of self-amplifying rna vaccines alphavirus vectors for cancer therapy liljeströ m p: self-replicating semliki forest virus rna as recombinant vaccine cancer therapy using a self-replicating rna vaccine induction of immune responses and protection in mice against rabies using a self-replicating rna vaccine encoding rabies virus glycoprotein self-amplifying rna vaccines give equivalent protection against influenza to mrna vaccines but at much lower doses vaccination using synthetic mrna and sarna expressing influenza virus ha was evaluated in parallel, observing that a similar protection was achieved using -fold less material of sarna compared to mrna immunogenicity of semliki forest virus based self-amplifying rna expressing indian hiv- c genes in mice efficient induction of t cells against conserved hiv- regions by mosaic vaccines delivered as self-amplifying mrna liljeströ m p: intradermal electroporation of naked replicon rna elicits strong immune responses electroporation of rna stimulates immunity to an encoded reporter gene in mice enhanced delivery and potency of self-amplifying mrna vaccines by electroporation in situ expression kinetics and innate immune response after electroporation and lnp-mediated delivery of a self-amplifying mrna in the skin comparison of the expression kinetics and immunostimulatory activity of replicating mrna, nonreplicating mrna, and pdna after intradermal electroporation in pigs effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging improving the repeatability and efficacy of intradermal electroporated selfreplicating mrna improved methodology to reduce variability of sarna in vivo expression delivered by ep in combination with rnase inhibitors rapidly produced sam(j) vaccine against h n influenza is immunogenic in mice cd tcell priming upon mrna vaccination is restricted to bonemarrow-derived antigen-presenting cells and may involve antigen transfer from myocytes selfamplifying mrna vaccines expressing multiple conserved influenza antigens confer protection against homologous and heterosubtypic viral challenge induction of protective immunity against toxoplasma gondii in mice by nucleoside triphosphate hydrolase-ii (ntpase-ii) selfamplifying rna vaccine encapsulated in lipid nanoparticle (lnp) a cationic nanoemulsion for the delivery of next-generation rna vaccines inside out: optimization of lipid nanoparticle formulations for exterior complexation and in vivo delivery of sarna induction of broad-based immunity and protective efficacy by selfamplifying mrna vaccines encoding influenza virus hemagglutinin co-administration of gm-csf expressing rna is a powerful tool to enhance potency of sam-based vaccines combination of sarnas expressing influenza virus np and gm-csf enhanced recruitment of apcs at injection site, increasing antigen-specific cd t-cell responses potent immune responses in rhesus macaques induced by nonviral delivery of a self-amplifying rna vaccine expressing hiv type envelope with a cationic nanoemulsion immunogenicity and protective efficacy induced by selfamplifying mrna vaccines encoding bacterial antigens self-amplifying rna vaccines for venezuelan equine encephalitis virus induce robust protective immunogenicity in mice first demonstration that lnps can be used to deliver a live-attenuated veev rna vaccine mannosylation of lnp results in improved potency for selfamplifying rna (sam) vaccines neutral lipopolyplexes for in vivo delivery of conventional and replicative rna vaccine new tripartite lnp formulation composed of rna, cationic polymer (pei), and anionic liposomes designated as neutral lipopolyplexes (lpps) with high capacity to transduce dcs this system a single dose of ng of sarna encoding zika virus antigens completely protected mice against lethal virus challenge cationic hdl mimetics enhance in vivo delivery of self-replicating mrna the skin you are in: design-of-experiments optimization of lipid nanoparticle self-amplifying rna formulations in human skin explants effects of cationic adjuvant formulation particle type, fluidity and immunomodulators on delivery and immunogenicity of sarna immunogenicity of rna replicons encoding hiv env immunogens designed for selfassembly into nanoparticles polyethylenimine-based polyplex delivery of self-replicating rna vaccines self-replicating rna vaccine functionality modulated by fine-tuning of polyplex delivery vehicle structure big is beautiful: enhanced sarna delivery and immunogenicity by a higher molecular weight, bioreducible, cationic polymer a transamplifying rna vaccine strategy for induction of potent protective immunity novel sfv bipartite vector based on trans-amplification of a mrna providing the replicase in trans from a non-replicating mrna. this system has the potential to be universally applicable for expression of different antigens induction of an ifn-mediated antiviral response by a selfamplifying rna vaccine: implications for vaccine design improvement of in vivo expression of genes delivered by self-amplifying rna using vaccinia virus immune evasion proteins self-amplifying rna sars-cov- lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice sarna vaccine against sars-cov- able to induce highly specific neutralizing antibodies and cellular responses in mice an alphavirusderived replicon rna vaccine induces sars-cov- neutralizing antibody and t cell responses in mice and nonhuman primates this work was supported by the following grants: instituto salud carlos iii financed with feder fundspi / , gobierno de navarra. departamento de salud / (co-financed at % by the european regional development fund through the feder operational program - of navarra: "european union. european regional development fund. a way to make europe"). mcbb received a fundació n eché bano fellowship, ghc received a fellowship from gobierno de navarra, departamento de educació n, and nsp received a fellowship "ayudas predoctorales de investigació n biomé dica ac". key: cord- -twp bb authors: becker, pablo d.; guzmán, carlos a. title: community-acquired pneumonia: paving the way towards new vaccination concepts date: journal: community-acquired pneumonia doi: . / - - - - _ sha: doc_id: cord_uid: twp bb despite the availability of antimicrobial agents and vaccines, community-acquired pneumonia remains a serious problem. severe forms tend to occur in very young children and among the elderly, since their immune competence is eroded by immaturity and immune senescence, respectively. the main etiologic agents differ according to patient age and geographic area. streptococcus pneumoniae, haemophilus influenzae, respiratory syncytial virus (rsv) and parainfluenza virus type (piv- ) are the most important pathogens in children, whereas influenza viruses are the leading cause of fatal pneumonia in the elderly. effective vaccines are available against some of these organisms. however, there are still many agents against which vaccines are not available or the existent ones are suboptimal. to tackle this problem, empiric approaches are now being systematically replaced by rational vaccine design. this is facilitated by the growing knowledge in the fields of immunology, microbial pathogenesis and host response to infection, as well as by the availability of sophisticated strategies for antigen selection, potent immune modulators and efficient antigen delivery systems. thus, a new generation of vaccines with improved safety and efficacy profiles compared to old and new agents is emerging. in this chapter, an overview is provided about currently available and new vaccination concepts. the mucosa of the human respiratory tract represents a primary target for a large number of microbial pathogens. typically, colonization is an asymptomatic process, resulting from the interplay between bacterial factors and host clearance mechanisms. clinical illness may result from either the local release of bacterial toxins or the systemic dissemination of the pathogen after breaching the mucosal barrier. in the course of respiratory infections adaptive immune responses could be significantly impaired. this might lead to more severe forms of disease or to super-infections, which in turn complicate the clinical management of the patient. the most severe forms of respiratory infection tend to occur in very young children and among the elderly, in whom immune competence is eroded by immaturity or immunesenescence, respectively. in addition, patients who are immunocompromised, as a result of disease or therapeutic interventions, have the greatest risk of developing a fatal infection. despite the availability of new antimicrobials and effective vaccines, community-acquired pneumonia remains a common and serious illness. in fact, it is a leading contributor to the nearly million deaths occurring each year due to respiratory infections, especially in children from developing countries [ , ] . the main causative agents of pneumonia differ according to the patient age and the geographic area. in addition, there are relatively few comprehensive studies on the specific aetiology of pneumonia [ ] due to (i) overlaps in the clinical manifestations of the different syndromes, (ii) difficulties in establishing the precise aetiology, and (iii) frequent occurrence of co-infections. however, streptococcus pneumoniae, haemophilus influenzae, respiratory syncytial virus (rsv), and parainfluenza virus type (piv- ) have been identified as the main agents responsible for acute respiratory infections in children, whereas influenza virus related pneumonia is the leading cause of disease-related deaths in the elderly. in addition, the availability of new and more sensitive diagnostic tests have contributed to the identification of hitherto unknown lower respiratory pathogens, such as the human metapneumonovirus (hmpv) and novel coronaviruses causing the severe acute respiratory syndrome (sars). significant efforts have been invested in the last two decades to develop new diagnostic tools, to elucidate the molecular mechanisms of microbial pathogenesis and to understand host clearance mechanisms. this resulted in an improved knowledge on host responses to infection and immuno-pathogenesis, which in turn have facilitated the establishment of new prophylactic and therapeutic interventions. however, despite our accomplishments in vaccine development, there are many pathogens for which vaccines or adequate therapies are not available or the existent ones are suboptimal. the main approach applied for vaccine development has radically changed in recent years. whole cell vaccines are systematically being replaced by subunit vaccines, in which purified antigens or their coding genes are exploited in combination with new adjuvants and/or delivery systems. as a result, many of the vaccines under development will exhibit consistently improved stability, safety and efficacy profiles. they will also be amenable for mucosal administration, thereby mimicking natural infections. influenza a viruses are the most commonly responsible for severe respiratory illness in humans, followed by influenza b. the population's susceptibly to infection is renewed annually, because of the rapid antigenic variation of this virus. the antigenic variation is due to the accumulation of point mutations in the two major surface glycoproteins of the virus, haemagglutinin (ha) and neuraminidase (na). this can lead to an antigenic drift of the virus, which often leaves current influenza vaccines outdated and ineffective. antigenic shift can also occur due to the segmented nature of the viral genome that favours the emergence of re-assorted strains, in which an entire glycoprotein can be acquired from a different animal influenza virus. both types of variation represent a critical bottleneck for the establishment of a robust vaccination strategy against influenza. in fact, when an influenza virus with the capacity to spread from person-to-person and a complete new glycoprotein subtype suddenly emerges, a worldwide pandemic outbreak can result [ ] . the earliest vaccines against influenza were whole cell vaccines obtained in the s by inactivating viruses grown in the allantoic cavity of embryonated chicken eggs with formalin. while contemporary inactivated influenza vaccines are still produced in embryonated eggs, improvements in manufacturing have resulted in a highly purified and less-reactogenic detergentsplit product. three viral strains are selected on the basis of the previous year's surveillance data on the most prevalent subtypes, therefore, vaccine composition may vary from year-to-year. vaccination has a high benefit:cost ratio, since influenza-related illness (e.g., hospitalizations and deaths) are effectively prevented [ ] . the world's total vaccine production is approximately million doses, with a maximum capacity of million doses. however, the world health organization (who) estimates that there are about . billion people at high risk for severe influenza outcomes (e.g., elderly over years of age, infants, health care workers, children and adults with underlying cardiopulmonary disease). furthermore, the global infrastructure would not be able to handle the timely manufacturing and distribution of a vaccine for a pandemic outbreak [ ] . one alternative would be to lower the quantity of antigen per dose and add adjuvants to the vaccine formulation, but this needs to be tested in clinical trials [ ] . another solution would be to improve current vaccine production technologies (i.e., egg-derived vaccines). however, there is the limited number of egg producers and viral strains can emerge, which could not be easily adapted to embryonated eggs. to overcome these problems, several pharmaceutical companies have embarked themselves on projects for the development of vaccines produced by growing the virus on cell lines. the influenza virus can be adapted to grow on a variety of mammalian cell lines, including vero, per.c- , and madin-darby canine kidney (mdck) cells [ ] [ ] [ ] . this strategy would also improve the possibility of up-scaling vaccine production in face of a pandemic spread. alternatively, it would be possible to develop a vaccine against any influenza virus, such as the avian h n strain, by using reverse genetics techniques [ ] (see below in advances in vaccinology). cold adaptation was found to be a reliable and efficient procedure for the derivation of live attenuated viral vaccine strains for humans. cold-adapted (ca) virus strains can grow in primary chick kidney cells or embryonated eggs at - °c, however, they exhibit a reduced replication at °c. the process of genetic re-assortment with the transfer of the six internal genes from a stable attenuated ca master donor strain of influenza a or b to the new prevailing wild-type epidemic strain has been used to generate attenuated cold-reassorted vaccines with the proper level of attenuation, genetic stability and immunogenicity, which show low or absent transmissibility [ ] . medimmune and wyeth have developed along these lines a trivalent live ca vaccine (flumist) for intranasal spray delivery, which was licensed in . in contrast, to parenterally-administered vaccines, this formulation triggers immune responses resembling those observed after natural infections [ ] . despite the moderate hemagglutination-inhibiting antibody titres observed in vacinees, flumist showed % efficacy over a -year period in children, including protection against antigenic variants that circulated during the second year [ ] [ ] [ ] . this ca vaccine also stimulated the production of nasal iga, as well as t-cell and interferon responses [ ] . the cell-mediated immunity against virus matrix and nucleoprotein antigens may favour viral clearance and early recovery from illness [ ] . the advisory committee on immunization practices has recommended its use only in persons from to years of age, since side-effects were observed in young children (wheezing, nasal congestion) and there are no data available for elderly [ , ] . despite its remarkable genetic stability, this vaccine has to be kept at - °c. thus, a new heat stable derivative has recently been developed, which showed good efficacy in clinical trials [ ] . a live vaccine based on a master virus strain developed at the institute of applied microbiology (austria) by growing wild influenza virus in vero cells at °c was also demonstrated to be safe, well-tolerated and immunogenic after intranasal immunization in young adults [ ]. a number of subunit-or dna-based vaccines are also in various stages of development. an influenza vaccine formulated in virosomes has been commercialized by berna biotech (inflexal ® v); it contains the surface spikes of the three currently circulating influenza virus strains inserted in vesicle membranes of the three corresponding virus types (for more details see section "pseudoviruses as antigen delivery systems") [ ] . this company has also developed a virosome-based nasal formulation. however, it was withdrawn from the market due to the presence of sideeffects (i.e., bell's palsy), which was assumed to be linked to the presence of the escherichia coli heat labile toxin (lt) as adjuvant. two companies, yeda and bionvax, are also developing a peptide-based influenza vaccine for nasal administration, which showed protective efficacy in humanized mice [ ] . a subunit vaccine containing recombinant ha protein produced using a baculovirus system was successfully tested in a phase ii trial in to -year-old volunteers. an epidermal dna-based influenza vaccine, which contained the ha gene from a/panama/ / delivered by particle-mediated epidermal delivery was also tested in humans by powderject [ ] . serum haemagglutination-inhibition antibody responses were observed in volunteers receiving a single dose of , or g of dna, with the strongest and most consistent responses in subjects vaccinated with the highest dosage. some immunization approaches aim at the development of a universal vaccine with a broad spectrum of protective activity against different influenza strains [ ]. among them, the use of the highly conserved transmembrane m protein of the virion can be mentioned. a recombinant particulate vaccine has been engineered by genetically fusing copies of the m to the hepatitis b core antigen (hbc). the m -hbc fusion protein spontaneously assembled into virus-like particles (vlp), which provided complete protection against a lethal challenge with influenza virus a in mice [ , ] . promising results were also obtained after vaccination with a m peptide conjugated with a neisseria meningitidis outer membrane protein complex (ompc) in monkeys [ ] . the human piv (hpiv) consist of four serotypes, with hpiv- being the second leading cause of bronchitis and pneumonia in infants. no vaccine has been licensed to date against piv, however, several approaches are currently under investigation. the initial attempts to provide protection by using vaccines based on formalin-inactivated viruses failed. subsequent work demonstrated that the glycoproteins haemagglutininneuraminidase (hn) and f, which are responsible for virus attachment and fusion, are able to stimulate the elicitation of neutralizing antibodies in animals. however, their poor immunogenicity in naïve subjects led to the currently favoured approach, which is based on the use of live attenuated piv. live attenuated piv vaccines have been developed from both human and bovine strains, which are amenable for delivery by the intranasal route. candidate vaccines should be able to replicate and induce a protective immune response in young infants, even in the presence of maternally acquired antibodies. two main attenuated strains have been studied in detail. one is the hpiv- strain cp , which was selected after passages of the virus in african green monkey cells at low temperature. the other is a bovine piv (bpiv)- strain, which is antigenically related to the hpiv- , and replicates poorly in humans. both cp and bpiv- have been evaluated in phase i/ii trials in sero-positive and sero-negative children and in young infants. they were found to be over-attenuated in sero-positive children, but immunogenic in sero-negative children and infants [ ] . however, the magnitude of the anti-hn response was lower in children who received the bpiv- vaccine [ ]. this prompted the engineering of chimeric bovine/human piv- candidates (e.g., hpiv-nb strain in which the human nucleocapsid is replaced by the bovine counterpart, or a bpiv- strain that expresses the f and hn proteins of hpiv- ). attenuated, chimeric viruses that contain piv- cp internal genes with the f and hn genes from either piv- or piv- have also been tested in hamsters [ ] . berna biotech is also developing a virosomal formulation of the piv- [ ] . using the successful approach of the influenza vaccine, a formalin-inactivated candidate against the respiratory syncytial virus (rsv) was tested in children in the s. the consequence was the hospitalization of % of vaccinees and two deaths [ ] . moreover, vaccinated children also suffered more severe disease on subsequent exposure to the virus, as compared to unvaccinated controls [ ] . this demonstrated that the elicitation of a strong immune response is not sufficient to confer protection against disease, and can even lead to immuno-pathological reactions. thus, it is essential to stimulate the "right" type of immune response. in the particular case of rsv, host responses play an important role in the pathogenesis of the disease, thereby making the development of a preventive vaccine extremely difficult. in addition, naturally acquired immunity to rsv is neither complete nor long-lasting, and recurrent infections often occur [ ] . however, older children and adults are usually protected, suggesting that protection against severe disease develops after several consecutive infections. passive immunization with rsv-neutralizing immune globulins was also shown to prevent rsv infection in newborns with underlying cardiopulmonary disease [ ] . this demonstrates that antibodies play a major role in protection against this disease, whereas t-cell immunity targeted to internal viral proteins appears to contribute to clearance. although live attenuated vaccines seem to be preferable for immunization of naïve infants, subunit vaccines may be of choice for elderly, high-risk children and pregnant women. candidate subunit vaccines based on purified f proteins (pfp- , - and - ) were demonstrated to be safe and immunogenic, even during pregnancy [ ] . maternal immunization using a pfp-based vaccine could be an interesting strategy to protect infants younger than months of age [ ] . however, no significant protection was reported in a phase iii trial performed on children - years of age with cystic fibrosis after vaccination with a subunit vaccine based on pfp- [ ] . a formulation based on surface glycoproteins f and g together with the virion matrix protein m from rsv-a was tested in healthy adult volunteers in the presence of either alum or polyphosphazene as an adjuvant. short-live neutralizing antibody responses to rsv-a and rsv-b were detected in - % of the vaccinees, suggesting that annual boosting will be needed [ ] [ ] [ ] . the central domain of the g protein of rsv-a is relatively conserved among viruses from the groups a and b. thus, a recombinant vaccine candidate, bbg na, was developed by fusing the g na domain to the albumin binding region of streptococcal protein g. this candidate was shown to be moderate immunogenic in adult human volunteers, but its clinical development was interrupted due to the appearance of purpura in vaccinees [ ] . the main two difficulties associated with the generation of live attenuated vaccines against rsv are over-or under-attenuation of the virus and limited genetic stability. temperature-sensitive (ts), ca and cold-passaged (cp) mutant viral strains have been generated. despite the attenuation shown in adults and sero-positive children, cpts mutants still caused moderate congestion in the upper respiratory tract of sero-negative infants ( - months old) [ ] . recombinant rsv vaccines with deletions in non essential genes (e.g., sh, ns or ns ), which also carry cp and ts mutations in essential genes are currently being evaluated [ ] . through recombinant dna technology chimeric viruses were engineered, which contain the genes of hpiv- surface glycoproteins f and nh together with those of rsv glycoproteins f and g in a bpiv- genetic background. one of these candidates was found to be attenuated and able to induce the elicitation of immune responses against both hpiv- and rsv in rhesus monkeys [ ] . similarly, a bpiv- genome was engineered to express hpiv- f and hn proteins and either native or soluble rsv f protein [ ] . the resulting strain, which induced rsv neutralizing antibodies and protective immunity against rsv challenge in african green monkeys, needs to be tested for safety and efficacy against rsv and piv- in infants. this emerging disease was originally described in the guangdong province of china in . even when the global outbreak of sars was under control in , new infections were reported in persons who had contacts with animals in and [ ] . the typical sars-cov-like virus is not transmitted from animals to humans. however, under certain conditions the virus can evolve into the early human sars-cov, which has the ability to be transmitted from animals to humans or even humans to humans, thereby leading to localized outbreaks of mild disease. the early human sars-cov, under selective pressure in humans, may further evolve into the late human sars-cov, which can cause local or global outbreaks of typical sars [ ] . sars can be easily grown in cell cultures [ ] . thus, there is an urgent need for vaccines, not only to prevent naturally occurring epidemic outbreaks, but also as a tool against the threat of biological weapons. several structural proteins are expressed by sars-cov, including nucleocapsid, envelope and spike (s) proteins [ ] . the latter is a type i trans-membrane glycoprotein, which is responsible for virus binding, fusion and entry, and being the major target of neutralizing antibodies [ , ] . the extracelullar domain of the s protein consists of two subunits, s and s [ ] . the s subunit possess a receptor-binding domain (rbd), which is responsible for viral binding to one of its receptors [ , ] . vector-based vaccines expressing the s protein, as well as dna vaccines encoding full-length s protein have been assessed in preclinical studies [ , ] . when modified vaccinia virus ankara (mva) coding for full-length s protein was administered by either intranasal or intramuscular route, neutralizing antibodies were elicited [ ] . however, vaccination of ferrets resulted in liver damage after challenge, raising some concerns about the safety of this approach [ ] . vaccines formulated using different synthetic peptides encompassing linear b cell epitopes from the s protein, which were identified using sera from convalescent patients, stimulated high antibody titres. nevertheless, none of them triggered the elicitation of neutralizing activity. on the other hand, some studies demonstrated that although antibodies against s protein of the late sars-cov (urbani strain) exhibit neutralizing activity, they can also enhance infection by an early human sars-cov isolate (gd t ) and the civet sars-cov-like viruses. a derivative of the s protein with a truncation at amino acid (aa) fails to cause antibody dependent enhancement of infection, but retains the ability to induce neutralizing antibodies. these findings suggest that the elimination of the putative heptad repeated (hr , aa - ), which is implicated in viral fusion, might abrogate the stimulation of virus infection-enhancing antibodies [ , ] . the use of the nucleoprotein of the coronavirus in a dna vaccination protocol also led to the stimulation of a protective response [ ] . in contrast, protection was not achieved when a recombinant piv- expressing the nucleoprotein alone or together with the matrix protein was used [ ] . this demonstrates that the selection of the delivery system and immunization strategy play a critical role in vaccine efficacy. the human adenoviruses are divided into six subgroups (a-f). the adenovirus can cause large-scale epidemics of acute respiratory disease, and dissemination is especially favoured under conditions in which persons are housed communally. the subgroup a viruses, such as ad , have been associated with pneumonia in immunocompromised patients. neutralizing antibodies directed against the capsid (hexon and fiber proteins) seems to be the main effector mechanism to prevent re-infections by adenovirus [ ] . until , military recruits in usa were administered enteric-coated capsules containing live viruses from the serotypes and . the virus, which was not attenuated if delivered by respiratory route, was able to replicate in the gastrointestinal tract without causing disease, thereby stimulating protective responses in the respiratory tract [ ] . when the vaccine went out of production, outbreaks of respiratory diseases caused by adenovirus reemerged among the military recruits [ ] . since serotypes , , and cause the % of adenovirus associated respiratory disease in young children, the development of a tetravalent vaccine similar to the above mentioned might solve the problem in children [ ] . however, the implementation of a vaccine (live or attenuated) against adenovirus should be carefully evaluated, since recombinant adenoviruses are proposed both as vaccine vectors and as tools for the transfer of foreign genes in gene therapy protocols. polysaccharide-based vaccines against s. pneumoniae in , macleod et al. [ ] reported the protective efficacy of a capsular polysaccharide (ps) vaccine in military personnel during an outbreak of pneumococcal pneumonia. the immunization with purified ps showed a drastically reduced reactogenicity, in comparison with the previously used inactivated whole cell vaccines. this was a major breakthrough, not only in terms of safety, but also because it demonstrated that a specific virulence factor can be purified and effectively implemented for the prevention of an infectious disease, thereby paving the road for modern non toxoid-based subunit vaccines. although the serological correlates of immunity are poorly defined, type-specific anti-capsular antibodies are responsible for protective immu-nity. however, immunity is serotype specific, rendering extremely difficult the development of a universal vaccine. this is in part due to the elevate number of serotypes, the regional variations in dominant serotypes and the lack of updated sero-prevalence data for certain regions. these problems have been partially solved by the use of ps-based polyvalent vaccines. the currently licensed formulations contain serotypes of s. pneumoniae, which cover approximately % of serious pneumococcal disease, but only in western industrialized countries. relatively good antibody responses ( - %) are elicited in healthy adults - weeks after a single intramuscular or subcutaneous immunization [ ] . unfortunately, they are poorly immunogenic in children aged less than years, in immune compromised individuals (e.g., aids patients) and in elderly people with concomitant disease, and they do not induce good immunological memory. randomized controlled trials in healthy elderly and young men also failed to show a beneficial effect against pneumonia [ ] . however, vaccination is recommended for healthy people over years of age to confer protection against invasive disease [ ] . ps-based vaccines can be also used in pregnant women to stimulate the production of antibodies, which are transferred to the foetus via the placenta or to the newborns by breast-feeding. however, it is still a matter of controversy whether maternal vaccination can indeed protect newborns against pneumococcal infections [ ] . the second generation of ps-based conjugate vaccines stimulates stronger antibody responses, even in infants, young children and immune deficient individuals, as well as immunological memory. these vaccines also suppress nasopharyngeal carriage of the pathogen and reduce bacterial transmission in the community leading to herd immunity, which adds considerable value to their implementation. the introduction of these vaccines in usa in resulted in a dramatic decline in the rates of invasive pneumococcal disease [ , , ] . a significant reduction in the incidence rates among non vaccinated individuals was also observed as a result of herd immunity [ , ] . however, the licensed seven-valent vaccine does not contain some of serotypes that cause severe disease in developing countries (i.e., serotypes and ). new conjugate vaccines including more serotypes, such as the ninevalent vaccine (wyeth) and two -valent vaccines (glaxosmithkline and sanofi-pasteur), should provide better serotype coverage. new approaches to develop protein-based subunit vaccines against s. pneumoniae are currently being pursued by different research groups. this is expected to enable the generation of a universal vaccine conferring protective immunity against a large number of serotypes, as well as to avoid the complexity of manufacturing a conjugate vaccine [ ] . there are different pneumococcal candidate antigens, such as the pneumolysin, neuraminidase, autolysin, pneumococcal surface protein a (pspa) and adhesin a (psaa), which are in an early phase of clinical development [ ] . in addition, several promising candidates have been identified, which are currently being tested in pre-clinical experimental models [ ] . among them, the two iron uptake abc transporters of s. pneumoniae (piaa and piua), which trigger protective immunity against invasive pneumococcal disease in mice. through the screening of s. pneumoniae genomic expression libraries with sera from convalescent patients, bacterial surface proteins were identified (e.g., bvh- and bvh- ) that promote the elicitation of protective anti-pneumococcal antibodies in mice [ ] . a recombinant hybrid protein, bvh / v, has successfully been tested in toddlers and elderly volunteers. this candidate vaccine should be able to trigger serotype-independent responses, since the bvh and bvh antigens are common to all serotypes of s. pneumoniae. the major obstacle for developing an effective vaccine against h. influenzae capsular ps was related to the inherently poor immunogenicity of this t-cell-independent antigen. antibody responses against ps are age-related, with extremely poor immunogenicity in infants during the first months of life. unfortunately, this age group exhibits the highest risk for invasive infections caused by h. influenzae. a ps-based vaccine against the h. influenzae type b (hib) was licensed in the united states in , for children more than months old [ , ] . the protective efficacy after licensure studies showed the inefficacy of this vaccine not only in infants, but also in older children [ ] . this problem was solved by the generation of a conjugate hib vaccine. to this end, the hib ps (i.e., polyribosylribitol phosphate; prp) was covalently linked to an immunogenic carrier protein, thereby leading to t-cell-dependent responses against the ps. different conjugate hib vaccines currently exist. these vaccines are hboc, prp-t and prp-omp, which make use of the mutant diphtheria toxin crm , the tetanus toxoid and the outer membrane protein from group b n. meningitidis as carriers, respectively. all of them trigger similar immune responses at the recommended doses. however, the dynamic of the elicited response may vary for each of them [ , ] . efficacy studies of these vaccines showed that they confer protection not only against meningitis, but also against pneumonia [ ] [ ] [ ] . although hib vaccines are highly effective, their cost is still prohibitive for the world's poorest nations. however, with the establishment of the global alliance for vaccines and immunization (gavi), we have moved consistently ahead in making them also available for developing countries. gavi has approved the establishment of a hib initiative to support countries wishing to sustain hib vaccination, as well as those exploring whether their introduction could be considered a priority in the near future. although the introduction of conjugated ps vaccines has significantly decreased the prevalence of invasive hib disease, paediatric infections due to non typeable h. influenzae (nthi) are still highly prevalent. nthi is most often associated with otitis media, sinusitis and bronchitis. in addition, nthi is an important cause of lower respiratory infection in adults with chronic obstructive pulmonary disease (copd). thus, the development of a vaccine against nthi is considered an important goal in public health. in contrast to hib, vaccines against the non-encapsulated nthi strains must be directed against alternative virulence factors. the lipoproteins d and p are widely distributed and antigenically conserved among h. influenzae strains, and also trigger the elicitation of protective immunity in animals vaccinated by mucosal route [ ] [ ] [ ] [ ] . thus, their incorporation in vaccine candidates might facilitate the generation of a universal vaccine against all typeable and non typeable h. influenzae. even in the age of vaccine availability, b. pertussis continues to be a major cause of childhood morbidity and mortality (i.e., approximately million cases and , deaths occur annually worldwide). since the late s, the incidence of whooping cough has dramatically decreased in most developed countries, as a result of widespread immunization. the first vaccine formulations, which are still in use, consist of preparations based on killed b. pertussis. the frequent incidence of minor adverse effects (e.g., fever, protracted crying and local erythematous reactions), as well as concerns raised by reports of serious neurological side-effects, resulted in a decline in vaccine acceptance and use [ ] . this in turn led to a re-emergence of whooping cough and its complications. this serious problem prompted the development of a new generation of acellular vaccines. in japan was the first country to successfully introduce acellular vaccines against whooping cough in its immunisation programme [ ] , leading to a consistent reduction in the reported side-effects. in the mid 's a major phase iii trial of acellular vaccines was undertaken in sweden, at a time when the banning of the whole cell vaccine had resulted in a pertussis epidemic in that country [ ] . the first vaccine trials contained chemically detoxified pertussis toxin (pt) and filamentous haemagglutinin (fha), or detoxified pt alone. the results of these trials showed that whilst producing good antibody responses, the vaccines failed to give an adequate level of protection in infants. the mono-component vaccine conferred no protection against infection, whereas the use of the two component candidate only gave incomplete protection against infection [ ] . the results obtained in japan and sweden stimulated vaccine companies in the usa and europe to establish vigorous research programmes aimed at the development of a new generation of acellular vaccines with higher efficacy. currently available vaccines have incorporated chemically or genetically inactivated pt and additional virulence factors, such as fha, the outer membrane protein pertactin (prn) and fimbrial proteins (fims). the efficacy studies of this second generation of acellular vaccines have demonstrated that they confer levels of protection equivalent to the whole cell vaccines. the advent of improved techniques for antigenic characterisation and the introduction of acellular vaccines containing genetically defined components also resulted in a reduction of lot-to-lot variation in comparison with conventional whole cell vaccines and the acellular formulation originally introduced in japan. however, despite the wide implementation of vaccination campaigns in infants and children, the disease continues to be endemic. in addition, in countries with high vaccine coverage we are now observing a consistent increment in the cases of pertussis in adolescents and adults [ ] [ ] [ ] . these patients can then transmit the disease to infants, thereby now representing a primary reservoir for bacterial transmission and cycling in the community. the above-mentioned observations can be explained by one or more of the following factors: (i) improved detection techniques, (ii) major awareness on the possibility that bacteria may affect these age groups, (iii) vaccinedriven antigenic changes in circulating isolates, and (iv) reduction in vaccine efficacy over time. in this context, concerns have been raised about genetic variation between the strains used for vaccine preparation and circulating isolates. this seems to be true, since the currently used whole cell and acellular vaccines are prepared with strains that were isolated before mass vaccine introduction and show clear mismatches with respect to circulating strains. there is a steady tendency to decrease diversity in recent isolates, together with clonal expansion during epidemic outbreaks [ , ] . over time, at least two surface proteins (pt and prn) may have changed sufficiently to allow for an increase in the incidence of disease. unfortunately, our global information on antigenic variation and disease in adults and adolescent is extremely limited. thus, despite widespread introduction of pertussis vaccines, it is essential to continue surveillance studies and collection of circulating strains. the present view is that successful control of pertussis in the community may require routine immunization of adolescents and adults with the new acellular vaccines, perhaps in combination with the diphtheria and tetanus toxoids (dtap). this intervention might help in turn to reduce the burden of disease and transmission to infants. chlamydia pneumonia is an intracellular bacterium transmitted person-toperson via respiratory droplets. this pathogen is a common cause of pneumonia, with infections usually being oligosymptomatic or asymptomatic in young age groups. however, the rate of asymptomatic carriage in the normal population is unknown. there is also a tremendous gap in our understanding of host response to infections caused by c. pneumoniae. most of the studies have been focused on the development of efficient diagnostic methods. however, less work has been done on vaccine development, and there is a paucity of knowledge on the microbial components which may serve as target antigens. in fact, at present there are no licensed vaccines against c. pneumoniae. however, the potential of different antigens, such as the major omp [ ] have been assessed in experimental animal models. nevertheless, mice vaccinated with omp using a protocol based on priming with dna and boosting with recombinant vlp showed only partial protection [ ] . recent studies also suggested that ctl responses play a role in protection and clearance [ ] . animals immunized with a mini-gene encoding seven h- (b)-restricted ctl epitopes fused to a endothelial reticulum-translocation signal showed protection following intranasal challenge with a virulent c. pneumoniae [ ] . the current view is that multi-component vaccine will be required in order to induce a protective response [ ] . using the promising approach of reverse vaccinology combined with proteomics (see section "reverse vaccinology"), the whole-genome of c. pneumoniae was screened searching for vaccine candidate antigens among exposed and immune accessible surface proteins [ ] . the selected candidates were then expressed in a heterologous system and used in immunization studies. approximately proteins were able to trigger the elicitation of c. pneumoniae-binding antibodies. when tested in secondary screenings, six of them were also able to neutralize bacteria in vitro, and four inhibited systemic dissemination of c. pneumoniae in a hamster model [ ] . moraxella catarrhalis is the third most common bacterial etiologic agent of otitis media in children. furthermore, m. catarrhalis is an important cause of respiratory infections in patients with copd. thus, different studies have been carried out to characterize potential protective antigens. in this context, two major omp (cd and e) have been identified, which are considered prime candidate antigens for vaccine development. these proteins are expressed on the surface and show a high degree of conservation among circulating strains. both omp triggered the elicitation of bactericidal antibodies and protective immunity in preclinical models [ ] . additional candidates are the uspa protein [ ] , which seems to be required for bacterial colonization of the human upper respiratory tract, the iron-induced omp b and lbp, and the iron-repressed omp b [ ] . a conjugate vaccine based on detoxified lipo-oligosaccharide was also tested in mice by intranasal route with encouraging results [ , ] . some of these candidates are planned to be tested in clinical studies soon [ ] . mycoplasmas are commensal microorganisms, as well as opportunistic pathogens. mycoplasma pneumoniae is one of the causative agents of acute and chronic human respiratory diseases and the main responsible for primary atypical pneumonia, accounting for approximately - % of all community-acquired pneumonia [ ] . there is a considerable underreporting for m. pneumoniae-associated diseases. this is in part due to the wide diversity of clinical manifestations, the difficulties associated with its cultivation from clinical specimens and the lack of adequate diagnostic tools. no vaccines are currently available against this pathogen. however, studies conducted in human volunteers in the late s demonstrated that a formalin-inactivated whole cell vaccine and an acellular extract were able to confer moderately protective immunity against m. pneumoniae [ ] . unfortunately, immune pathological reactions were observed following challenge with live organisms. therefore, studies are still needed to understand the underlying mechanisms to the observed autoimmune responses [ ] . more specifically, we need to elucidate the specific role played by humoral and cellular response in protection against m. pneumoniae. m. pneumoniae is one of the smallest self-replicating prokaryotic pathogens (approximately kb). the complete genome sequence is now available. this is expected to expand our knowledge on the physiological and virulence properties of this agent, as well as new hints for vaccine development. a previously unrecognized bacterium was isolated after the outbreak of legionnaires disease in , which was designated legionella pneumophila [ , ] . the spreading of l. pneumophila is increasing due to the use of air-conditioners and humidifiers, since infections can occur by inhalation of aerosolized contaminated water sources. several approaches have been developed in the fight against this facultative intracellular pathogen. infection and immunization induce a rapid increase of antibody titres. however, antibodies do not seem to play a significant role in host resistance, particularly after aerosol challenge [ ] [ ] [ ] . some authors also suggested that these antibodies can promote bacterial phagocytosis, thereby favouring invasion and subsequent intracellular replication [ ] . in contrast, cellular responses appear to be important for protection. different vaccine candidates were tested in the past. heat-, acetone-and formalin-killed l. pneumophila vaccines were not able to confer protective immunity in guinea pigs, whereas animals immunized with l. pneumophila membranes survive an aerosol challenge with virulent bacteria [ , ] . additional work demonstrated that also purified antigens, such as the major secretory protein [ ] , the major cytoplasmatic membrane protein [ ] , the peptidoglycan-associated lipoprotein [ ] , omps [ ] and flagella [ ] can confer protection against challenge with virulent l. pneumophila. finally, different live attenuated mutants of l. pneumophila were used in animal infection models with promising results [ ] . cystic fibrosis (cf) patients are particularly susceptible to severe bacterial infections of the lung, being pseudomonas aeruginosa one of the most prominent etiologic agents. thus, significant efforts have been invested to develop a vaccine against this pathogen. surface ps are among the antigens that were most intensively assessed. berna biotech have developed an octavalent vaccine against the eight most prevalent serotypes based on o-ps conjugated with the exotoxin a [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a consistent reduction in the number of cf patients with chronic p. aeruginosa lung infection was observed in a cohort receiving the basic immunization protocol, followed by yearly boosters over a period of years [ , ] . the conjugate vaccine induced the production of specific igg antibodies and increased the number of igg memory b cells. it is still unclear if cellular responses might contribute to the overall protection conferred by this vaccine. however, strong proliferative responses of lymphocytes with a th phenotype were observed in vaccinated individuals in response to the carrier exotoxin a protein [ ] . alternative vaccination strategies are currently being tested in clinical trials. among them, formulations based on a fusion protein between the outer membrane proteins f and i, which have been administered by parenteral and mucosal routes [ , ] . these formulations were demonstrated to be safe in volunteers and conferred increased protection against p. aeruginosa in cf patients. cell-surface alginate, flagella, components of the type iii secretion system, inactivated toxins and proteases are other proposed target antigens [ ] . some of them are already in clinical trials alone or in combination [ ] . when pasteur returned from his summer holidays in to continue with his studies on chicken cholera, he inoculated chickens with an old culture of pasteurella multocida, which was left during the whole summer on his bench. the animals that received the preparation were protected against a challenge performed with a fresh isolate. thus, pasteur developed the hypothesis that pathogens could be attenuated by exposure to environmental insults (e.g., high temperature, oxygen and chemicals) [ ] . the strategy was then successfully extrapolated for developing anthrax vaccines in livestock in the s, with significant economic benefits. this was followed by the generation of attenuated vaccines against rabies and other important pathogens towards the end of the nineteenth century. pasteur's approach for "attenuating" or "inactivating" a pathogenic organisms still constitutes a cornerstone in vaccine technology [ ] . this exemplifies that until recently the major achievements in vaccinology have been facilitated by technological (e.g., adjuvants, delivery systems, reverse vaccinology, genetic engineering) rather than immunological advances [ ] [ ] [ ] . however, it is expected that the impressive knowledge accumulated in recent years in the fields of immunology, immune pathology and microbial pathogenesis will pave the road to a new golden era in vaccinology, in which knowledge and technology will enable rational vaccine design. in the th century, pertussis vaccines progressed from crude bacterial preparations to the highly purified antigens used for acellular vaccines. a similar quantum jump in technology allowed the development of subunit vaccines against influenza, hib and s. pneumoniae, as well as the production of antigens by recombinant dna techniques (e.g., genetically inactivated pt). despite the fact that these techniques enable the production of almost any foreseeable antigen, the identification of suitable targets still remained as a main bottleneck for vaccine development [ ] . the advent of genomics and its exploitation in the vaccinology field have rendered possible the implementation of a systematic and holistic approach for the screening, identification and prioritisation of candidate antigens. this new approach, called "reverse vaccinology" [ ] , does not require cultivation of the original pathogen, thereby being amenable for highlypathogenic or non culturable micro-organisms. it is possible to predict and select the most promising candidates by the analysis of genomic sequences in silico, which will then be cloned and expressed in heterologous systems. the resulting proteins are then used to perform immunological and/or functional studies to select the most promising candidates (e.g., able to induce the production of microbicidal or neutralizing antibodies, capacity to confer protective immunity). flanking studies are usually carried out, such as molecular epidemiological analysis to assess their degree of conservation among circulating strains, or transcriptional profiling to evaluate their expression during natural infections [ ] . the time-consuming process in which highly expressed components of an in vitro cultivable organism are identified (one at a time) and separated (different components between them) is one of the disadvantages that reverse vaccinology has solved. the conventional method usually requires - years to arrive to a clinical trial, whereas reverse vaccinology reduces the process to approximately years. reverse vaccinology also allows the identification of hundreds of potential candidates in a few days, in comparison with the small number of antigens that conventional approaches have provided after decades of research. moreover, reverse vaccinology offers the possibility to select potential candidates independent of their expression levels or purification easiness. the reverse vaccinology approach has proved its usefulness in the field for both viral and bacterial pathogens (e.g. hepatitis c virus, group b meningococci, group b streptococci) [ , ] . reverse vaccinology has also become an essential tool for several vaccine development projects against agents causing community-acquired pneumonia (e.g., c. pneumoniae, streptococci). the potential and speed of genomic-based approaches was also shown when the nucleotide sequence of the coronavirus causing sars was made available in less than one month. in addition, the increasing number of available genomes from bacteria and viruses would allow comparative genomic studies, thereby providing hints on conserved protein families and/or functional domains. this would facilitate the generation of vaccines using immunogens covering multiple micro-organisms [ ] . despite the incredible potential of reverse vaccinology, this approach also has some important limitations (tab. ). among them is the fact that it is not be possible to identify non-protein antigens (e.g., ps, glycolipids), which are the cornerstone for many successful vaccines (e.g., pneumococcal and hib vaccines). currently available influenza vaccines (see above) are based on inactivated viruses, and, more recently, attenuated ca viruses and virosomes. all these vaccines exploit the same starting material (wild-type virus), which is inactivated or attenuated. the last approach consists in the co-infection of chicken eggs with the new isolate and a master attenuated strain, and subsequent selection for re-assorted viruses with the desired genotype/phenotype. however, the virulence of certain virus strains, such as the h n , renders difficult the implementation of this traditional strategy. the use of reverse genetics represents a valid alternative for the generation of vaccines against rna respiratory viruses, such as the influenza virus, piv and rsv. it consists in the production of the virus from cloned dna [ ] , thereby allowing the development of vaccines against any pandemic viral strain. in some cases (e.g., avian h n ) an additional mutagenesis step would be required to attenuate its virulence [ ] . then, the new ha and na segments would be transferred into an appropriate influenza a virus master strain adapted to grow in a cell line. the final re-assorted virus will have the antigenic specificity of the pandemic strain and the growth characteristics of the master strain [ , ] . this technology would also allow production of the influenza vaccine in cells that are co-transfected with plasmids encoding for different frag- [ ] . therefore, the complete genome is inside the cell and virus can be produced and assembled. one of the main advantages is that a plasmid encoding for ha and na can be easily replaced. therefore, re-assortment and selection become unnecessary. this method would considerably reduce the time for vaccine production, from many months to only a few weeks. another advantage would be the simple manipulation of the genome (contained in plasmids), which would enable detoxification of specific virulence factors. similar approaches can be implemented for other viruses, such as rsv, piv and sars-cov. however, intellectual property and liability issues are still obstacles for the industrial development of reverse-genetics-based vaccines [ ] . furthermore, since the resulting viruses are considered genetically modified organisms, additional problems may arise from the regulatory stand point [ ] . most of the infective agents are either limited to the mucosal membranes, or need to transit across them in order to cause disease. therefore, it is highly desirable to elicit an efficient immune response at the local site in which the first line of defence is laid. the stimulation of a pathogen-specific response at the portal of entry is expected to impair infection (i.e. colonization), thereby reducing the risk of transmission to susceptible hosts. parenterally administered vaccines mainly stimulate systemic responses, whereas vaccines given by the mucosal route mimic natural infections, thereby leading to efficient mucosal and systemic responses. thus, there is a considerable interest in the development of mucosal vaccines. however, antigens administered by this route are usually poorly immunogenic. different strategies are being pursued to overcome this bottleneck, among them can be cited the use of (i) advanced synthetic delivery systems, (ii) live attenuated bacterial or viral vectors, (iii) bacterial ghosts, (iv) pseudoviruses and (v) mucosal adjuvants [ ] [ ] [ ] [ ] . advanced synthetic mucosal delivery systems particulate antigens are more immunogenic than those in solution, due to their vulnerability to degradation by enzymes and extreme ph. thus, it would be helpful to incorporate them into a protective vehicle. often, these vehicles do not serve only to protect them, but can also enhance their uptake, promote targeting to antigen presenting cells and serve as adjuvants [ ] . the most commonly exploited delivery systems are: (i) gelatine capsules, which are dissolved at alkaline ph in the intestine but not in the stomach, (ii) muco-adhesive polymers that are highly viscous inert ps, (iii) eldexomer and carboxymethyl cellulose, which have been used for oral, nasal and vaginal delivery, (iv) lipid-based structures with entrapped antigens, such as immune stimulating complexes (iscoms) and liposomes, and (v) biodegradable micro/nano-spheres based on biocompatible materials such as starch, copolymers of lactic or glycolic acid [ , ] . some of these approaches are currently being explored to develop vaccines against agents causing community-acquired pneumonia. encouraging results have been obtained, among others, using surface antigens from s. pneumoniae encapsulated in micro-spheres [ ] and a iscom-adjuvanted vaccine obtained by reverse genetics against the influenza virus, in preclinical models [ ] . attenuated viruses and bacteria can be used not only as vaccine candidates per se, but also as delivery systems for heterologous antigens. thus, many attenuated microorganisms have been exploited as a scaffold for the development of subunit vaccines against other agents, under the premise that the expression of the recombinant antigen(s) does not increase their pathogenic potential for humans or animals. the most frequently exploited bacterial vectors are attenuated derivatives of salmonella enterica and shigella spp., and the bacille calmette-guérin (bcg). for example, vaccination with an attenuated salmonella expressing the oprf-opri was also shown to be able to confer protection against p. aeruginosa in a murine experimental infection model [ ] . in addition, it was also demonstrated that a recombinant bcgbased vaccine expressing the pspa confers protection against s. pneumoniae in an infection animal model [ ] . the use of commensals represents an alternative to attenuated organisms (e.g., lactobacilli). in this context it was demonstrated that oral administration of lactobacillus expressing proteins from coronavirus can protect against a gastric infection [ ] . thus, this approach has been also proposed to combat sars. promising results were also obtained using x chlamydia psittaci [ ] . on the other hand, different attenuated viruses, such as mva, bovine or attenuated hpiv- and adenovirus can be used as delivery systems for heterologous antigens [ , ] . in fact, mva has recently been exploited for antigens of the sars associated coronavirus [ ] . an alternative approach to the use of live attenuated carriers is given by the use of bacterial ghosts. ghosts are generated by the conditional expression of the lethal lysis gene e from bacteriophage phix in gram-negative bacteria [ ] [ ] [ ] [ ] [ ] . this leads to the formation of a trans-membrane tunnel through the bacterial cellular envelope [ ] . due to the high internal osmotic pressure, the cytoplasm content is expelled through the tunnel, thereby leading to an empty bacterial cell envelope [ ] . the presence of envelope components in the ghosts provides a strong danger signal through the activation of pattern recognition receptors [ ] . in addition, bacterial ghosts are efficiently taken up by antigen-presenting cells, stimulating their maturation and activation [ ] . bacterial ghosts retain all morphological, structural, and antigenic features of the cell wall and can be used as vaccine candidates per se. ghosts can also be externally loaded with purified antigens. alternatively, ghosts can be generated from recombinant bacteria expressing heterologous antigens, hence avoiding the difficulties associated with the purification steps. this technology also offers the possibility to manipulate the topology of the recombinant antigen (e.g., the antigen can be bound to the inner membrane, secreted into the periplasmic space or associated to the surface). encouraging results has been obtained in preclinical models using ghosts expressing chlamydial antigens [ , ] . promising results have been reported using different types of pseudoviruses, such as virosomes and virus-like particles (vlp), which are non-replicating viral-like structures. virosomes are based on the principle of reconstituting empty viral envelopes through integration of viral envelope proteins in liposomes. they offer the versatility of liposomes in terms of lipid composition, with the advantage of including viral membrane proteins. virosomes are produced by disassembling the viral membrane envelope with detergents. then, the viral nucleocapsid is removed by ultracentrifugation before reconstitution (fig. ) . in contrast, vlp exploit the capacity of recombinant viral coat proteins to spontaneously self-assemble, thereby mimicking at structural level the viral capsid. vlp can be isolated after protein expression in eukaryotic cells or by in vitro assemblage from subunits produced in an heterologous system [ ] . their main advantages are the lack the viral genetic material with an "intact" envelope, and the fact that they are significantly more immunogenic than soluble proteins. they can be used as vaccines per se, as well as a delivery system for protein-or nucleic acid based vaccines, or as carriers for small molecules. foreign antigens can be expressed on their surface, or can be simply encapsulated. in addition, amphiphilic adjuvants can be incorporated into their membranes, thereby offering the advantage of combining an adjuvant and the antigen in one entity without a covalent attachment. pseudoviruses are especially attractive for mucosal vaccination protocols, since they offer the opportunity to use the natural route of transmission of the agents. induction of serum antibodies, secretory iga, t helper and ctl responses, and protection against mucosal pathogen challenge has been reported from studies in animals and humans [ ] [ ] [ ] . the virosomes generated using the influenza virus retain membrane fusion properties very similar to the naïve virus. therefore, they are able to deliver material to the cytosol of target cells, offering the possibility to access the mhc class i-restricted pathway of antigen presentation to prime ctl activity [ ] [ ] [ ] . bacterial toxins and their derivatives are among the first molecules that have been used as mucosal adjuvants. they are characterized by the presence of an a moiety with enzymatic activity, and a b moiety that mediates toxin binding to the target cells. cholera toxin and the closely related escherichia coli heat-labile toxin showed potent adjuvant activity when co-administrated with different antigens by the mucosal route [ ] [ ] [ ] . however, their use in humans is hampered by their intrinsic toxicity. thus, mutated derivatives were developed, in which the a subunit was modified to remove the adp-ribosylating activity. the resulting polypeptides retain their adjuvanticity, in the absence of detectable toxicity [ ] [ ] [ ] . however, additional studies have demonstrated that even these derivatives can lead to potential severe side-effects, such as retrograde homing of adjuvant and antigen to neural tissues [ ] . this might explain, at least in part, the side-effects observed after intranasal vaccination against influenza with a virosomes-based formulation containing heat-labile toxin (i.e., bell's palsy), which in turn led to its retraction from the market. however, chimeric derivatives lacking the targeting moiety for neural tissues (i.e., b subunit) are now available [ ] . they might allow the exploitation of the high potential of these molecules for the development of vaccines against respiratory pathogens. in fact, preclinical studies provided the proof-of-concept for the usefulness of derivatives of bacterial toxins in the generation of acellular vaccines against microorganisms, such as s. pneumonia and h. influenzae [ , ] . other bacterial components were also explored for their activity as adjuvants. the monophosphoryl lipid a retains much of the immune stimulatory properties of lps, without the inherent toxicity [ ] . on the other hand, extracellular matrix binding proteins, such as the fibronectin binding protein i of streptococcus pyogenes, also exhibit adjuvant activity [ ] . this offers the possibility of using them as dual antigen/adjuvant moieties in the same formulation. recent reports also demonstrate that vaccine formulations containing adamantylamide dipeptide, a non-toxic compound obtained by linking the l-alanine-d-isoglutamine residue of the muramyl dipeptide to the antiviral drug amantadine, confer protection against non typeable h. influenzae in preclinical models [ ] . the innate immune system plays a critical early role in host defence against pathogenic microorganisms through the recognition of pathogenassociated molecular patterns [ ] . this is achieved through the stimulation of pattern-recognition receptors (prr) that sense a broad range of exogenous and endogenous danger signals [ , ] . toll-like receptors (tlr) represent the best-characterized family of prr. natural and synthetic tlr agonists are being used as immune modulators to optimize responses after vaccination. since the identification of the tlr , many mammalian tlr homologues have been identified (i.e., in humans and in mice) [ ] . each tlr member binds specifically to different ligands (tab. ), alone or in combinations (e.g., heterodimers formed by tlr with either tlr or tlr ). an example of tlr agonist is bacterial dna, but not vertebrate dna, and synthetic oligodeoxynucleotides containing unmethylated cpg motifs. they act on tlr , thereby inducing a strong th responses by activation of dendritic cells [ , ] . cpg motifs have been successfully used as adjuvants in preclinical studies of different candidate vaccines against agents causing community-acquired pneumonia [ ] [ ] [ ] . another important adjuvant with tlr-binding capacities is the mycoplasma-derived macrophage-activating lipopeptide malp- , which act a the level of the tlr heterodimer / [ , ] . malp- promotes a global activation of cells from the innate and adaptive immune system [ , ] , such as macrophages, dc, t-and b-lymphocytes [ , ] . when coadministered with an antigen by either the parenteral or the mucosal route, malp- promotes the elicitation of humoral and cellular responses at systemic and mucosal level [ ] . preclinical studies suggested that malp- could be exploited in vaccine formulations against the sars-associated coronavirus, m. catarrhalis and influenza virus, among others (unpublished data). dna vaccination offers some advantage over the normal antigen vaccination, such as the fact that it is not necessary to express any antigen. in contrast, it is the biosynthetic machinery present in the cells of the vaccinees that takes care of this work. furthermore, since eukaryotic cells are in charge of protein synthesis, their glycosylation and folding are optimal. however, the large-scale purification of dna might be associated with high costs. this can be solved by the use of attenuated or inactivated bacteria or viruses as delivery systems [ ] . this approach can also lead to an enhanced induction of antibodies, which is otherwise poor using conventional naked dna vaccines. we have recently demonstrated that bacterial ghosts can be also exploited as a delivery system for dna vaccines for both in vivo and ex vivo applications [ ] . the potential of this approach is demonstrated by the fact that it is possible to optimize performance by a broad range of manipulations, such as (i) choice of optimal promoters, (ii) use of codon optimized genes for expression in mammalian cells, (iii) addition of nuclear localization signals or ubiquitination signals to improve expression and processing, and (iv) co-delivery of dna constructs coding for immune modulatory molecules [ ] . in addition, by the presence of immune stimulatory cpg motifs, the dna vaccine constructs has built-in adjuvant properties. this vaccination approach is particularly suited for the stimulation of cellular immune responses [ ] . interestingly, several reports suggest that dna vaccines may represent a valid alternative to prime the neonatal immune system, even in the presence of passive transferred maternal antibodies [ , ] . in fact, promising results were also obtained in preclinical models of community-acquired pneumonia, such as influenza [ ] and s. pneumoniae [ ] . furthermore, dna coding for vaccine antigens appears to induce excellent immunological memory, which can be reawakened by later immunization or exposure to the pathogen. the knowledge generated in several basic disciplines, such as immunology and microbial pathogenesis, has allowed the identification of critical bottlenecks for establishing a successful vaccination strategy. it is expected that in the coming years we will develop customized approaches to address each of them, in order to stimulate efficient protection against infective agents under specific clinical settings (i.e., newborns, aging individuals, immunocompromised patients). the importance of immunological memory b lymphocytes that have differentiated into plasma cells are the producers of antigen-specific igg antibodies. bone-marrow (bm) plasma cells have a short life, therefore, the bm reservoir needs to be replenished by the stimulation of memory b cells [ , ] . the maximal life span of bm plasma cells is still debated. only few factors have been identified that control the differentiation of antigen-specific b cells toward short-or long-life plasma cells or to memory b cells [ ] . beside the requirement of cd + t cells, the nature of the antigen [ ] and the dose are also important. higher antigen doses, as well as rapid vaccination schedules (closely spaced vaccine doses) tend to favour the rapid induction of short-term effectors, whereas lower doses of antigens preferentially support the induction of immune memory [ ] [ ] [ ] [ ] . it was demonstrated that neonatal vaccination (priming) and infant boosting might be effective even when pathogen exposure occurs very early in life. in children in whom vaccine-induced hib antibody titres have fallen to undetectable levels, memory is readily demonstrated [ ] . however, immune memory per se is not enough to protect against pathogens that required high levels of neutralizing antibodies. the delay between memory b-cell reactivation and differentiation may limit the ability to interrupt pathogen invasion. therefore, it is important to establish vaccination protocols in which the population is boosted at different ages in order to maintain the required levels of antibodies. this is particularly important in diseases in which antibodies play a central role in microbial clearance or toxin neutralization. in the particular case of community-acquired pneumonia, we should consider that aging individuals are neglected in many vaccination programs. however, the strategies proposed for elderly would be different from those used for small children, since the main factors affecting vaccine efficacy are immune senescence and immaturity, respectively. the attempts to give a rational solution to this issue are discussed in the next sections. the immune system in children immune responses to bacterial and viral antigens usually increase with age in a stepwise manner [ ] . prompt immunization after birth is required to induce active immunity against diseases that may occur early in life. unfortunately, this strategy is limited by the relative immaturity of the neonatal and infant immune system. some factors implicated in this poor response are the limited switch from igm to igg antibodies, impaired complement-mediated reactions and deficient organization of the splenic marginal zone. vaccination studies performed in newborn mice suggested that limited germinal centre reactions may results from the delayed devel-opment of follicular dc and limit plasma cell differentiation [ ] . it was also showed that the neonatal bm has a limited capacity to support the establishment of long-life antibody-secreting plasma cells [ ] . thus, the responses to glyco-conjugates and to most t-cell-dependent antigens are usually affected [ ] . therefore, only few and highly immunogenic vaccines show significant protective efficacy after a single dose in infants. the limited igg responses are extended all over the first year of life. in addition, the immune responses, particularly antibodies, elicited in the first year of life after vaccination rapidly decline [ ] . however, the problem observed in infants in terms of magnitude and duration of immune response does not seem to affect efficient priming. in fact, the immune memory generated in neonates may be recalled later in life [ ] . nevertheless, strategies to generate strong and long-lasting protective responses in infants are still needed. this is in part due to the presence of maternal antibodies, which inactivate and clear the vaccine antigens, thereby rendering difficult the stimulation of an immature immune system [ ] . in addition, the effects of adjuvants reported in adults cannot be extrapolated to neonates [ ] . a potential strategy to overcome these problems would be to implement vaccination during pregnancy, to provide the required antibodies by placenta and later by maternal feeding [ , [ ] [ ] [ ] . this could be complemented with an early priming of the "immature" immune system of the newborn by dna vaccination, followed by a boost during the second half of the first year or later in life [ ] . poly-pathology and multiple organ failure is the rule rather than the exception in aging individuals. thus, many systems are affected (e.g., endocrine, cardiovascular), and the immune system is not an exception. the mechanisms involved in the immune senescence process, which in turn may lead to poor response to vaccination, are not fully understood. however, it is clear that responses against certain vaccines are more affected by immune senescence than others (e.g., ps-based vaccines against s. pneumoniae) [ ] . in contrast, the responses to a boost dose of the anti-tetanus vaccine are hardly affected by age [ ] . a rapid decline of antibody responses, together with a relative restriction of the t-cell repertoire is characteristic of the immune senescence process. this restriction and the reduction in the pool of naïve cells can explain the poor cd + t cell responses against antigens that are cross-reacting with proteins which were seen earlier in life. in contrast, t-cells responses of healthy elderly individuals to new antigens are often unaffected. nevertheless, the overall response to vaccination in the elderly is less efficient than in young adults, making more vigorous approaches necessary (fig. ) . in the case of influenza, the actual strategy is annual re-vaccination. however, there are concerns regarding the capacity to increase antibodies with proper specificity against re-assorted viruses in aging adults who have been repeatedly infected or immunized. after exposure to a new, but cross-reacting antigenic variant, such individuals may respond by producing antibodies. however, these antibodies could be primarily directed against influenza strains, which were encountered earlier in life. figure . factors affecting the responses in young adults and aging individuals after vaccination. the process of immune senescence impairs host response to both infection and vaccination. this critical issue needs to be considered during vaccine design and will require the development of special approaches. for example, individuals previously exposed to the "old" h n influenza strain (i.e., years ago), may respond differently from naïve adults who are vaccinated with a "new" h n strain which have accumulated different mutations. the former might produce antibodies against the ha of the "old" h n strain rather than to the cross-reacting epitopes of the new strain [ ] . this is phenomenon is the so-called "original antigenic sin" [ ] . on the basis of this observations, it was proposed that variations in vaccine efficacy might be due to differences in the antigenic distance between the vaccine strains and the epidemic strains responsible for influenza outbreaks [ ] . however, this hypothesis was not confirmed by epidemiologic studies [ ] . even more, individuals aged years or older who were annually vaccinated showed a significantly reduced mortality risk. therefore, until now, it seems that the antigenic sin does not represent a major practical obstacle in influenza vaccination and additional strategies may not be required. despite the broad availability of vaccines against agents causing community-acquired pneumonia, they still represent an important cause of death, human suffering and economic losses. however, we have dramatically expanded our knowledge on the pathophysiology of diseases caused by respiratory pathogens, their virulence factors and the effector mechanisms responsible for their clearance. it is becoming clearer which microbial components are attractive as vaccine targets, as well as the type of immune response needed to confer protection against disease. thus, it is now possible to address vaccine development using rational rather than empiric approaches. this is facilitated by powerful bioinformatics tools for the accurate prediction of epitopes and proteasome trimming [ ] [ ] [ ] , as well as by the availability of a broad palette of immune modulators and delivery systems. therefore, we can predict that new and improved vaccines against the etiologic agents of community-acquired pneumonia will considerably reduce the global impact of this disease in 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cleavages available on the www this work was supported in part by grants from the dfg (gu / - ) and the bmbf ("pathogenomik" -competence center for genome research of pathogenic bacteria "pathogenomik", u b) to cag. we are particularly grateful to d. felnerova from etna biotech, who provided us with a micrograph from a transmission electron microscopy of a virosome, and to m. höfle for critical reading of the manuscript. key: cord- -hqmyund authors: tang, yi-wei; li, haijing; wu, huiyun; shyr, yu; edwards, kathryn m. title: host single-nucleotide polymorphisms and altered responses to inactivated influenza vaccine date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: hqmyund we analyzed the relationship between host gene polymorphisms and responses in recipients of inactivated influenza vaccine, who were classified into poor, normal, or adverse response groups. the frequency of the mannose-binding lectin- codon allele was significantly different among the types of responders, with a decreased odds ratio for the development of poor or adverse responses (p = . ). there was no statistical relationship between responses and either tumor necrosis factor-α or interleukin (il)- promoter polymorphisms among the response groups. when poor and normal responses were combined, the - a allele in the il- promoter conferred a significantly decreased risk of the development of adverse responses (p = . ). these data indicate that host polymorphisms play a role in determining responses to influenza vaccine. populations, occasional serious adverse events, in addition to local pain and tenderness at the injection site, have been noted [ ] . vaccine responses are determined not only by the chemical nature of the antigen and the manner in which it is delivered but also by environmental factors and host genetic factors. identifying the predictors of individual variability in immune responses to vaccination and the factors that contribute to an increased risk for adverse reactions would enhance our understanding of vaccine responses. it has been demonstrated that host polymorphisms affect the development and progression of certain diseases and disorders by either encoding altered gene products or causing changes in transcriptional regulation. the mannose-binding lectin (mbl)- gene encodes a calcium-dependent protein that plays an important role in innate immunity; circulating mbl- levels are largely the result of several single-nucleotide polymorphisms (snps) in the exon gene and promoter region. variant mbl- alleles have been associated with increased susceptibility to several infections [ ] [ ] [ ] . tumor necrosis factor (tnf)-a and interleukin (il)- are important cytokines that are associated with the regulation of cellular immune responses. several polymorphisms in their promoter regions have been shown to directly affect their gene transcription and are associated with the development and progression of autoimmune and infectious diseases [ , ] . furthermore, the imbalance in th /th cytokine production may contribute to the adverse responses induced by vaccines when natural infection occurs [ ] . the present study focused on host snps in immunogenetic genes: codons (rs ), (rs ), and (rs ) in the mbl- exon gene; Ϫ (rs ) and Ϫ (rs ) in the tnf-a promoter region; and Ϫ (rs ), Ϫ (rs ), and Ϫ (rs ) in the il- promoter region. using immune responses and adverse events assessed in an earlier influenza vaccine trial that enrolled subjects [ ] , we defined poor and normal immune responses and the presence or absence of adverse events as the variables of interest. archived serum specimens were retrieved, genomic dna was extracted, and snp alleles were determined by either ( ) polymerase chain reaction (pcr) followed by restriction fragment-length polymorphism (rflp) analysis or ( ) real-time allele-discrimination taqman pcr. subjects who experienced either poor influenza virus-specific antibody responses or adverse events to vaccines were compared with those who had brisker immune responses and no adverse reactions. subjects, materials, and methods. a large number of healthy volunteers had been enrolled in a national institutes of health-funded, double-blind randomized controlled trial at vanderbilt university to compare the safety, immunogenicity, and efficacy of standard trivalent inactivated influenza vaccine (h n , h n , and b) with experimental bivalent cold-adapted live attenuated vaccine (h n and h n ) in the s [ ] . only recipients of the inactivated vaccine were included in the present study (institutional review board approval number ). the recipients were categorized on the basis of their vaccine-induced responses into the following groups: ( ) poor responders, defined as vaccine recipients with a prevaccination hemagglutination-inhibition (hi) antibody titer of ! : who achieved a ! -fold increase in hi titer to both the h n and h n vaccine components after the first vaccination; ( ) adverse responders, defined as vaccine recipients with a temperature у . Њc after the first vaccination; and ( ) normal responders, defined as vaccine recipients with a у -fold increase in hi antibody titer to both the h n and h n vaccine components and a temperature of р . Њc after the first vaccination. all subjects categorized as poor and adverse responders who had archived serum specimens available were included in the present study. approximately the same number of subjects was selected from normal responders, who were matched for age, sex, and race to the poor and adverse responders. genomic dna was extracted from serum by use of a qiaamp blood kit (qiagen), in accordance with the manufacturer's instructions [ ] . dna extracted from ml of serum ranged from to ng (on the basis of measurement of optical density at nm), and ∼ ng of dna was used in pcr amplification. pcr was used to amplify a -bp region of exon that contains codons , , and and to detect and determine the alleles in these codons. polymorphisms were determined by rflp analysis through the digestion of pcr products with the restriction enzymes bani, mboii, and mlui (new england biolabs), followed by separation on % gel with ethidium bromide staining [ ] . a real-time allele-discrimination pcr assay was used to detect and discriminate alleles Ϫ and Ϫ snp in the tnf-a promoter region and Ϫ , Ϫ , and Ϫ snp in the il- promoter region, by use of the abi prism sequence detection system (applied biosystems). pcr amplification was performed by denaturation at Њc for min, followed by cycles of denaturation at Њc for s, and then annealing and extension at Њc for min. after pcr, the genotype of each sample was attributed automatically by measuring allele-specific fluorescence with the abi prism sequence detection system, by use of the sds software for allelic discrimination (version . . ; applied biosystems). nucleotide sequences of primers and fluorophore taqman mgb probes were designed using primer express software (version . ; applied biosystems) (for tnf-a Ϫ , -aaatcagtcagtggcccagaa- , -tcattcaaccagc-ggaaaact- , -fam-ctccctgctccgatt-mgb- , and -vic-ctccctgctctgatt-mgb- ; for tnf-a Ϫ , -gaaatggaggcaataggttttgag- , -gtaggaccct-ggaggctgaac- , -fam-ccgtccccatgcc-mgb- , and -vic-ccgtcctcatgcc-mgb- ; for il- Ϫ , -agctgaagaggtggaaacatgtg- , -caagcagccc- differences in age, sex, and race among the poor, normal, and adverse responders were examined using epiinfo software (version ; centers for disease control and prevention). because very few subjects were determined to be homozygous for all tested snps, all alleles with heterozygous and homozygous mutations were combined for statistical analysis. a multinomial logistic regression was performed for a global test in which the response groups were analyzed simultaneously to obtain an overall p value for the groups. unconditional univariate logistic regression was used to test the association between host polymorphisms and varied vaccine-induced responses as well as to estimate odds ratios (ors) and % confidence intervals (cis). the wild type of the snps was treated as the reference category for the or calculation. the probability of the logistic regression was modeled for an abnormal response (either poor or adverse) for mbl- and for an adverse response for tnfa and il- . all p values presented in this report are -sided, and was considered to indicate statistical significance. p р . all of the calculations were done using sas software (version . ; sas institute). results. vaccinees enrolled in the parent vaccine trial who had received inactivated influenza vaccine and who fell into of the above-defined response categories were re-reviewed. all poor and adverse responders with available archived serum specimens were included. approximately the same number of subjects was selected for the normal response group, who were matched by age, sex, and race with the other groups. a total of subjects were included in the present study- , , and in the poor, normal, and adverse response groups, respectively. no significant differences were noted in age ( , , , and mean ‫ע‬ sd . ‫ע‬ . . ‫ע‬ . . ‫ע‬ . years), sex (proportion male, . %, . %, and . %), or race (proportion white, . %, . %, and . %) among the poor, normal, and adverse response groups, respectively. we first analyzed the snps in the mbl- exon gene; the frequency of each allele in codons , , and is listed in table . the differences in each allele among the poor, normal, and adverse responders were analyzed by multinomial logistic regression. there were no significant differences in codon and alleles; however, a significant difference in allele frequency in codon was detected ( ; ). an two host snps in the tnf-a (Ϫ and Ϫ ) and in the il- (Ϫ , Ϫ , and Ϫ ) promoter regions were determined by real-time allele-discrimination taqman pcr. the multinomial logistic regression analysis did not discern any significant differences in allele frequency among the poor, normal, and adverse responders (table ) . we then focused on testing our hypothesis that imbalanced th /th cytokine production is associated with an adverse response, indicated by a temperature у . Њc. we combined the subjects in the poor and normal response groups and compared them with adverse responders by unconditional univariate logistic regression analysis. in comparison to the poor/normal response group, the gra polymorphism in the il- promoter Ϫ allele indicated a significantly decreased risk for the development of adverse responses (or, . [ % ci, . - . ]) (table ) . these data suggest that il- promoter polymorphisms may be associated with adverse systemic responses to influenza vaccine. discussion. in humans, the immune response to vaccination is heterogeneous despite the use of a constant formulation, route of administration, and dosage. although the majority of vaccinees generate brisk immune responses with no adverse reactions, ∼ % experience either hyporesponsiveness or adverse events [ ] . the present study explored the possibility that host gene polymorphisms influence inactivated influenza vaccineinduced immune responses by comparing the frequencies of snps in the mbl- gene and in the tnf-a and il- promoter regions among different groups. we found a significant difference in allele frequency in the mbl- codon among the poor, normal, and adverse responders, suggesting that the allele polymorphism is independently associated with poor and adverse responses to influenza vaccination. mbl- is a member of the collectin family and is important in the initiation of the lectin pathway of complement activation and in opsonization [ ] . the variant alleles in the mbl- gene are associated with mbl- deficiency, especially in individuals homozygous for the variant alleles [ ] . host polymorphisms can affect the development and progression of certain diseases and disorders by encoding altered gene products, resulting in poor immune responses. the variant codon allele is more prevalent than codons and in white populations. individuals with allele a in codon demonstrate an even lower mbl- protein concentration than individuals with the t allele, and the mbl- protein produced is incapable of activating the classic complement pathway [ ] . of the polymorphisms within the mbl- gene, codon has independently been found to be associated with increased susceptibility to infection [ , ] . when natural disease occurs after receipt of inactivated vaccine, th /th cytokine imbalances have been demonstrated for respiratory syncytial virus infection and measles [ ] . inflammatory responses must be finely tuned: too strong a response produces adverse events after vaccination, whereas too weak a response attenuates the immune responses. the th -like tnfa is a potent immunomodulator and proinflammatory cytokine that has been implicated in the pathogenesis and development of various infectious diseases. in contrast, the th -like il- is a potent anti-inflammatory cytokine that plays a role in down-regulating cell-mediated and cytotoxic inflammatory responses. several polymorphisms in the promoter regions of the genes for these cytokines have been reported that affect the transcriptional regulation of the genes. il- responses to influenza vaccination have been reported to vary significantly among age groups and vaccines [ ] . we hypothesized that snps in promoter regions result in imbalanced th /th cytokine production, which leads to the occurrence of adverse events after the administration of inactivated influenza vaccine. although a multivariate analysis did not show that the allele frequencies of snps in the tnf-a and il- promoter regions among the poor, normal, and adverse response groups altered responses, a specific analysis between adverse and nonadverse (poor or normal) responses indicated that the Ϫ gra polymorphism in the il- promoter region conferred a significantly decreased risk for the development of adverse responses. previous studies have indicated that the gra snp at Ϫ is important in il- regulation; homozygous individuals (g/g) had higher il- expression after in vitro stimulation [ ] . the snp at Ϫ has been associated with increased susceptibility of infection, severity of illness, organ dysfunction, and mortality [ , ] . these findings support the present data by suggesting that the Ϫ allele polymorphism in the il- promoter region may be associated with adverse responses induced by influenza vaccine. by use of genetic sequencing of the human genome, scientists are relating polymorphisms in various host gene alleles to variable host immune responses to infectious agents and vaccines. because most previous vaccine trials have focused on humoral antibody responses, archival serum specimens are available for study. several earlier studies as well as the present one have demonstrated that substantial quantities of human genomic dna are present in such clinical samples as serum and cerebrospinal fluid [ ] . with appropriate approval by institutional review boards, retrospective studies using the large quantity of archived serum specimens from previous vaccine trials and prospective studies can be conducted to assess many genetic factors. there were several limitations to the present study, including ( ) the small quantities of dna available from serum specimens, ( ) only adverse event (fever) was assessed, and ( ) a small number of host snps and not the entire genome were assessed. in spite of these limitations; however, this study suggests that additional studies should be conducted to confirm these findings. influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly guillain-barre syndrome and the - influenza vaccine association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis mannose-binding lectin in severe acute respiratory syndrome coronavirus infection analysis of candidate-host immunogenetic determinants in herpes simplex virus-associated mollaret's meningitis variation in the tnf-alpha promoter region associated with susceptibility to cerebral malaria association of tnf , a tnf-alpha promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study responses to influenza vaccination in different t-cell subsets: a comparison of healthy young and older adults a randomized controlled trial of cold-adapted and inactivated vaccines for the prevention of influenza a disease mannose-binding lectin (mbl) deficiency: variant alleles in a midwestern population of the united states hutchinson iv. an investigation of polymorphism in the interleukin- gene promoter distinct and overlapping functions of allelic forms of human mannose binding protein mannose-binding lectin gene polymorphism is a modulating factor in repeated respiratory infections association of il- polymorphism with severity of illness in community acquired pneumonia pneumococcal septic shock is associated with the interleukin- - gene promoter polymorphism we thank jennifer doersam, jamie rickmyre, sandra yoder, shufang meng, jody peters, amondrea blackman, pam palmer, sharon tollefson, and william dupont for their excellent technical assistance and barney graham, david persing, and bonnie lafleur for their helpful suggestions and review of the research proposal and/or manuscript. key: cord- -ibca xu authors: xie, tian; wei, yao-yao; chen, wei-fan; huang, hai-nan title: parallel evolution and response decision method for public sentiment based on system dynamics date: - - journal: eur j oper res doi: . /j.ejor. . . sha: doc_id: cord_uid: ibca xu abstract governments face difficulties in policy making in many areas such as health, food safety, and large-scale projects where public perceptions can be misplaced. for example, the adoption of the mmr vaccine has been opposed due to the publicity indicating an erroneous link between the vaccine and autism. this research proposes the “parallel evolution and response decision framework for public sentiments” as a real-time decision-making method to simulate and control the public sentiment evolution mechanisms. this framework is based on the theories of parallel control and management (pcm) and system dynamics (sd) and includes four iterative steps: namely, sd modelling, simulating, optimizing, and controlling. a concrete case of an anti-nuclear mass incident that sparked public sentiment in china is introduced as a study sample to test the effectiveness of the proposed method. in addition, the results indicate the effects by adjusting the key control variables of response strategies. these variables include response time, response capacity, and transparency of the government regarding public sentiment. furthermore, the advantages and disadvantages of the proposed method will be analyzed to determine how it can be used by policy makers in predicting public opinion and offering effective response strategies. social networking services (sns) such as microblog and wechat have become the most prominent carriers and tools for disseminating public sentiment. public sentiment reflects the ideas, attitudes, opinions and emotions that people express on certain noticed issues. positive public sentiment is often a necessary prerequisite for political action, and vice versa; negative public sentiment may become a major obstacle for a correct decision (burstein, ) . in this situation, public sentiment plays an increasingly important role in policy making processes in many areas such as public health, food safety, and large-scale projects where public perceptions can be misplaced (jiang, lin, & qiang, ) . typical examples include incidents like "the adoption of the mmr vaccine has been opposed due to the publicity indicating an erroneous link between the vaccine and autism" (godlee, ) , "china's milk powder industry had seriously declined due to the public sentiment of the sanlu milk powder incident" (chen, ) , " g mobile phone masts have been vandalised caused by a false link with the coronavirus", and "nuclear fuel processing project has been cancelled because of an anti-nuclear public sentiment in guangdong province of china" (weiwei li; . lacking relevant popular science knowledge, most people never say 'yes' to any large-scale project or product involved in a public sentiment. in addition, as long as the projects are planned or "fears" are turned into "crisis," negative public sentiment will be sparked immediately (xiong, liu, & cheng, ; lee and chun ) . worse yet, if public sentiment is not guided and controlled in time, its effects will spread from the virtual network to the physical world and cause further secondary disasters, such as mass incidents. the interdisciplinary online-offline effects which make the situation more complex and uncertain, acutely threaten social stability and national economic development. there have been significant studies of public sentiment triggered by different incidents. in the public health areas, several epidemiologic studies found that mmr vaccine was not associated with an increased risk of autism even among high-risk children whose older siblings had autism. despite strong evidence of its safety, some parents are still hesitant to accept mmr vaccination of their children due to the fake scientific report and news (destefano & shimabukuro, ) . to characterise and evaluate the dissemination of information to the community during a suspected tb outbreak, a model of effective information dissemination in a crisis was developed through a study of information dissemination during the incident (duggan, ) . in the food safety area, lyu identified and compared the crisis communication strategies that organizations used to respond to a congenetic melamine-tainted milk crisis and public opinion in two chinese societies (i.e., mainland china and taiwan) and found that sanlu (a mainland china-based organization) and kingcar (a taiwan-based organization) demonstrated inverse pattern of the strategy adoption (lyu, ) . in the large-scale project area, jeong used situational communication crisis communication theory and attribution theory to explain the public's responses to a corporation that caused an oil spill accident (jeong, ). sun investigated nuclear power in china from the perspectives of electricity preference and social perceptions and assessed the public willingness-to-pay (wtp) to prevent a local nuclear power plant. research results indicate that the chinese pay most attention to the development of nuclear energy when they are worried about nuclear accidents (sun, zhu, & meng, ) . following the anti-nuclear event in jiangmen in guangdong province, china, li analyzed the causes and characteristics of nuclear public sentiment in depth and presented some relevant measures for preventing negative public sentiment in the era of new media (weiwei li; . above all, most relevant studies include some significant ideas for our purposes. however, existing research methods are mainly qualitative rather than quantitative and post-mortem analysis rather than real-time decision-making. two bottlenecks, a lack of prior knowledge and the dynamic of public sentiment evolution, have caused this situation. on the one hand, the original events (large-scale projects, major incidents or scandals) which trigger the relevant public sentiments occur with low frequency, and the triggered public sentiments are diverse and non-duplicated. thus, the key parameters (such as "posting rate of microblog") of prior knowledge (such as decision models and data) for describing or predicting the scenarios of public sentiment systems are difficult to obtain in time. on the other hand, interdisciplinary and dynamic original events lead to the features of feedback, nonlinearity and instability of the relevant public sentiment systems. this also means that the key parameters of the decision models are adjusted over time. therefore, it is more difficult for policy makers to build a quantitative evolutionary model and make an effective response decision based only on previous decision methods. . insightful lessons from the perspectives of system dynamics (sd) and parallel control and management (pcm). . . lesson from the perspective of sd modelling. the sd research method focuses on system structure and simultaneously takes selectivity (decision-making processes based on information), self-discipline (multi-feedback processes) and non-linearity (time delay) into account (torres, kunc, and o'brien ; rashwan, abo-hamad, and arisha ) . it is a perspective and set of conceptual tools that helps us to learn about the structure and dynamics of complex systems. additionally, the rigorous modeling method of sd enables us to build formal computer simulations of complex systems and use them to design more effective policies and organizations (john sterman, ) . several scholars have used sd models to study the diffusion mechanism of different complex systems, which are helpful for us to develop insightful lessons. the sd methodology is used to develop some increasingly complex models to investigate the process of innovation diffusion and enhance insight in the problem structure and increase understanding of the complexity and the dynamics caused by the influencing elements (maier, ) . a diffusion model is employed to extend commonly used epidemic models in the study on analyzing dynamics of intra-organizational innovation implementation processes. the sd modelling and simulation results of this study provides structural explanations and offers insights into why, given various intra-organizational networks, management policies can make different results on whether and how innovations are adopted in an organization (wunderlich, größler, zimmermann, & vennix, ) . furthermore, by applying system dynamics, yu proposed a simulation model to study the public sentiment diffusion mechanism in dangerous chemical pollution water emergencies and discover effective response strategies (yu le-an et al. ) . his research shows that the sd modelling methods are useful for describing, understanding and simulating the complex public sentiment diffusion systems and for making relevant optimization policies. more importantly, compared with decision-making theories based on large amounts of historical data, sd has the advantage of being able to predict the future based only on system structure. therefore, when one lacks historical data, most complex social systems can be described and analysed using sd method (morgan, howick, & belton, ). public sentiment system is no exception. to solve the first bottleneck (lack of prior knowledge), the structure of the public sentiment system can also be represented by multi-feedback relationships, and its system functions can be achieved and controlled by sd method. moreover, by now many mature statistical technologies, such as regression analysis, are available to help decision-makers obtain key parameters and build sd simulation models to respond to possible disasters and social crises. . . lesson from the perspective of pcm theory. with the advent and development of cloud computing and the internet of things (iot), the capacities of perception, computing, storage, transmitting and analysing have been greatly improved. in this era of new information and communication technology (ict), artificial society and pcm theory have been proposed by the chinese academy of sciences and the national university of defense technology to provide an available decision-making paradigm to respond to the dynamically evolutionary scenarios of interdisciplinary disasters and social crises. this theory uses multi-agents modelling and simulation technology to build an artificial social system equivalent to the real system. by simulation computing in the artificial system, decision-makers are able to recognize the evolutionary mechanisms of disaster scenarios. moreover, by comparatively analysing the artificial and the real systems, the simulation model of the artificial system is modified to parallel the actual situation in real time. lastly, with the knowledge obtained from the simulation results, the evolutionary processes of disasters can be controlled and optimized by making the appropriate decisions in time (f. wang, ) . inspired by pcm theory, the public sentiment system, a type of crisis system, can be considered a real system carrying public attitudes and opinions. thus, the second bottleneck (dynamic of public sentiment evolution) may be resolved based on both pcm theory and sd method. after building and running the artificial sd simulation system parallel with the public sentiment system, evolutionary scenarios can be predicted and effective response policies can be provided by decision-makers. therefore, a novel decision-making method "parallel evolution and response decision framework for public sentiment (perdfps)" based on the theories of pcm and sd is proposed as a specific contribution of this paper. this method is structure-dependent rather than data-dependent and can be implemented in real-time, which makes it helpful to simulate, analyze and guide the evolution processes of dynamic public sentiment in the case of lack of historical knowledge on less-frequently occurring original events. the paper is organized as follows for achieving the contribution. section proposes the perdfps framework. and section " methodology" is developed to abstract and present the perdfps methodology with a roadmap (table ) helping decision makers effectively use the novel method. furthermore, the section " empirical research" shows a whole case study to deduce the principle and verify the effectiveness of the perdfps method using the anti-nuclear mass incident in jiangmen, china. lastly, theoretical discussions, the limitations and future studies are presented in section . dynamics. pcm theory holds that social mass incidents are essentially unpredictable. however, one can now build artificial societies parallel to real-world societies and deduce their evolution in a computational experimental environment. in addition, with dynamic and real-time data, decision-makers are able to perceive coming disaster scenarios and response demand using artificial simulation models and create effective and timely coping strategies, policies and response solutions to control the real situations (f. wang, ) . therefore, the "parallel evolution and response decision framework for public sentiment (perdfps)" is proposed as a guideline for achieving this function. moreover, within this framework (fig. ) , according to the "scenario-response"-based emergency management paradigm (bañuls, turoff, & roxanne, ) , the public sentiment system (pss) can be divided into two subsystems: the real scenario system (rss) and the simulation decision-making system (sds). as the source of real-time information, real scenarios are the basis for decision-making as well as the targets of public sentiment control. the original events, sentiment disseminators and sentiment regulators are the essential elements of the real scenario system. the original events (such as public health emergencies, food safety scandals and large-scale projects) may easily trigger relevant public sentiment. public sentiment disseminators include the media, netizens, and others. the media triggers and influences the processes of public sentiment propagation through reporting and directing the news. in addition, netizens use social networks to express and communicate their own opinions, which results in the continued diffusion and evolution of public sentiments. because the collective behaviours of the netizens comprehensively reflect their attitudes towards source events, their support or opposition are essential factors for the government in making an efficient response decision. generally, the government response departments dealing with the emergencies or large-scale projects assume the greatest responsibilities as public sentiment regulators. by taking measures such as holding lectures and seminars, press conferences on the events, and by releasing positive news, they supervise, guide and even control the development of the public sentiments. based on pcm theory, the simulation decision-making system is a closed-loop functional circuit consisting of four steps: sd modelling, simulating, decision-making and parallel controlling. in the first step, using real-time perception (which includes the processes of information collecting, analysing and abstracting), the characteristic elements of the public sentiment system, including source events, sentiment disseminators and sentiment regulators, can be extracted from real scenarios. based on the sd method, the causal loop diagram model is suitable for describing the relationships between the elements of public sentiment systems. in addition, after setting the parameters and calculating rules, the stock flow diagram model, which is based on the qualitative causal loop diagram model, can be built to describe the dynamic system structure and simulate its functional mechanism quantitatively. in the second step, simulation results of public sentiment diffusion and evolution at a future time can be obtained by importing real-time status data perceived from real scenarios into the stock flow diagram model and running it. next, to keep simulation models consistently parallel with real scenarios, decision-makers should dynamically adjust the parameters and structures of the stock flow diagram models by observing and analysing the running effect of models and the real-time status of the scenarios. in this way, the validity and reliability of the simulation results of the sd stock flow diagram model can be gradually improved. in the last step, given a reliable sd model, different response strategies or policies can be tested, verified and optimized in the simulated environment. roadmap is useful for decision makers to know when and how to use a modelling and simulation method for dealing with practical problems (davis, eisenhardt, & bingham, ) . for implementing the perdfnps method, we develop a roadmap that describes the principles and steps shown in table ( thompson, howick, & belton, ) . no matter how the perdfnps method is carried out, the steps do not occur in neat sequence. specifically, the steps of sd modelling and parallel controlling constitute an iterative process for cultivating a rational decision model. table roadmap for implementing the perdfnps method. step description  identify the type of the decision problem: the sentiment triggered by a periodic heated topic with enough investigable historical data or by an unconventional heated topic?  selecting the relevant decision methods, data-dependent or structuredependent?  reality hypotheses of the model: the simulation results of the model are consistent with the real scenarios.  validity hypotheses of the response strategies: testable relationships between the response strategies and the controllable degree of public sentiments.  analysis of real scenario system from perspectives of structure.  causal loop diagram modelling: abstracting the elements and the causal relationships between them from the scenario system; defining the circular causality loops of relevant modules of system.  stock flow diagram modelling: selecting levels, selecting rates and describing their determinants; selection of parameter values. simulating . parallel controlling  reality testing: contrasting the simulation results to verify the reality hypotheses of the model.  model improving: to achieve the rationality and consistency of the model.  response strategies setting.  response strategies testing: simulation and discussion of strategy effects for verifying the proposed validity hypotheses. . identify the decision problem. as a structure-dependent decision-making method, the perdfnps framework is applicable for response to the public sentiments without enough historical data. therefore, in the implementation stage, the decision makers must firstly determine what type of decision problem it is: is there enough historical data for building a model for this event? or is the perdfnps method suitable for the decision problem? if public sentiment is triggered by a periodic heated topic with enough investigable historical data, the decision-making model can be constructed based on the data-dependent statistical methods. for example, the training data used for cultivating the model of its public sentiment system for the presidential election every four years, can be obtained from the social network services including twitter, facebook (h. wang, can, kazemzadeh, bar, & narayanan, ) . conversely, if the public sentiment is triggered by an unconventional or non-periodic heated topic such as a nuclear-related event without sufficient investigable historical data, the sd simulation model can be cultivated to describe and solve these kinds of problems based on the structure-dependent perdfnps methods. two kinds of hypotheses are proposed for the perdfnps method according to the requirements of simulation reality and decision validity. ( ) dynamic simulation reality hypotheses: hypothesis , the simulation results of the cultivated sd model are rational and consistent with the real scenarios. the rationality of the cultivated sd model and the consistency between its simulation results and the real evolution trends of the public sentiment are essential to achieve scenario rehearsal and response effectively in the decision-making processes (thompson et al., ) . thus, we firstly present hypothesis . then, we begin to cultivate our sd model and verify the hypothesis for a specific case through comparison between the real scenarios and the simulation results (section . ). of course, the rational initial general sd model is very important because it determines the efficiency for reality improvement of the descendant models. the best method for verifying the dynamic reality of a simulation model is testing the consistency between its simulation results and the real evolution trends in a real-time case. however, it is difficult to achieve this process in our empirical research. therefore, the special hypothesis for the subsequent section of empirical research is proposed. hypothesis , information of the past scenario of the public sentiment, is unknown but can be gradually revealed through evolutionary process. the goals of the empirical research are to verify whether a reliable simulation model that parallels the real public sentiment scenario can be developed and whether the response strategies based on the validity hypotheses can be optimized. for comparing the simulation results with the real-time scenarios, we assume that the data of the scenario is unknown but will be gradually revealed through evolutionary process in the experiment. the public sentiment event is considered a real-time scenario to verify the rationality and consistency of the parallel simulation models to be cultivated. hypothesis and can be validated by reality testing (section . ). ( ) decision validity hypotheses the public sentiment system is a complex social network system. the various decision response behaviours of government drive the evolutions and diffusions of public sentiments and ultimately decide the results of the original events through affecting the attitudes of people. therefore, four testable decision validity hypotheses are proposed to explore the effective response strategies for guiding the evolution of the public sentiment with the perdfps method. firstly, the government's response measures, including social stability risk assessment, answering questions from the public, giving lectures on the original event-related knowledge and so on, is significant for guiding the evolution processes of the public sentiment events. higher degree of government response results in a more controllable public attitude to the public sentiment event (zhang, qi, ma, & fang, ) . the amount of times the government responds to an event can reflect the degree of government response. therefore, the relevant hypothesis is proposed as follows. hypothesis is that, firstly, the more times the government responds, the more controllable the public attitude to the public sentiment event is. secondly, response time represents the time-delay in the government response processes. another important factor is controlling the evolution processes of the public sentiment events. a timely response means higher response speed which leads to a lower heat rate and a higher controllable degree of public sentiment (yu le-an, li ling, wu jia-qian, ) . similarly, hypothesis is proposed as follows. hypothesis is that a higher response speed (or short response time) of government leads to a higher controllable degree of public sentiment. furthermore, the degree of information transparency and the degree of popularity of original event-related knowledge also make significant influences to the evolution trends of the public sentiment event. the degree of information transparency represents the disclosure degree of the original event-related information, and the degree of popularity of original event-related knowledge reflects how the knowledge related to safety and risk was popularized locally. scholars analyzed the response processes of public sentiment events and believed that these two factors are positively related to the controllable degree of large scale project-related public sentiment (weiwei li; . hypotheses and are proposed as follows. hypothesis details that a higher degree of information transparency leads to a higher controllable degree of public sentiment. hypothesis is that a higher degree of popularity of original event-related knowledge leads to a higher controllable degree of public sentiment. ( ) analysis of real scenario system from perspectives of structure. the sd approach emphasizes how causal relationships among system structures can influence the functions, behaviours and evolution processes of a system. analysis of the boundary and structure of a and regulations, and holding a press conference) to guide the development of original events and control the diffusion of triggered public sentiments in a timely manner. furthermore, the netizen module: original events may affect social and economic benefits, damage the ecological environment and even directly threaten our life safety. therefore, they quickly garner attention from people and trigger various public opinions or sentiments. there are both advantages and disadvantages for various people, and thus netizens are divided into three groups: opponents, supporters and neutrals. these groups express their own opinions and sentiments online and this kind of behaviour turns original events into controversial issues. in addition, the main attitudes of netizens influence and even decide the response behaviours of the relevant media and government departments, as they reflect public opinion. ( ) causal loop diagram modelling the sd approach describes a system as a series of simple processes with positive and negative circular causality loops (forrester & senge, ; sterman ) . the feedback function of a positive loop is self-reinforcing and amplifying; the feedback function of a negative loop is dampening. the public sentiment systems evolve continually under the influence of the interactions between the feedbacks. based on the structure and mechanism of real scenario public sentiment system of a concrete case, the qualitative causal relationships and the circular causality loops of relevant modules can be defined. the concrete examples of this process are shown in section . . . ( ) stock and flow diagram modelling causal loop diagrams aid in visualizing a system's structure and behavior, and analyzing the system qualitatively. to perform a more detailed quantitative analysis, a causal loop diagram is transformed into a stock and flow diagram. a stock and flow model helps in studying and analyzing the system in a quantitative way; such models are usually built and simulated using computer software such as vensim (rahmandada & sterman, ; martinez-moyano, ) . a stock is the term for any entity that accumulates or depletes over time. a flow is the rate of change in a stock. the key step to build the stock and flow diagram is converting the system description into level and rate equations (torres et al., ) . usually, the parameters and initial conditions of the equations can be estimated using statistical methods, expert opinion, market research data or other relevant sources of information (j sterman, ) . in a decision-making process for a non-duplicated public sentiment triggered by a major public health incident or a large-scale project, because the decision makers lack prior data and knowledge, the parameters of the initial equations of the -general sd model can be referenced from the developed models of historical cases which are similar with the current event in type, system structure and situation. by analyzing and refitting the real-time data gradually collected from the real scenarios, the parameters are modified and the equations are improved. to achieve the target of public sentiment simulation and control, the variables of the stock and flow model are classified into two categories, including state and control variables. the latter is classified into two subcategories, including parallel cultivating control and decision-making control variables. each kind of variable has relevant functions in the model. according to the literature (rahmandada & sterman, ) , the variables of the constructed sd model should meet the minimum and preferred reporting requirements for research reproducibility, communication and transparency. appendix shows an example of a constructed and defined sd model of all variables and their equations. model library scenario simulaiton decisionmaking system non-duplicated source events trigger diverse public sentiments and lead to a lack of prior knowledge (the parameters to describe or calculate the key variables) in order to make an effective response decision. in addition, even if we obtain some experience from the public sentiment triggered by a similar original event, due to their various spaces, locations and social environments, the evolution processes of the public sentiments are too different to build a simulation and decision model in one step using the most traditional methods. thus, in this paper, we use pcm theory to cultivate the sd simulation models dynamically and to keep the models parallel with real public sentiment systems in order to obtain accurate simulation results and reliable response solutions (f. wang, ) . the parallel controlling principles are shown in figure . ( ) -generation sd model constructing. the parallel simulation decision-making system enters into the emergence response state immediately after "scenario " of a new original event occurs. at this point, decision-makers should decide whether this event has ever occurred using a semantic searching and matching in an existing model library (wu et al., ) . if there is a public sentiment scenario very similar to the current scenario through the perspectives of event type and system structure, the relevant simulation model of the scenario is chosen as the initial model " -generation sd model." however, there are usually no extremely similar scenarios for a non-duplicated public sentiment event. in that situation, the parameters of the initial equations of the -generation sd model can refer to the developed models of historical cases which are similar with the current event in type, system structure and situation. regarding the historical cases, it is very difficult to find the most similar one in an emergency situation. we can still improve and cultivate our models iteratively by adjusting them in parallel with the real-time scenarios even if the selected historical case is not identical to the current public sentiment event. of course, the more similar a selected historical case is, the less iteration there will be, so the improved decision-making model will be obtained quickly. ( ) reality testing. we can validate hypothesis and by rationality and consistency testing. the rationality and consistency between the models and real public sentiment systems should be discussed by (wooldridge, ) . ( ) model improving. by analyzing and refitting real-time data gradually collected from real scenarios, the parameters are modified and the equations are improved. in the entire parallel controlling process, the real time information from the various stages of the real scenarios (from scenario to scenario n) should be collected, integrated, and analyzed at a specific time-interval, "△t". at the same time, modifying processes such as parameter refitting and data updating can be used to continually improve the simulation models from " -generation sd model" to "n-generation sd model," ensuring that simulation results are consistent with the real scenario in the parallel evolution processes. regarding the parameter fitting into the model cultivating process, we constructed the -generation sd model and the other n-generation sd models in different ways. take the case shown in the empirical part of this paper as an example. on the one hand, most of the equations of the -generation sd model based on the literatures (tian, ), (weiwei li; , (zhang, ) and (yu le-an, li ling, wu jia-qian, ) are obtained using parameter fitting with the data of similar cases such as the "sanlu milk powder incident" from "people.com." on the other hand, for the n-general models, we do not have to fit the parameters to improve the equations. only real-time data needs to be collected to draw the curves of the key state variables such as "increment of posts" and "increment of news" during the time-interval (△t = hours). after contrasting the simulation results of the n-generation model and the scenario n, the parameters of the equations are adjusted and the model is improved to approach the real scenario in an iterative and parallel way. when an n-general model and its simulation results are consistent with the real scenarios, it means that the model has passed reality testing and has become a cultivated model. optimal strategy can be obtained by adjusting the control variables. as shown in the stock and flow diagram model (fig. ) , the interactions between these system variables drive the evolutions and diffusions of public sentiments and decide the final results of the original events, as seen through the attitudes of people. in the end, when the entire emergency response process ends, the latest parameters and structure of the simulation model are recorded and archived in the model library for the next similar public sentiment. some advanced sd software tools, such as vensim, stella and anylogic, are available to help decision-makers construct, run and analyse the sd simulation models of the public sentiment systems and to create the optimized response policies and solutions in a graphic and visual way (rahmandada & sterman, ) . according to the evolutionary cycle and system boundary of the specific public sentiment system, and after setting the main calculating parameters such as simulation step size and run time, the simulation process can be implemented. to propose suitable response solutions, the relevant decision analysing process should include two aspects: ( ) response strategies setting. the control variables can be tentatively set by the decision makers to represent different response strategies. however, most of the variables, especially state variables, are uncontrollable from the perspective of government. next, taking the public sentiment of anti-nuclear mass incident as an example (section ) and according to the "validity hypotheses of the response strategies" table . the r strategy is the baseline strategy extracted from this real case; the other response strategies, including a ~a , b ~b , c ~c , d ~d and e, are set with different specific values of the key control variables, respectively. ( ) response strategies testing. the effects of different strategies can then be tested by reviewing the simulation results with the cultivated n-general sd model. policy-makers would be able to control the system state variables and create an effective and optimized response strategy to guide the public sentiments by adjusting or resetting the values of the four key controllable variables in the sd simulation models. some practical measures, such as enhancing the credibility and degree of information transparency of government, are available to improve the control performances of public sentiments. however, increasing these variable values always brings higher response times or costs. thus, balanced considerations should be included in the decision-making processes. . case analysis (including "indentify the decision problem" and "research hypotheses") in this section, the anti-nuclear mass incident sparked by a nuclear fuel processing base project near jiangmen city in guangdong province is taken as a case study to verify the feasibility and validity of the perdfps method proposed in section above. the announcement of the result of the social stability risk assessment of the nuclear project is considered the source event of the media module of this public sentiment system. in addition, by choosing and analyzing the increment data of baidu news and xinlang blog posts (fig. ) during july - , , and the relevant government response measures (table ), the key parameters of the initial stock flow diagram model and the initial data of the simulation process of public sentiment evolution can be extracted gradually. due to the actual evolution cycle of public sentiment lasting only days, it is too short to prepare enough data and develop a reasonable simulation trend. the simulation model is thus set for running hours per day. therefore, the simulation cycle is translated or expanded from days to hours. the main data used in this paper is collected from references and websites (weiwei li; cnsa, ) . moreover, some variables or parameters of the model should be adjusted according to the demand of the actual simulation experiment. for example, some unstructured behaviour data, such as government response behaviours, should be ranked into the qualified and controllable variables quantity of government response or increment of government response. note that the data shown in figure are considered unknown information at the beginning of the simulation experiment and will be revealed gradually during the simulation processes. in fact, this primary data is used to verify the reality and validity of our parallel simulation model by describing the real scenarios and comparing them with the artificial ones. therefore, the decision problem of this case is structure-dependent and can be described and solved with the perdfnps method based on the hypotheses proposed in section . the jiangmen government released the report of the social stability risk assessment of the nuclear project. the deputy general manager answered questions from the public. th three lectures on nuclear knowledge are given by the jiangmen government. th professor zhou participates in the propaganda lectures on nuclear knowledge, telling the public that the project is safe. th announcement of the determination of the nuclear project is delayed for ten days. the nuclear project is terminated by the government. . -genernal sd modelling. plans by the china national nuclear corporation (cnnc) to build a nuclear fuel-processing base near public sentiment in that city. taking this anti-nuclear mass incident as a concrete case, we can analyze the boundary, structure and evolution mechanism of the public sentiment system of this event and build a relevant qualitative causal loop diagram model. this model is divided into three main modules: the media module, the netizen module and the government module. ( ) the causal loop diagram of the media module is a positive feedback loop r (fig. ) . once the nuclear event does occur, the relevant news is released on an ongoing basis. in addition, as the event evolves, the quantity of news and the participation degree of media both increase. at the same time, due to media influence, the increased participation degree of media always leads to a higher rate of public sentiment and results in more news increments. the evolution processes of the nuclear public sentiment system are driven by a multi-feedback mechanism, which leads to some delays in the propagation and response processes. thus, the nuclear public sentiment system should be described as a number of nonlinear and multi-feedback relationships, such as the exponential dependence relationship between interactive system variables such as increment of government response, increment of posts and news increment. taking the incident of "anti-nuclear public sentiment in jiangmen city, guangdong province" as an example, and according to the main causal loops in section . , the sd stock flow diagram model of the nuclear public sentiment system is shown in figure . ( ) stock flow diagram model of the media module. because the source event doesn't last long, the variable increase rate of news can be defined as a lookup or fitted function and described directly according to the captured short-term history data. the variable quantity of news is influenced by the variable heat rate of public sentiment, which is constrained by the variables participation degree of government, participation degree of media and participation degree of netizens. referring to the evolution mechanism of public sentiment (yu le-an, li ling, wu jia-qian ; zhang ), the functional relationships of the variables can be defined respectively as the formulas ( - ). in these formulas, we assume that there is no delay in the transmission processes of baidu news, and the variable type of source event is boolean (its value equals if the source event is trigged). ( ) stock flow diagram model of the netizen module. the posting reaction of the netizens after receiving news information causes a time-delay effect and the variable increment of posts is also influenced by the variable heat rate of public sentiment. the variable increase rate of posts can be defined as a lookup or fitted function and described directly according to the captured short-term history data. the function of increment of posts is then constructed as a formula ( ). referring to the evolution mechanism of public sentiment ( figure . at the beginning of the simulation, we lack sufficient data to fit certain parameters of the -generation model. the variables "increase rate of posts" and "increase rate of news" are defined temporarily as formulas ( ) and ( ) (in the appendix) by collecting and polynomial fitting the hours of data (from july th to august rd ) of the similar public sentiment triggered by the changsheng vaccine event from ef.zhiweidata.com (figure -①), because these two events have the similar "happening, developing, declining, and extinction" life cycle. ) -generation model testing and improving. in the parallel iteration processes, the time-interval for improving the models is set as "△t = hours." we constructed the improved n-generation formulas based on the "minimum sum of squared errors" algorithm with the real data during n△t, and we tested the reality of the model based on "goodness-of-fit formula" with the real data during (n+ )△t. considering the scenario during the th hour as scenario (testing period of -generation model t testing- = △t), assume that the real scenario evolves under the combined influences of the media, netizens, and government. after importing the initial data of the first hour of this case and simulating it for steps (hours), the evolutionary trends of the variables "increment of posts" and "increment of news" can be drawn into figure -②. the simulated time series (orange curves) are both obviously too steep to match the real situations (blue curves). the model reality is tested by calculating the goodness-of-fits ( , gof∈ (-∞, ] ) between the real time series and the simulated time series of the key state variables. in this formula, y is the observed posts/news increment value, y * is the simulated value (wooldridge, ) . we use "gof dp " and "gof dn " to indicate the gof of the "increment of posts" and "increment of news" by the day respectively. after programming and calculating, gof dp and gof dn are - . and - . respectively. therefore, the simulation results show that the " -generation model" is very different from scenario during t testing- . to ensure that the simulation results are consistent with real scenarios, we developed a "minimum sum of squared errors" algorithm to improve the models. with this algorithm, each polynomial coefficient of (n- )-generation "increase rate of posts" and "increase rate of news" formulas is traversed within a certain range, and the sum of squared errors between the observed real value series and the simulated value series is calculated for different coefficient combinations through multiple iterations. then, the optimal coefficient combination with the minimum sum of squared errors is selected to be the polynomial coefficients of the "increase rate of posts" and "increase rate of news" formulas of the n-generation model. after inputting the hours of scenario- data, the -generation variables "increase rate of posts" and "increase rate of news" are defined as formulas ( ) and ( ) in the appendix (also shown in figure -②). ) -generation model testing and improving. considering the testing period of the -generation model as t testing- = h ( △t), the real scenario continues to evolve from scenario to scenario for h(△t ). after simulating for h, the calculated gof values and drawn evolutionary trend curves (figure -③) of the variables "increment of posts" and "increment of news" both show that the -generation model is much better than -generation model. however, gof dp and gof dn are still as low as - . and - . respectively, which means the -generation model cannot simulate the real scenario during t testing- or help us to predict the evolutionary trend of the public sentiment. therefore, after calculating the "minimum sum of squared errors" between the observed hours of scenario- data and the simulated value series, the optimal coefficient combination is selected as the polynomial coefficients of "increase rate of posts" and "increase rate of news" formulas of the -generation model shown in figure -③. similarly, models are tested and improved from -generation to -generation after a set number of parallel iterations. the variables "increase rate of posts" are defined as formulas from ( ) to ( ) and the variables "increase rate of news" are defined from ( ) to ( ) in the appendix. the simulated trend curves are becoming more and more similar to the real curves, and the gof dp and gof dn of the models are increasing in the iteration processes (figure -④~⑧) . finally, the cultivated -generation model is obtained with high gof values (gof dp = . and gof dn = . ). although the simulated curves are still smoother than the real scenarios, the overall public sentiment trend is consistent with the real scenarios as shown in the stock flow diagram model (fig. ) , the interactions among these system variables drive the evolution and diffusion of public sentiments and ultimately decide the results of the nuclear project by affecting the attitudes of the public. however, most of the variables, especially state variables, are uncontrollable from the perspective of government. next, we chose the four controllable variables, degree of popularity of nuclear knowledge, to explore the effective response strategies for guiding the evolution of the public sentiment in this event. the series of different response strategies is shown in in this table, "times of government response" represents how many times the government responds to public sentiment in an event. its initial value is (times), because in this case, measures were taken to respond to the nuclear-related public sentiment event by the government. we tentatively adjusted its value from to to present the different strategies a -a . the number of "response time of government response" represents the time-delay in the government response processes. its initial value is set to (hours). we tentatively adjusted its value from . to to represent the different strategies b -b . the "degree of information transparency" values represent the disclosure degree of the nuclear project-related information. its initial value is set to . . we tentatively adjusted it from . to . to represent the different strategies c -c . the "degree of popularity of nuclear knowledge" value represents how the knowledge related to nuclear safety was popularized locally. its initial value is set at . the variables "degree of information transparency" and "degree of popularity of nuclear knowledge" are considered relative rather than absolute because they are hard to exactly quantify. furthermore, our research focuses on reality testing (how the evolutionary trends of the public sentiment are simulated by the parallel cultivated sd model) and response strategies testing (how the evolutionary trends of public sentiment are influenced by different strategies). this consideration is reasonable. we can simulate the public sentiment scenarios and test the effectiveness of the improved response strategies by increasing or reducing their initial values. ) simulation and discussion of strategy effects (response strategies testing) after running the cultivated " -generation model" with different values of control variables for hours, the results indicate the corresponding effects of different strategies a ~d on public attitude to the nuclear project, respectively, and find the most appropriate response strategy for guiding nuclear public sentiment. the effects of the four controllable variables on public attitudes are shown in the in the two right-most columns of table . ( ) the separate effects of the four control variables on public attitudes compared with the baseline scenario r , the percentages of support posts under the control of strategies a and a , respectively, rose to . % and . %. this means that the approval rating of the nuclear project will rise only by greatly increasing the "times of government response." it is reasonable because the government is one of the project sponsors. enhancing the measures of response and disposal by means of open information, popular science propaganda, and so on is helpful for increasing the approval rating and guiding public sentiment in the right direction. this result also indicates that hypothesis is tenable in this case. usually, the project approval rating increases as response speed or information transparency increases. however, the simulation results of the eight strategies from b to c show that the adjustments to the variables "response time of government" and "degree of information transparency" cause no obvious change in the public opinion. therefore, these two variables are not the most important and hypothesis , is not entirely tenable in this case. the percentages of support posts under the control of strategies d and d are, respectively, reduced to . % and . % from . %; in addition, the percentages of support posts under the control of strategies d and d rise to . % and . % respectively. this simulated result obviously indicates that a lower "degree of popularity of nuclear knowledge" by the public leads to a lower approval rating of nuclear projects. as the project was supported by government-owned corporations, this result also indicates that a higher "degree of popularity of nuclear knowledge" leads to a higher controllable degree of nuclear project-related public sentiment. professor zhou also publicly declared that "the whole nuclear fuel processing in the jiangmen base project does not relate to the nuclear fission reactions" (weiwei li; . however, due to the high threshold of nuclear technologies, it is very difficult for most people to quickly and effectively understand the safety of nuclear energy and accept nuclear projects. the blind worries regarding crises stemming from nuclear projects are one of the main reasons for triggering and spreading negative public sentiment and ultimately terminates potential projects. therefore, spreading long-term and effective popular science propaganda about nuclear safety is necessary and important for the government to accumulate and improve the local degree of popularity of nuclear knowledge and ensure project success. hypothesis is therefore tenable. above all, adjusting the key control variables appropriately leads to completely different public attitudes and event results of the jiangmen anti-nuclear mass incident. in the response processes, a higher approval rating of the nuclear project can be achieved using response strategies with higher degrees of response strength, response speed, information transparency and popularity of nuclear knowledge. the variable "popularity of nuclear knowledge" has the most remarkable effects on public attitudes than the other three variables. the approval rates ahead the disapproval rates leading to the project establishment only by appropriate increase (about six times as the initial value) of the "degree of popularity of nuclear knowledge." these conclusions also indicate that only adjusting three other intermediate control variables, such as the "times of response" even up to ten times as the initial value, is still not enough to reduce the people's unreasonable fears and change the event result. the control variable "degree of popularity of nuclear knowledge" plays a decisive role in the evolution process of the public sentiment. however, it is not easy to increase the degree of popularity of nuclear knowledge in a short period of time. as an advance control variable, this indicator must be improved by education and advocacy activities for months or even years before the nuclear related public sentiment event actually occurs. ( ) the effects of the flexible combination strategies on public attitudes additionally, some flexible combination strategies adjust two or more control variable values at the same time, such as strategy e with the higher "times of government response" and "degree of popularity of nuclear knowledge." these strategies are better than the strategies adjusting only one control variable value once for guiding the public sentiment. therefore, the evolution trend of the variable "quantity of support posts" impacted by the combination strategy e is compared with the trends impacted by the strategies r , a and d respectively in figure . the comparison shows that the quantity of support posts increases significantly with the strategy a (much more times of government response) or the strategy d (much higher degree of popularity of nuclear knowledge). however, it can be seen in table that the percentages of support posts are still lower than percentages of support posts with the two single strategies separately in the end of simulation. however, the comparison also shows that the quantity of support posts increases much more significantly and rapidly using strategy e rather than a or d . it can also be shown in figure that strategy e leads to a higher percentage of support posts than percentage of opposite posts, which presents a reversed public sentiment simulation result that the approval rating is higher than disapproval rating for this nuclear-related project. therefore, the government is able to guide and control the evolution and development of public sentiments more effectively in such a scenario by using the combination strategies. taking the anti-nuclear mass incident as a case study, the parallel simulation models of public sentiment regarding the incident are constructed and improved into the final realistic model gradually and iteratively, and the different strategies for responding to real scenarios are simulated with the improved model to discover the effects of the control variables on public sentiments and provide relevant policy implications. the empirical research verifies the effectiveness of the perdfps framework and shows that the methods proposed in this paper can be very useful in providing decision support for guiding and controlling public sentiments during future nuclear energy developments. traditional sd and pcm are integrated into the perdfps framework to solve the unconventional and unrepeatable public sentiment decision-making problems without any sufficient historical data in this paper. the novel approach cultivates the models to simulate real scenarios with real-time data rather than historical data in a parallel way. compared with the data-dependent "predictresponse" decision-making methods, the structure-dependent "scenarioresponse" perdfps framework has more advantages. first, the parallel evolution and response mechanisms of the public sentiment are both considered in the iterative model-improving processes to achieve the consistency between the simulation results and the real scenarios. second, in the case of a lack of historical data, the decision-making model can be constructed and cultivated gradually in the parallel-improving processes if the initial structure of the public sentiment system is describable. moreover, the effects of various response strategies can be simulated and optimized by adjusting the key control variables of the cultivated model. it would be helpful for the government to enact better policies or decisions in future public sentiment events. the parallel interactions between the real scenarios and simulation models will be truly achieved and the decision effectiveness of the perdfps framework will be completely verified in the practical evolution and response processes of the public sentiments. however, due to the limited conditions (most original events such as nuclear-related large-scale project occur rarely), it is impossible to develop a simulation experiment according to a real-time scenario of a nuclear public sentiment in this paper. this research aims to propose the basic parallel decision-making framework to help governments guide and control public sentiment. thus, the empirical research component of this paper uses the historic case of the jiangmen anti-nuclear mass incident to demonstrate and discuss the principle and effectiveness of the perdfps framework. meanwhile, in order to carry out concise empirical research, details are not completely considered as the key variables of the multi-generational parallel simulation models. therefore, in the next step, more elements, features, and other details of public sentiment systems, such as opinion leaders and their impacts on the public sentiment, should be designed and simulated to make decision-making models more realistic and effective in practical response processes. furthermore, due to the similarity of the system structures, the novel approach can be used in the public sentiment events not only in the nuclear related field but also in many other fields, such as food safety and healthcare. once events break out, the public sentiment systems can be constructed rapidly using little initial data and improved on iteratively and dynamically with real-time data. using this framework, decision makers are able to describe, rehearse and make the proper decisions to guide or control the evolution trends of the public sentiment effectively. therefore, in future studies, the perdfps framework and the relevant sd models can be extended to some other social heat incidents with similar public sentiment system structures such as "vaccination scandals" and "food-safety scandals." the percentage of support posts drops to % and the percentage of the opposite posts rises to %. the curves shown in figure are consistent with the real nuclear project case that was eventually cancelled. therefore, the cultivated -generation model can simulate and match the evolutionary real scenarios very well. meanwhile, the simulation results also indicate that the dynamic reality hypotheses proposed in section . are tenable. references anton collaborative scenario modeling in emergency management through cross-impact public opinion, public policy, and democracy. handbook of politics: state and society in global perspective sham or shame: rethinking the china's milk powder scandal from a legal perspective emergency management typical case study report developing theory through simulation methods the mmr vaccine and autism constructing a model of effective information dissemination in a crisis tests for building confidence in system dynamics models wakefield's article linking mmr vaccine and autism was fraudulent public-opinion sentiment analysis for large hydro projects public's responses to an oil spill accident: a test of the attribution theory and situational crisis communication theory reading others' comments and public opinion poll results on social media: social judgment 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modeling the paper is supported by national natural science foundation of china (no. ); young the following etable is developed to clearly explain all the variables defined in our models. the variables of the sd model are classified into two categories including state and control variables. and the latter is classified into two subcategories including parallel cultivating control and decision-making control variables. each kind of variables has relevant functions in the model. ② the parallel control variables including "increase rate of posts" and "increase rate of news" are used to cultivate the n-general sd model. the key state variable "increment of posts" is directly decided by the real-time state variables "heat rate of public sentiment", "reaction time of netizens" and the parallel control variable "increase rate of posts". as shown in the rewritten section . , the variable "increase rate of posts" of the initial -genernal sd model (△t= hours) is defined temporarily as the formula ( ) by collecting and fitting the hours of data (from july to august , ) of similar public sentiment triggered by a similar event from ef.zhiweidata.com. and the formula ( ) is finally adjusted into the formula ( ) for the cultivated -general model (△t= hours) after parallel iterative cultivating.③ the decision-making control variables including "times of government response", "response time of government", "degree of information transparency" and "degree of popularity of nuclear knowledge" are controllable from the perspective of government. these variables can be tentatively set by the decision makers to represent different response strategies. and then the effects of different strategies can be tested by reviewing the simulation result with the cultivated n-general sd model and the optimal strategy can be obtained by adjusting the control variables. key: cord- -h qwxdtn authors: speckhard, anne title: prevention strategies and promoting psychological resilience to bioterrorism through communication date: journal: risk assessment and risk communication strategies in bioterrorism preparedness doi: . / - - - - _ sha: doc_id: cord_uid: h qwxdtn nan terrorism as a psychological weapon . dread not only in its immediate victims but in its wider witnessing audience. continually redefining itself in response to counterterrorism measures. the progress and portability of high-tech weaponry and the ability to communicate information quickly (over internet and telephone) has advanced the ability of small groups to create virtual command centers that can operate simultaneously and cover multiple world regions, and in doing so enact events of worldwide mass terrorism. moreover biological, chemical, and nuclear wmd -all previously weapons of states -are increasingly coming within the grasp of smaller groups of actors, and terrorists have made clear their desire to obtain and use such weapons. in the fight against terrorism societies must prepare themselves for all variants of terrorist attack and institute policies that prevent widespread dread and panic, and promote resilience in the larger civil society. the defense against terrorism is in reality four-tiered. firstly, it involves investing huge amounts of resources into hardening defenses in terms of securing buildings, airports, and civil military installations. this defense is important in securing key resources. yet it has been called a placebo response by some because in reality, the entire nation can be a target, total defense is illusory and any death will achieve media coverage -thereby radicalizing public opinion and demonstrating the ineffectiveness of the security forces (mackenzie, ) . secondly, tier of defense against terrorism includes infiltrating and destroying terrorist groups -by discovering and thwarting their plans ahead of time and raising questions about their methods and ideology within the groups (atran, ; post, ) . society that help shape interactions amongst the population including nongovernmental organizations (ngos), media, and those intermediary institutions (e.g., professional associations, religious groups, labor unions, citizen advocacy organizations, etc.), which build links between the population, provide information, analyses and political responses, and that give voice to various sectors of society and enrich public participation in democracies. of course civil society never acts in a vacuum or completely independent of government hence this paper focuses often upon how the two overlap, including through laws, policies, and instructions from one governing the other, as well as their interaction through the media, the public health service, hospitals, medical institutions, universities, think tanks, foundations, the legal system etc. civil society being broadly defined here as the formal and informal structures of the emerging threat of global terrorism is one that is dynamic and prevention strategies and promoting psychological resilience thirdly, for winning the "war on terrorism" societies can defend against terrorism by working to understand and diminish the reasons for popular support of terrorist groups -"debranding" the ideology and looking for and addressing the root causes. lastly and most importantly for this paper is the need for society to anticipate the responses of its own citizenry to terror attacks of all kinds and build resilience into it so that the psychological effects of terrorism are minimized. with the advent of groups like al-qaeda and the interest in creating terror attacks involving mass casualties and the use of self martyrdom missions many in the terrorism field have begun to speak of a "new terrorism". whether or not we are seeing a real break with old terrorists' methods and goals it is certainly true that today's terrorists function in a completely new global environment. with the erosion of strict borders between countries (particularly in the european union) and even world regions (since the fall of the soviet bloc), the advance and portability of high-tech weaponry including biological, chemical, and nuclear hazards, and the ease and speed of communication through the internet and telephones for purposes of recruitment, training, and planning terror attacks -terrorists now have a global playing field in which even small groups of individuals can motivate, plan, and enact mass terrorist events. moreover biological, chemical, and nuclear wmd -all previously weapons of states -not small groups, are increasingly coming within the grasp of smaller groups of actors, and terrorists have made clear their desire to obtain and use such weapons. use wmd including bioterrorism (schweitzer, ) . in addition to the much publicized words of osama bin laden in which he stated it was a sin not to make use of such weapons, shamil basayev, leader of the chechen terrorist groups has also avowed his willingness to attack his enemies with the same agents he believes his people have been attacked with including i am indebted to thelma gillen of the uk mod for this brilliant idea of attempting to "debrand" an ideology, much like one might attempt to debrand a trademark. martha crenshaw severely criticizes this conceptualization, which she credits to simon and benjamin. qaeda and its affiliates. chillingly they have avowed their willingness to the most well-known and perhaps most feared global terrorists are al-a. speckhard the new terrorism bioterrorism. abu musa'ab al suri one of the contemporary al-qaeda ideologues also advocated the use of wmd and criticized osama bin laden for not previously using them (paz, ) . likewise aum shinrikyo, a nonal-qaeda linked group, that was active in japan aimed to develop such weapons and actually shocked the world with the first mass chemical attack when they dispersed sarin gas in the tokyo subway, injuring hundreds. likewise two al-qaeda affiliate groups were thwarted in their attempts to use a ricin like substance in london and paris, and a failed biological attack was carried out in the united states when a small anti-government group attempted to contaminate a salad bar with salmonella. so clearly there is an avowed willingness by today's terror groups to resort to the use of bioterrorism. as far as intelligence analysts have been able to piece together when groups are searching for wmd they have thus far resorted to utilizing state sponsorship, trying to buy materials on the world black market, or resorted to illicit pilferage. when these activities have not worked, terror groups have adjusted their strategies by putting their resources into internal reuven paz writes, "abu mus'ab al-suri -a former leading trainer and scholar of al-qaeda, published two significant documents calling for a new organization of global jihad: "the islamist global resistance." one was a nine-page letter published in december , and the other was a huge book totaling , pages about the strategy of global jihad in his open letter to the state department, al-suri talks at length about the importance of using wmd against the united states as the only means to fight it from a point of equality. he even criticizes osama bin laden for not using wmd in the september attacks: "if i were consulted in the case of that operation i would advise the use of planes in flights from outside the u.s. that would carry wmd. hitting the u.s. with wmd was and is still very complicated. yet, it is possible after all, with allah's help, and more important than being possible-it is vital." al-suri states that "the muslim resistance elements [must] seriously consider this di cult yet vital direction." reuven paz writes, "abu mus'ab al-suri -a former leading trainer and scholar of al-qaeda, published two significant documents calling for a new organization of global jihad: "the islamist global resistance." one was a nine-page letter published in december , and the other was a huge book totaling , pages about the strategy of global jihad in his open letter to the state department, al-suri talks at length about the importance of using wmd against the united states as the only means to fight it from a point of equality. he even criticizes osama bin laden for not using wmd in the september attacks: "if i were consulted in the case of that operation i would advise the use of planes in flights from outside the u.s. that would carry wmd. hitting the u.s. with wmd was and is still very complicated. yet, it is possible after all, with allah's help, and more important than being possible-it is vital." al-suri states that "the muslim resistance elements [must] seriously consider this di cult yet vital direction." research and development (hoffman, ) . the aum shinrikyo group recruited and hired highly trained russian scientists and set up a highly specialized laboratory. likewise, information discovered in afghanistan makes clear that al-qaeda was engaged in a serious effort to develop a usable chemical and biological weapons capability. seized materials include films of tests carried out by al-qaeda operatives showing that the group achieved their goals enough to having reached the stage of limited testing of agents on live subjects. in the united kingdom and france terrorists groups have utilized crude recipes for biological agents such as ricin (hoffman, ; schweitzer, ) . in the thwarted uk case the group planned to smear small amounts of ricin on the door handles of random vehicles and thereby hoped to create mass hysteria from a few deaths rather than enact a mass killing. terrorists have certainly observed that it is possible to undermine public confidence and create mass anxiety responses by simple and even limited dispersal of a biological agent. the huge public reaction to the anthrax attacks in the united states (and indeed worldwide when one considers the providers) included: shutting down buildings; strangling the mail system; workers donning masks and rubber gloves while processing mail; quarantining areas, requiring employees to take precautionary strong antibiotics, public and private stock piling of medicines, and widespread anxiety about anthrax. these responses made clear that one could both undermine public confidence and create widespread mass distress with relatively few anthrax attacks (hoffman, ; speckhard, a) . likewise the fact that it took months and $ . million to decontaminate the hart senate office building and nearly $ million to do the same in the boca raton postal facility demonstrated the high costs of responding to the dispersal of minute quantities of a biological agent. terrorists learned from these events that rendering an important facility inoperable by virtue of biocontamination can have widespread and devastating social, psychological, and economic repercussions (hoffman, ) . the so-called chechen cell of north africans in paris were discovered preparing ricin for an attack on the russian embassy in paris and the london group had already prepared large quantities of ricin for an unspecified attack. attacks throughout europe which were taken seriously by emergency number of us embassies that were also affected, as well as the many hoax a. speckhard mass anxiety with bioterrorism . . undermining public confidence and creating ricin scandal it became apparent that the terrorists were more interested in their ability to undermine public confidence and to potentially create mass hysteria with a bioterrorism attack than in actually enacting a mass killing event. likewise the algerian cell's interest in ricin appears to be based in their inability to achieve sufficient media impact using strictly conventional attacks (hoffman, ) . terrorism relies upon making a strong media impact and bioterrorism has that potential. while the war in afghanistan made significant inroads in taking out the main al-qaeda leadership, its ideology still flourishes and in the absence of a strong centralized leadership the movement has continued unabated. recruitment, training and perhaps most important, a motivational ideology is daily transmitted globally via the internet bringing on board a disparate (but unified by a common ideology) group of disenfranchised, alienated, frustrated, and even traumatized individuals living in both conflict and non-conflict zones willing to sign on to support and even enact terrorism. in terms of western security, this is particularly troublesome in europe where radicalization among disenfranchised muslim communities appears to be a swelling phenomenon. the unresolved conflicts and human rights violations in chechnya, palestine, kashmir, and now in afghanistan, iraq, and guantanamo bay as well, all give fuel to the al-qaeda ideology, which argues that islam is under attack by corrupt western powers and militant jihad including attacks on civilians and self martyrdom operations is not only justified, but a duty. with the current ease of communication, lessening of technological barriers, convincing ideology, and a ready pool of recruits, it appears that the occurrence of a mass bioterrorism attack may simply be only a matter of time. given this state of affairs can we anticipate the responses of our citizenry to bioterrorism and if so how can government and civil society prepare and increase societal resilience to such attacks? to answer this question we must acknowledge that our citizenry is far more resilient than they are often given credit for. the common view espoused by government officials and policymakers is an expectation that people will panic in the face of a mass terrorist event and that chaos will ensue. experience with terrorism, however, does not bear that out (wessely, ) , although most of our research is based on conventional versus bioterrorism. in nearly all civil disasters that have been studied by researchers, holding aside the moments of escaping an imminent danger such as a fire, earthquake, hurricane, or bomb -researchers repeatedly have found volunteer their resources, and risk their lives to help each other. society actually becomes more civil during times of disaster as social cohesion increases rather than decreases under threat: in the short-term attachment behaviors generally increase, social cohesion increases, and the heroic is often called forth in ordinary people (speckhard, a) . we have numerous studies that bear this out. / is a good example. after-the-event interviews of persons involved revealed that people did not take a "me first attitude", disabled and injured persons were not run over by panicking hordes or left behind, abandoned (furedi, ) . on the contrary the / evacuation was self-generated, orderly, and without panic, and those who were hurt or disabled were carefully and calmly assisted and taken to emergency services (glass and schoch-spana, ) . the explosion of the chernobyl nuclear power plant gives us another window in which to view the responses of citizenry in times of mass disaster. when the reactor was about to explode the plant operators did not abandon their posts and flee like cowards to protect themselves. instead they heroically fought to the last moments to shut down the reactor, to contain the damage -many giving their lives to do so. the madrid train bombings give us another example. witnesses state that after the first explosions passengers helped one another and calmly began to exit the scene. only when repeated blasts occurred did some begin to run. even then, when it became clear that emergency vehicles were having a hard time getting to the platform, taxi drivers waiting for customers did not flee in fear, but volunteered themselves as makeshift ambulances and ferried wounded persons to emergency care (speckhard, b) . the same happened in the moscow subway bombingspeople walked calmly for long distances in the darkness, helping the wounded to evacuate. when terrorists bombed a crowded rock concert outside of moscow -the band agreed to bravely play on, the audience followed instructions not to stampede in fear and the wounded were carefully attended to. in the moscow theater where hostages were held by chechen suicide terrorists the hostages also remained calm for the most part during their ordeal and helped one another (speckhard, a; speckhard et al., ; speckhard et al., a, b) . even in the beslan hostage taking crisis where hundreds of children were held hostage, water and toilet privileges were withheld, and shooting occurred in front of the hostages -there were only limited outbreaks in times of threat. total strangers help each other, open their homes, that citizenry has become quite attachment and community-oriented extremely heroic behaviors were displayed (speckhard, c) . the london public transportation bombings also met with relative calm, with londoners quickly resuming confidence in the use of public transportation (wessely, ) . even under sustained and intense terrorist threat such as what has occurred in israel during the second intifada (uprising) we witness that the vast majority of the population there has habituated to the threat, certainly making adjustments in daily living to avoid as much as possible their potential for being killed by terrorists -but yet carrying on with life all the same. while the psychological costs of living under a sustained and intense terror threat are still not well understood (likely causing hyperarousal states in many, bodily distress, etc.) we do also see that most of the israeli population have on their own found ways to be resilient in the face of it. terrorists aim at creating widespread horror, dread, and fear, and to divide society. however, it is safe to say that despite this when under threat one can see in civil society, at least in the short term that attachment behaviors generally increase, social cohesion increases, and the heroic is often called forth in ordinary people. while this is true of terrorism that involves bombs and destruction we know less about societal responses to bioterrorism and we may find that the lessons learned from one type of terrorism may not transfer as well to another. we still have not studied well the potential effects of a mass bioterrorism event in which the dread caused by the spread of an invisible, contagious, and potentially lethal pathogen may be horrendous and in fact lead to less resilient responses. in this case the spread of horror from the terrorist attack will be amplified not only by the mass media but also by the many vectors of contagious contact all of which are difficult for ordinary individuals, much less medical professionals to understand and cope with. likewise trying to contain a potentially lethal contagious disease, that has a presymptomatic incubating period, and that can be spread through contact with others is extremely challenging and requires societal cohesion and compliance with rapidly responding and well-informed government authorities. in this case quarantines and the means of enforcing them, panic-driven hoarding of medicines and overwhelming the medical care services with "worried well" and psychosomatic individuals are issues that we know from other disasters might well occur and, which might severely impact societal resilience. thus we cannot say in all cases we expect society to be resilient. when it comes to poisons, invisible toxins, and fear inducing contagious illness, we have to look at other incidents to draw lessons. of hysteria. for the most part the adults present calmed the children and some when a threat is invisible and difficult to comprehend, some individuals may be expected to respond with fear, aggression, hysteria, and even psychosomatic symptoms if the fear of a potential toxic exposure becomes overwhelming. such responses have been witnessed in many events and are well documented in the literature. after the goiania radioactive incident (brandao mello et al., ) , following chernobyl (bromet et al., ; green et al., ; havenaar et al., ; speckhard, b) , and after the sarin attacks in the tokyo subway, medical systems were briefly overwhelmed by thousands of individuals who feared that they had the symptoms of poisoning, many who became psychosomatically ill. the explosion of the chernobyl nuclear power plant was perhaps the most serious "invisible" threat to date. likewise the bhopal and goiania and other similar incidents give us additional information making clear that there are unique psychological and fear responses to "invisible" toxins, poisons, and contaminants, as well as the widespread dread of pervasive and random threat that accompanies conventional terrorism. not only is disease contagious but psychosocial phenomena can also spread as infectiously through populations as biological agents, sometimes wreaking as much havoc with health as the disease agents themselves. the processes whereby emotions, attitudes, beliefs, and behavior are spread, transmitted, and even leap between populations, similar to other contagious outbreaks like measles, chicken pox, or even the common cold is referred to as psychological or psychosocial contagion. psychosocial contagion moves from person to person, often times requiring only a single exposure. categories of contagion important for understanding potential societal responses to bioterrorism include emotional, behavioral and aggression contagions. mood, fear, and anxiety states can be transmitted quickly through a population as humans tend to synchronize their facial expressions, voices, and postures with those in their immediate environment taking on fear and distress states when they witness these in those around them (behnke et al., ; hatfield et al., ; hsee et al., ; mcdougall, ) . this is particularly true of children. this synchronization can occur in response to viewing live footage in the mass media and may be one of the modern day mechanisms for rapid transmission of emotional contagions. behavioral contagions can also occur. for instance individuals exposed to violations of rules often increase their likelihood to engage in a similar or fear of the unseen and unknown -poisons, toxins, identical behaviors (i.e., speeding, delinquency, criminality, teenage smoking, youth sex, substance abuse) (connolly, ; ennett et al., ; jones, ; jones and jones, ; rowe et al., ) . these two contagionsemotional and behavioral likely explain the psychology behind the hysterical buy out of duct tape in the united states when word got out that the homeland defense report had made mention of duct tape as a useful means of protecting oneself from chemical and biological attacks. similarly it can explain in part why many americans were massively noncompliant and failed to heed public health officials instructions not to stockpile cipro (flaxocin) the antibiotic used to treat anthrax, instead of following a general panic among many to buy out unnecessary antibiotics and perhaps by doing so deprive those truly in need of them. if the anthrax attacks had been widespread, this may have caused significant hardship for some. as of this writing, we see the dread and dismay caused as avian flu makes its way westward, with many citizens overly worried and rejecting poultry products and others noncompliant due to economic concerns of losing livelihoods, with the long-term health and economic consequences still unknown. certainly governments must plan ahead for how they would handle issues of quarantine if it were needed and work beforehand with the public to get their participation and acceptance for plans, as well as with the media, police, military, or national guard units that would be responsible for reporting on and enforcing quarantine so that as much as possible contentious issues are dealt with and anticipated beforehand. even rehearsing how a decontamination unit would function in a mass terror setting is important for small but crucial issues like deciding does everyone who goes through the unit have to strip naked and if so can provisions be made for segregating the sexes -a difficult issue for those for whom modesty is a key value; how do decontamination units handle the need to give up contaminated items including car keys -raising the issue of how does one get home; or how to handle the surrender of contaminated mobile phones -creating stresses and tensions for family members who can no longer check on and reassure their loved ones. small but crucial issues like these if anticipated and thought through beforehand, with useful remedies built into the response scenarios can be arranged for the least stressful responses. new models of readiness are necessary to counter this threat especially when it pertains to biological terrorism because biological contamination raises unique and difficult issues, differing dramatically from other types of terrorism. as psychological contagion is a very real response to the potential of toxic exposure, medical systems should prepare ahead for massive onslaughts of the "worried well". the severe acute respiratory syndrome (sars) virus crossing from asia to canada in a very short time -shutting down an entire city; the cyrptosporidium epidemic of in the state of wisconsin; and the current epidemic with avian flu makes clear that huge number of people can be affected when a bio-threat spreads quickly through a community and that these threats raise difficult psychological and medical issues (glaser, ) . governments and media must work together preparing ahead of time on how to communicate calmly in such crises in a manner that will offer useful preventative measures, minimize the potential negative effects of psychosocial contagions (including citizenry becoming noncompliant and aggressive), prevent mass sociogenic illness from occurring, and prevent overwhelming of the medical systems by those whose emotional state has put them in need of medical care. in the case of bioterrorism as we shall see this is no easy task. in its extreme form psychosocial contagion can spawn mass hysterical contagions or mass sociogenic illness -that is, the rapid spread of illness signs and symptoms, which has no physical basis for the symptoms and no known exposure to a pathogen (bartholomew and wessely, ; cohen et al., ; kerckhoff, ; marsden, ) . hysterical contagions involve the spread by contact, including mass media exposure, of reported symptoms and experiences usually associated with clinical hysteria (hallucinations, nausea, vomiting, fainting, etc.) in the absence of exposure to a pathogen. such illnesses often begin with exposure of a limited group to a biological contagion or chemical toxin with the others around these persons or learning of them responding hysterically with some form of nervous excitation, including a significant loss or alteration of function, and physical symptoms with no basis in physical etiology. these types of illness often affect members of a cohesive group although they can leap across groups when common links are made in reality or imagination. such links are often made through the mass media in which one quite limited group of individuals is actually exposed to a biological or chemical toxin and has real symptoms but other groups fear that they too have been exposed. study of these types of contagions has found that exposure to the verbal reporting of symptoms rather than exposure to the symptoms themselves was enough to pass it on to others (colligan and murphy, ) , which makes it clear that responsible and nonhysterical news reporting is very necessary to contain such contagions. often there is a sensitizing issue that makes populations vulnerable to psychogenic illness. in belgium in , a mass sociogenic illness occurred in response to tainted coca cola that gave off harmless fumes, but caused psychogenic symptoms in schoolchildren mass sociogenic illness and members of the general public. this may have occurred because the belgian public had been sensitized by serious food scares during the previous year involving dioxin contamination in the food (nemery et al., ) . research has also shown that when one groups feels under attack by an enemy it is much easier for the symptoms to spread as the "victim" group finds it easy to believe that they have been poisoned by their enemies (bartholomew and wessely, ) . this occurred in palestine (modan et al., ) , kosovo (hay and foran, ) , and recently in chechnya. recent evidence indicates people do not even have to be present at a terrorist event to experience posttraumatic symptoms (speckhard, a, b; speckhard and mufel, (speckhard, a, b; speckhard and mufel, ) . in these studies, media and, particularly television exposure, was an important predictor of stress or traumatic symptoms in the face of terrorism second to geographic distance from the attacks. we must recognize that graphic images have the potential to be traumatic in themselves in terms of their potential to create a "witnessing" experience of trauma and their constant replay can also become traumatic as a potential victim also proved to cause stress symptoms (dixon et al., ), something that can also occur via televised images. terrorists' goals are to spread horror in behalf of their political cause and they reach their goal of maximum psychological impact through their manipulation of the mass media. the media, which is in a sense symbiotic with all the horrors of the world, generally responds within minutes of any terror attack and coverage begins immediately. in the case of a mass terror attack, the "talking heads" follow shortly thereafter. in most cases of mass terrorism it will be through these channels that the interventions to the resiliency of civil society -by what they communicate or fail to communicate. journalist robert frank points out that in a disaster situation people often "only recall from the peak moment, in the peak intensity, and far less attention is paid to the more accurate picture that emerges over time." this then according to frank, "creates a predisposition to think a certain way before the facts are fully presented and afterward then only to listen and retain those that confirm what was previously believed." unfortunately journalists are under pressure to get stories quickly and report the news with insufficient information. frank goes on to state, "it is however, very, very seductive to news-workers to appear knowledgeable when you are not" (speckhard, b) . certainly the emotional and behavioral response of citizenry to an event of bioterrorism will depend in part on how well and calmly government communicates the events to citizens and directs them in useful activities rather than leaving an information vacuum for the media experts or "talking heads" to fill with emotionally fearful information. if governments wish to avoid such consequences and compete with the unbridled freedom of mass media to form public opinions they must be prepared and have their own "sound bites" and "talking heads" prepared well ahead of a disaster, otherwise the mass media will fill the vacuum. while the practice of journalists presenting incomplete stories with only half fashioned facts is unlikely to disappear, government and the public health systems have a responsibility to prepare ahead of time and be ready to provide psychological triage -both through the media and in person for the worried well and psychosomatic individuals who will likely overwhelm the medical facilities. in the short term government and experts credibility is crucial. once that is lost it is very difficult to calm arousal states in individuals who will not believe competing information from that they already took on board. terrorists thrive on creating a mental environment in which citizens live in fear and dread of the next attack. civil society can do a lot to fight this type of psychological tactic. one of the most important ways is for those in charge of information to be well prepared and to speak in a reassuring manner about what is both known and unknown, giving essential information but not creating a sense of constant danger. this is a difficult but necessary balance to strike. in providing psychological reassurance over the media, government needs to think ahead of time to taking advantage of the new technologies as well -particularly the internet. in today's world we must recognize that many people will instantly log onto the internet in search of information and that rumors will abound. public health officials should have already prepared and be ready to launch (or have already launched) reliable and useful information via the internet and through all other channels of mass media to reassure the public and instruct them for the most protective responses. likewise government can make use of mobile phones, computers, and hand held devices that can receive transmissions with messages specifically aimed at them by virtue of where they are presently located. in the case of a contagious outbreak it is possible to transmit information regarding advisories of where not to travel and information about which hospitals and clinics in the area are free versus overwhelmed and general health care advice for that regional area (hopmeier, ) . in this case credibility is crucial. government must be very careful from the beginning to not lose the trust of the public in announcing what is known and still unknown, and to address psychosomatic responses in a meaningful way that differentiates them from the actual illness in question. while state control of media is an anathema to those who hold dear the rights of free press and freedoms of speech, the media can take actions collectively to self censure sensationalist reporting that continues to ratchet up fears. government spokespersons can put fears in perspective reminding people not to generalize from one event to all potential possibilities. for instance, following the february moscow subway suicide bombing that killed less than persons, moscow's carnegie center dmitri trenin stated, "every time i go down into the underground i wonder if i will finish my journey. now nine million people feeling they are playing russian roulette" (ostrovsky, ) . while this was a statement of his feelings, it reflects the sense of psychological contagion that can occur when nine million people fear an event that affected only a very small proportion of their total. statistically the dreaded terror event is much more unlikely to happen to them than many other ordinary horrors that they forget to fear. the same occurred with the sniper in the washington area in the fall of , with fear of a deadly but highly unlikely threat nearly paralyzing a huge metropolitan area. terrorists win when they can create a sense of dread of a pervasive and random threat -one that can strike anyone, anywhere at anytime. invisible threats -as involved in bioterrorism have the most likelihood of achieving this goal. the chernobyl disaster is probably the most well-known example of an abysmal failure by government to communicate and protect its citizenry and the effects of this failure are still felt today. twenty years later the prevention strategies and promoting psychological resilience population has still not recovered from fearing what their government failed to protect them from and many individuals disregard competing causes for illnesses such as alcoholism, pollution, stress, poor nutrition, etc., with nearly every birth defect, many serious illnesses -especially cancer, and even symptoms of minor distress in the region still suspected and blamed on chernobyl (speckhard, b, b) . most of the potential bioterrorist threats are clear -although the uncertainty lies as to where, when and how. thus it is possible for government to think ahead to what the population needs to know to respond calmly and with insight. indeed it may be wise to be already letting people know that smallpox vaccinations work even after exposure, that anthrax can be lethal upon direct exposure but is not spread infectiously. these bits of information can lay a foundation for calm responses, should the dreaded event occur, and create confidence that one could survive. in the wake of an actual mass terror event it is wise if the government has prepared ahead of time on who will speak and given some thought, not only to the facts that must be relayed, but also to that how the message is relayed is often as important as the message itself. the emotional tone of the message can create fear or calm. the israelis' success during the first gulf war ( ) when the population was being bombarded by scud missiles and directed to don gas masks (including putting them on small children), and retire to safe bio-sealed rooms in the event of a bioterror attack depended in large part on the preparation taken beforehand by government to disperse gas masks and to teach individuals how to take preventative measures to respond to bioterrorism. likewise israeli army spokesman, nahman shai, whose task it was to announce to the citizenry instructions to don gas masks and go to shelters, performed this duty in such a reassuring manner that he is still remembered fondly. in providing this anxiety inducing information at the moment of imminent attack his voice remained so calm and soothing as did his demeanor that he was later nicknamed the "the valium of the nation." likewise when the decision was made in israel to inoculate first responders (i.e., medical personnel, police officers, public health teams, and army soldiers) to a potential bioterrorism attack involving smallpox the fear surrounding doing so was addressed by the general director of the ministry of health, boaz lev, going on television and being the first to take the inoculation -showing by example that he had faith that it was worth the risks of doing so. this is a heroic example of how to communicate calmness in a crisis situation. often when a disaster or terrorist event occurs all the information is not known and the greatest psychological issue is about safety and what is next. frightened citizens want to know what to expect and they need things explained in a way they can understand. this is difficult for government officials who often do not have all the information they need to respond immediately and do not know if the threat is ongoing. in this case it is of paramount importance to tell the truth. short-term pacifications achieved with falsehood only create mistrust and blame later. it is far wiser to state clearly what is known and to admit what is still not known, making it clear that government is working hard to get the answers and nothing will be withheld to achieve maximum protection for the citizens. when explaining the risks of toxic and radioactive exposures it is important to speak in ways that put the dangers in perspective. far more people currently die in road accidents than in terror attacks, radiation exposure also occurs normally at the dentist, while flying, etc. people can understand and respond better when risks are explained in terms of comprehensible and clear comparisons. public health systems in the united states at least, have been losing funding in recent years. without the foresight of politicians, to have made preparatory investments of resources and personnel, they may not be ready to handle a huge public health epidemic, especially one caused by bioterrorism. the equipment and training alone needed to competently handle a bioterrorism attack (in terms of rapid identification and containment) must be anticipated ahead of time and the need for a central command and control, and clear lines of communication often across many agencies must also be determined well in advance. these are lessons we have learned from other terrorist and disaster events. in the japanese sarin attacks for instance the lack of emergency decontamination facilities and protective equipment resulted in a further secondary exposure of medical staff ( ambulance staff and staff in the main receiving hospital reported symptoms). the same occurred following chernobyl. in a training scenario involving multiple bombs and a potential chemical attack played out at the north atlantic treaty organization (nato) support facility in brussels in numerous personnel who were unaware it was not a real event, were called to the scene -ambulances, bomb detonation, decontamination units, etc. who interacted with guards already present at the unit. it became clear in analyzing the exercise that the various actors could not communicate well, as the handheld radios of the emergency workers did not coordinate with those in the nato support facility. it was also not clear who should take charge of the multiple units who converged upon the site and when the decontamination unit did not arrive (it went to another facility by accident) the entire "rescue" was delayed by hours. likewise the ambulatory contaminated "victims" ran to ambulances who refused to take them -because they were contaminatedand then ran through the neighborhood. had it been a real chemical or biological attack the contaminant or pathogen would likely have been widely spread. a similar public safety exercise known as topoff -for "top officials ," sponsored by the us department of homeland safety and state designed to test and improve us domestic response to terrorist incidents, carried out a fictional simultaneous attack against chicago using pneumonic plague and seattle using a radiological bomb in may . similar to the nato support facility exercise the real first responders were not alerted ahead of time that it was a fictional attack but once on the scene worked simulated crime scenes and treated volunteers pretending to be victims. nineteen federal agencies, as well as state and local emergency responders from illinois and washington, as well as from canada and the american red cross were involved. the exercise provided valuable lessons, including the realization that multiple control centers, numerous liaisons, and increasing numbers of response teams only complicated the emergency effort. likewise officials noted that it was essential to monitor and correct false media reports that might have inflamed the public to panic (miller, ) . certainly we know from such exercises that resources must be devoted not only to equipment but to careful planning of how to respond well technically and media-wise to terrorist threats, especially those involving radiological, biological, and chemical terrorism. most worrisome in a biological attack is the ability of government officials to detect unusual activity -as in new strange symptoms -and act early enough to contain the spread of lethal contagious disease within a geographical and population area in time to prevent mass casualties. this is extremely difficult to do as lethal contagious bioterrorist attacks will follow a trajectory beginning with exposure, to incubation, to latently symptomatic individuals to those who succumb and die. in the event of a biological terrorism attack public health officials working with government will be called upon to quickly identify if they are dealing with a bacteria, virus, or toxin and to identify it as quickly as possible and mount an efficient response. since biological infections have an incubation period an efficient response could mean cordoning off those who have been exposed and who are potentially dangerous transmitters (vectors) of the disease. this could mean quarantining symptomless individuals in an effort to make sure that those who have been exposed and could, but are not necessarily proven to, be incubating disease do not spread it to others. it is unclear in a liberal democracy if government officials would be able to establish quarantines that keep people in, much less out, of a zone that has been identified as "exposed" to a lethal biological agent. in these cases one might envision in the united states, the national guard or police troops called in to quarantine off a subpopulation of highly upset individuals who have families, cares, and responsibilities outside of the zone being quarantined (pollack, ) . certainly, in such a scenario, we can expect extremely strong fear and anxiety states to be transmitted quickly through the population and much rule-breaking behavior. whether or not this would mount to the point of contagious aggression is unknown as it has never been well tested. we do know, however, that the contagion of aggressive behavior has been shown to operate in both local and dispersed collectives, particularly within transitory and unpredictable angry crowds (mobs) (bandura, ; lachman, ; reicher, ) and we know from the recent riots in the islamic world that such aggressive contagions can easily be mediated and whipped up by the mass media. it is unlikely that democratic governments would ever desire or strive to shut down media reporting of a bioterrorist attack, yet we can learn from other societies that have taken this tact. after the initial stages of the beslan hostage-taking siege, ossetian authorities shut down broadcasts from the local televisions stations in an effort to defuse some of the local tension of televised broadcasting of the event. likewise psychiatric consultants brought in to help with the siege realized that mothers sitting at home with nothing more to do than agitate and shame their husbands for not going to rescue their children caught in the school building had to be addressed. they organized meaningful tasks for the mothers and opened a briefing center where every three hours or so they gave reports to the townspeople outlining everything they knew about the siege, potential negotiations, the state of the children inside, and so on. while it is unlikely that western countries would follow suit in shutting down television broadcasting, and even in beslan, cable networks, internet, and radio continued to broadcast in the area, it is useful to think ahead on how to work with the media and how to give citizens useful tasks to help them be empowered to be heroic in a crisis versus feeling helpless and frustrated with a sense of powerless inactivity. government decisions on when and what to tell, regarding attempts to contain the threat using quarantine strategies, whether or not to take action in a bioterrorism attack during the period of incubation when there are still no casualties, how to educate now and during the crisis, decisions about putting resources into the public health system, making sure medicines and vaccinations are available and dispersed fairly -are all public policy issues that should be ethically addressed well ahead of time. active public participation in such plans creates a societal investment in carrying them out. if this work has not been done ahead of time it may result in less complaint, less cohesive, and less resilient responses to terrorist threats. while civilians are more resilient that given credit for, a proportion of individuals will predictably suffer from symptoms of acute and posttraumatic stress when exposed to violence and death in a terrorist attack. while many of these responses are short-lived and resolve themselves through normal coping channels, some do not. the nature of a bioterrorism event, however, is less likely to result in acute posttraumatic stress states (unless there are massive numbers of deaths) than one might expect when the attack involves an explosion or other act of mass violence because the traumatic stressor is information versus a witnessed trauma. this is the difficulty inherent in dealing with "invisible" stressors, such as toxins, pathogens, and contaminants -they create fear, horror, and dread but there is often no clearly defined event to address, but instead an amorphous and undefined emotional horror. acute stress responses to a bioterrorism attack are much more likely to include psychosocial and behavioral contagions including hysteria, somatization, mass sociogenic illness outbreaks, and hysterical and possibly even aggressive demands for medical care, vaccines, and medicines than the acute posttraumatic responses often seen in response to an explosion or an act of violence. in all cases reassuring information and calm responses are the most helpful. there is a strong body of literature that demonstrates that intrusive psychological debriefing applied in a coercive manner in the immediate aftermath of traumas, is neither necessary or helpful, and sometimes even harmful, as most acute and posttraumatic symptoms to terror attacks decline overtime when normal coping channels are utilized (national institute of mental health, ). however, this is not to say that the "worried well" or psychosomatic individuals who appear asking for help should not receive psychological triage. in most cases traumatized individuals and those in high arousal states will respond well to having their posttraumatic and acute stress symptoms normalized -learning that it is a normal reaction to a traumatic event to feel fear, to even be dissociative if the fear is overwhelming, to experience intrusive thoughts and bodily arousal afterwards, and to engage avoidance strategies to little avail. learning that these are normal responses to trauma often helps individuals to move beyond them more easily versus get caught up in additional fears and shame over why they are not feeling or acting normal, as well as to diminish the avoidance responses that often occur in those who suffer from posttraumatic reexperiencing (speckhard, a; speckhard et al., a, b) . likewise those who have "caught" contagious psychological states and somatic symptoms are often also well served to receive medical care assuring them they are not a victim of the biological contaminate, as well as reassuring them that it is normal for some individuals to "catch" fear states and for these to evidence themselves in the body. in this way the individual is not shamed by having somatized their stress, something most individuals find distressing in itself, but also receives a logical explanation for what is happening in their body -an explanation that if judged as credible (and this is crucial) allows them to calm the bodily arousal that is supporting the negative symptoms. when a sense of humor and normality is introduced, the somatizing response often lessens. however if the person's real concerns are not taken seriously, and are ridiculed, he is accused of making up symptoms, ignored or told to go home as nothing is wrong, symptoms can often worsen. fear states can increase causing further somatization, shame can lead to strong avoidance, and isolation responses or the fear can drive aggressive responses. thus a balance must be struck between kindly understanding gentle humor to help somatizing individuals to understand how psychological contagions pass between persons, and firm reassurance that they are indeed not infected by the biocontaminate. of course those who are most distressed and less responsive to short-term triage should be identified and put in contact with helping professionals for longer term care with particular emphasis on those who are seriously dissociative and children with strongly embedded somatic symptoms. the best ways to ensure that acute stress responses to a mass terrorist event are minimized is to move survivors as quickly as possible to safety and reunite family members. this can be complicated in the event of a bioterror attack as it can be unclear for sometime if it is safe for persons to be reunited and it takes some time to establish when the critical period of attack (and ensuing contagion) is over. communications in all terror attack scenarios should be calm and clear and everything supportive that can be done to lower physiological states of arousal should be done. hyperarousal states. hence behaviors that are attachment oriented -calling family, seeking out contact with others, etc. should be encouraged in the aftermath of a terror event. the internet may also prove to be a very useful tool in this regard. following / , numerous researchers made use of the internet as a research tool and surprisingly found it also functioned as a therapeutic tool -that discussing the issues for many functioned as a social buffer. in terms of posttraumatic sequelae to bioterrorism there are unique longterm variants that must be taken into account. those exposed to poisons, invisible toxins, radiation, and so forth often worry less about their "traumatic" exposure, since that was often a non-event for them at the time it occurred, but only took on importance retrospectively when they learned since horrifying information is usually the central aspect of contamination stressor and there is often an absence of sensory details in the threat, the stressor may be said to be of a more cognitive, but equally horrifying form. indeed, victims of toxic disasters often experience horror in their imaginations of the future. for instance, the chernobyl victim who has a high radiation exposure as a child may continually see himself in the future as a cancer victim, or the pregnant woman exposed to a toxic contaminant may continually flash forward to the birth of deformed child, fearing to continue her pregnancy but loath to abort it. as a result survivors of toxic traumas develop a unique trauma-induced time distortion that is better understood as a "flash-forward" because it is the constant intrusion and reexperience in the mind of a horrifying, inescapable, and life-threatening event that the survivor expects to happen in the future as a result of having been exposed to a contaminant in the past. these flash-forwards are made up of repetitive and intrusive thoughts and images (similar to flashbacks) and create acute emotional distress and bodily agitation similar to the hyperaroused state typically observed with flashbacks (speckhard, b (speckhard, , b . this was quite common among those traumatized by chernobyl. they did not evidence clear posttraumatic arousal states to the memories of actual exposure but instead displayed them in response to involuntary horrific thinking about the future, experiencing intrusive and distressing states about getting cancer, dying young, bearing deformed children, etc. (speckhard, b (speckhard, , b . horrifying information about their potential exposure to a deadly contaminant or toxin, which now threatens to poison their entire future. pregnancy is known to often be a time fraught with worry about the health of the future child. one researcher wrote that the highest death toll from the chernobyl disaster was not caused by direct exposure to radiation but abortions were provided for many women in the direct exposure area. the decision to abort is an extremely difficult one for many persons, particularly those with a wanted pregnancy and in instances following toxic exposure is often made in a relatively short time period with the potential for deep psychological distress afterward including impacted grief, guilt, and traumatic responses (speckhard and mufel, ; speckhard and rue, ) . given that we can anticipate many of the bioterrorist threats we may face, we should prepare ahead of time, reassuring and giving accurate information concerning the potential desire to abort what may be a perfectly healthy pregnancy, so that unnecessary abortions of wanted pregnancies do not take place. researchers of toxic disasters often find that mothers are often more worried about their children's symptoms than children themselves (bromet et al., ) . finding a way to reach out to mothers and reassure them while giving them useful strategies for finding mastery for combating their fears, versus feeling powerless to protect their children, can also be very helpful. likewise we must address long-term fears of mothers-to-be when contamination of any type has occurred. girls and young women yet to bear children are often stigmatized after toxic exposures. not only do they often fear having deformed children, even many years afterwards, but they also often become the victims of the stigmatizing fears of others, which can diminish their ability to find suitable marriage partners. following chernobyl, the birthrate in contaminated areas as opposed to noncontaminated areas fell dramatically from to ; an effect which was attributed, as most likely, due to maternal anxiety about birth defects (world health organization, ) and many young men and women who had been exposed found they were shunned as marriage partners. worries of being exposed to a toxin, contaminant, or pathogen especially in girls and young women and in regard to childbirth nearly always is an issue that must be addressed even long after the fact of exposure. health-care workers are another vulnerable population. they form the front line in an often terrifying scenario caring for individuals who may in europe not directly exposed (knudsen, indeed when the israelis decided to inoculate their first responders against smallpox the only fatality was a family member of a doctor who ended up infecting his immune-suppressed wife. the horror of potentially contaminating one's own children was voiced by health-care workers after the sars outbreak, as well as by those caring for contaminated individuals directly after the chernobyl explosion, with workers saying that was the hardest part of the ordeal for them. we must take into account these very difficult situations our health-care workers will face, and consider ahead of time emergency protocols for childcare for those who take the very front lines in a serious bioterrorism threat, so that they can devote their complete energies to medical care and worry less about spreading lethal and not well understood disease at home. we must also find ways to support and honor heroic health-care workers like those who died taking care of sars patients, as well as those others who volunteered to help with the sars outbreak, knowing full well that to do so might be risking their lives. in the face of a massive bioterrorism threat we should give some thought to preparing psychological triage workers -mental health workers who have been trained ahead of time to sort through psychosomatic symptoms versus those needing immediate quarantine and treatment. given the thousands of psychosomatic and worried individuals who have overtaken the health-care systems in other similar situations, mental health-care workers can ease the burden on health-care workers and send the worried and hysterical patients home with some calming reassurance, sorting through those who also should be referred for additional psychological assistance. terrorists achieve their goals when they manage to derail political processes and move democracies to compromise their cherished values. as many terrorist attacks have been aimed at and timed with elections it is wise for governments to plan ahead how to respond when a candidate is killed, when a terror attack occurs during an election and so forth, so that the processes of democracy do not become derailed. the chaos of a bioterror attack can easily disrupt an election if voters are afraid to congregate in public places. it is wise to have thought ahead of time what the strategy would be for delaying or recounting an election in such a scenario. a. speckhard likewise when terrorists exploit the freedoms of liberal democracies it is tempting to surrender civil liberties in order to stop them. while some liberties may have to be suspended to effectively fight terrorism, it is are willing and able to do by virtue of their training, but this can be especially difficult for health-care workers who are also parents, or who have elderly relatives at home, and who fear exposing their own families. centers, such as at guantanamo bay, abusing prisoners in abu ghraib, etc. simply discredits our integrity and plays into the terrorist promoting ideologies that support violent responses against our abuses. these things unleash the terrorists' justifications for "defending" themselves through the use of wmd including bioterrorism. we need to always occupy the higher ground and continually remind, especially those who support the new "religiously" oriented terrorists, that islam in particular does not condone killing by poison and that many scholars of the koran takes a firm stance against the use of such weapons. but such statements fall on deaf ears if we ourselves are guilty of similar violations of morality and ethics. individuals need to feel that their world is somewhat predictable and that they have some mastery in it. bioterrorism involves an invisible threat that can create the opposite feelings: fear, horror, and dread as the actual contaminate spreads by biological contagion, and fear states including psychosomatic responses spread via psychosocial contagion. to defend against bioterrorism government initiatives should have been well thought out ahead of time, include preparatory education and a participatory process of the citizenry, preparation of experts who will be called on to help and work ahead of time with the media who will report the crisis, coupled with leaders whose words and actions inspire the belief that government is credible, calm, and acting in the public's behalf during a crisis. when leaders are honest, communicate calmly, and have prepared their societies ahead of time to respond well to terrorism, we can expect resilient responses. in times of threat and disaster populations often become attachment oriented and cohesive and heroic behaviors are frequently displayed. the best defense against terrorism, however, is to address first and foremost the root causes of it and end the motivations of terrorist leaders and sympathizers to kill and destroy. ultimately terrorism will not disappear until its root causes are addressed. social injustices that inflame terrorist rhetoric must be addressed as we all work for peace and security in this new uncertain world. until we achieve the ability to undermine terrorism by addressing its root causes, infiltrating terrorist groups and hardening our defenses we will have to continue to work toward strengthening civil society to be resilient to face this continuing threat. in the prevention strategies and promoting psychological resilience absence of achieving this we must prepare our societies with well thought out plans to be as resilient as possible. important to recognize that going too far in this direction -detaining thousands of aliens following / , practicing "torture lite" in detention genesis of suicide terrorism aggression: a social learning analysis protean nature of mass sociogenic illness. from possessed nuns to chemical and biological terrorism fears contagion theory and the communication of public speaking state anxiety the psychological 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methods of mitigation assessments of the emotional states of others -conscious judgements versus emotional contagion behavioral contagion and official delinquency: epidemic course in adolescence preferred pathways of behavioural contagion the june bug: a study in hysterical contagion legally induced abortions in denmark after chernobyl psychological perspective for a theory of behavior during riots terrorism and the placebo response. jane's defense weekly memetics and social contagion: two sides of the same coin the group mind communicating at topoff : a keystone in terrorism response the arjenyattah epidemic. a mass phenomenon: spread and triggering factors mental health and mass violence: evidence-based early psychological intervention for victims/survivors of mass violence: nih publication no the coca-cola incident in belgium russian authorities blame chechens as moscow subway train blast kills . financial times global jihad and wmd: between martyrdom and mass destruction risk assessment and risk communication strategies in bioterrorism preparedness social and psychological factors in the genesis of terrorism paul's riot: an explanation of the limits of crowd action in terms of a social identity model an epidemic model of adolescent cigarette-smoking the age of non-conventional terrorism innoculating resilience to terrorism: acute and posttraumatic stress responses in us military, foreign & civilian services serving overseas after toxic turmoil: psychological and societal consequences of ecological disasters soldiers for god: a study of the suicide terrorists in the moscow hostage taking siege unpublished interviews from the madrid training bombings civil society's response to mass terrorism: building resilience psycho-social and physical outcomes of technological disaster: information as a traumatic stressor unpublished beslan interviews universal responses to abortion? attachment, trauma and grief responses in women following abortion complicated mourning: dynamics of impacted post abortion grief research note: observations of suicidal terrorists in action posttraumatic and acute stress responses in hostages held by suicidal terrorists in the takeover of a moscow theater stockholm effects and psychological responses to captivity in hostages held by suicidal terrorists report on the nato russia advisory panel on mitigating the social and psychological consequences of chemical, biological and radiological terrorism. paper presented at the nato headquarters council meeting victimhood and resilience world health organization. health effect of the chernobyl accident and special health care programmes key: cord- - xo fiop authors: criscuolo, e.; caputo, v.; diotti, r. a.; sautto, g. a.; kirchenbaum, g. a.; clementi, n. title: alternative methods of vaccine delivery: an overview of edible and intradermal vaccines date: - - journal: j immunol res doi: . / / sha: doc_id: cord_uid: xo fiop vaccines are recognized worldwide as one of the most important tools for combating infectious diseases. despite the tremendous value conferred by currently available vaccines toward public health, the implementation of additional vaccine platforms is also of key importance. in fact, currently available vaccines possess shortcomings, such as inefficient triggering of a cell-mediated immune response and the lack of protective mucosal immunity. in this regard, recent work has been focused on vaccine delivery systems, as an alternative to injectable vaccines, to increase antigen stability and improve overall immunogenicity. in particular, novel strategies based on edible or intradermal vaccine formulations have been demonstrated to trigger both a systemic and mucosal immune response. these novel vaccination delivery systems offer several advantages over the injectable preparations including self-administration, reduced cost, stability, and elimination of a cold chain. in this review, the latest findings and accomplishments regarding edible and intradermal vaccines are described in the context of the system used for immunogen expression, their molecular features and capacity to induce a protective systemic and mucosal response. one of the ten greatest public health achievements of the last century was preventative vaccination [ ] . vaccines have successfully reduced the spread of diseases and mitigated mortality associated with infectious agents such as diphtheria, tetanus, polio, measles, mumps, rubella, and hepatitis b [ ] . in spite of the many successes achieved by vaccines, novel technologies and administration routes remain one of the main focuses in the vaccinology field. although many licensed vaccines are administered by injection, in certain cases, this administration route suffers from limitations. in particular, injectable vaccines require trained personnel for the administration of the vaccine and may require multiple doses or inclusion of an adjuvant. moreover, injectable vaccines may require specialized storage and transport conditions. from an immunological point of view, injectable vaccines are capable of eliciting robust systemic humoral responses while conferring weaker t cell-mediated immunity and mucosal protection [ , ] . importantly, t cell effector activity and mucosal immunity both contribute to prevention and control of infection from pathogens targeting the mucosa [ ] . to improve on this limitation, alternative vaccine delivery methods coupled with novel formulations and production systems have recently been proposed. numerous studies have focused on vaccines delivered to the mucosal interface or intradermally, demonstrating rapid and wide biodistribution of the antigen and the capacity to induce both protective mucosal (mainly mediated by secretory iga [siga] ) and systemic cellular and humoral responses [ ] [ ] [ ] . in this review, we discuss current advances and advantages of edible systems based on plants, algae, yeast, insect cells, and lactic acid bacteria and of the intradermal immunization route. . . the mucosal delivery and the immune response. the efficacy of the mucosal administration route is largely based on the fact that mucous membranes constitute the largest immunologic organ in the body. moreover, this interface is endowed with well-organized lymphatic structures, termed mucosa-associated lymphoid tissue (malt), containing both the innate and adaptive (t and b cells) arms of the immune system [ ] . furthermore, antigen-specific siga plays a pivotal role in protecting mucosal surfaces from both microbe adhesion and toxin activities [ ] . thus, the development of novel vaccine delivery platforms implementing the elicitation of pathogen-or toxin-specific siga, as well as systemic igg, is pivotal to improve vaccine effectiveness [ ] . to date, the most well-studied vaccine delivery platforms capable of eliciting both mucosal and systemic immunities are edible or intradermal vaccine formulations ( figure ). oral vaccines stimulate the generation of immunity in gut-associated lymphoid tissue (galt), which includes lymph nodes, peyer's patches (in which lymphocytes are the major component:~ % are b cells, while~ % are t cells), and isolated lymphoid follicles in the gastrointestinal tract (git). an effective immunization using oral vaccines is achieved when sufficient quantities of antigen are transported across the mucosal barrier by m cells into peyer's patches and subsequently presented to t cells by antigenpresenting cells (apcs) [ ] . briefly, professional apcs display peptide fragments of the antigen in the context of the major histocompatibility complex (mhc) on their surface, which leads to activation of cd + t cells [ ] . subsequently, activated cd + t cells support germinal center development, including b cell affinity maturation and class switching to iga, through providing cd /cd ligand interactions and cytokine secretion [ ] [ ] [ ] . moreover, through the expression of specific chemokine homing receptors (e.g., cxcr or ccr ), antigen-experienced b cells migrate to distant effector regions where they differentiate into plasma cells capable of secreting dimeric or polymeric figure : alternative methods of vaccine delivery. development of rationally designed vaccines starts with the identification of the gene encoding for the protective antigenic protein(s). subsequently, the antigen(s) can be incorporated into different edible systems, as plants, algae, insects, or yeasts, or used for intradermal formulations to induce a mucosal protective response. following the administration of the edible vaccine and the subsequent passage of the antigen(s) through the m cell compartment delivering it to dendritic cells, the individual's immune system triggers a response leading also to specific iga production and secretion. similarly, patches with coated microprojections or biodegradable needles activate langerhans cells and dermal dendritic cells in the skin dermis. these cells capture and present the antigen(s) to t and b lymphocytes, triggering both a mucosal and a systemic immunity. iga molecules that are transported into the intestinal lumen as siga [ , ] . in the context of edible vaccines aimed at eliciting pathogen-specific responses, it will be necessary to overcome mucosal tolerance. briefly, mucosal tolerance is achieved against certain foreign antigens, such as those contained in our food, and serves to prevent unnecessary and potentially detrimental immune responses in the gut mucosa. due to this phenomenon, an erroneous mucosal vaccine formulation could induce a t reg -based tolerogenic response instead of th -mediated protective immunity [ ] . this potential shortcoming can be circumvented using several strategies, including incorporation of an appropriate adjuvant in the vaccine formulation or using sufficiently high doses of antigen to promote induction of effector rather than regulatory cells [ , ] . moreover, in the context of edible-based vaccine immunizations, it will also be important to consider the characteristics of the git, in which several factors, including proteolytic enzymes, acidic ph, bile salts, and limited permeability, may hinder the induction of a protective immune response [ ] . to this end, conjugation of the vaccine antigen with specific ligands that enhance their uptake by m cells represents a focus of ongoing studies aimed at improving immunogenicity [ ] . moreover, antigen bioencapsulation avoids degradation and conformational alterations [ ] . in the following sections, we review the various strategies underlying the development of edible vaccines. in particular, we focused on plant, algae, insect cells, whole yeast, and lactic acid bacteria-based vaccines and describe the advantages and limitations of individual systems. plants have been extensively used for developing novel biopharmaceutical-producing platforms in recent years, as they promote proper folding of exogenous proteins and are economically sustainable [ , ] . this is also known in the context of "molecular farming," in which biomolecules of commercial value are produced in genetically engineered plants. there are several ongoing clinical trials using purified antigens transiently produced in tobacco plants (nicotiana benthamiana) for injectable vaccine formulations. for example, medicago recently completed a phase ii clinical trial using a plant-derived, virus-like particle (vlp) quadrivalent influenza vaccine and announced a phase iii clinical study in the last year (clinical-trials.gov identifier: nct ) [ ] . owing to the fact that plants are edible, the notion that they could serve as a delivery vehicle, as well as biofactories, led to their use for oral vaccination in the early s [ ] . in recent years, additional studies have sought to overcome the limitations of conventional vaccines through development of edible formulations [ , ] . since the inception of the idea, it has been evident that using plants to produce vaccines would have several advantages. first, plant vaccines would likely have a low production cost and could be easily scaled-up, as has been demonstrated by the biopharmaceutical industry. molecular farming gained visibility thanks to the success of zmapp, the experimental drug against the ebola virus that was produced in nicotiana plants [ ] . however, unlike biomolecule production, edible vaccine formulations do not need processing or purification steps before administration, which serves to further lower productionassociated costs. indeed, another advantage of this strategy is that plant cells would provide antigen protection due to their rigid cell wall. this is also known as the bioencapsulation effect and could increase bioavailability of antigenic molecules to the galts through preserving structural integrity of vaccine components through the stomach to elicit both a mucosal and a systemic immune response. additional strategies for antigen protection can be achieved through targeting biomolecule expression inside chloroplasts or other storage organelles [ ] or in the protein bodies of seeds [ , ] . this technology also offers advantages in terms of storage and cold chain-free delivery due to the high stability of the expressed antigens bioencapsulated within the plant and seed tissues. moreover, plant materials can be stored at elevated temperatures for longer periods and grown where needed, making this strategy even more attractive for developing countries [ ] . finally, plant-based oral vaccines are characterized by improved safety relative to traditional recombinant vaccine platforms, especially since contamination from mammalian-specific pathogens can be eliminated [ ] . indeed, some studies using lyophilized leaves have shown their advantages over fresh materials such as long-term stability, higher antigen content, and lower microbial contamination. as an example, freeze-dried ctb-ex -expressing (ctb: cholera toxin b subunit; ex : exendin- ) leaves were shown to be stable for up to months at room temperature, and lettuces expressing soluble antigen (pa; protective antigen from bacillus anthracis) were successfully stored for up to months at room temperature without showing antigen degradation [ ] . the antigen content in lyophilized leaf materials was also -fold higher than fresh leaves. an additional benefit of lyophilization was its ability to remove microbial contamination. while lyophilized lettuce had no detectable microbes, fresh leaves contained up to approximately cfu/g microbes when plated on various growing media [ ] . to date, vaccine antigens have been transformed into many edible species including lettuce, tomato, potato, papaya, carrot, quinoa, and tobacco [ ] . their proper folding and enhanced expression have also been tested in animal models, proving the immunogenicity of antigens produced in these systems [ , ] . to obtain high quantities of the protein of interest, both nuclear and chloroplast genomes have been successfully engineered. however, the latter option is preferred owing to some advantages including high levels of transgene expression (up to % of total soluble proteins (tsp)) [ , ] , bioencapsulation effect, and regulatory concerns since transgene containment is assured by the fact that plastids are not spread via pollen in most plants. moreover, incorporation of vaccine antigens into the chloroplast genome would also enable the expression of multiple genes in a single operon, which would be very attractive for multivalent vaccine development. likewise, this approach may enable the production of vaccines conferring protection against multiple infectious agents and would serve to further reduce costs associated with vaccine production and administration [ ] . unfortunately, there are some disadvantages undermining their applications. first, plastids are not suitable for production of antigens that require glycosylation for proper folding or those antigens in which a protective immune response requires glycan recognition. however, nuclear transformation represents a valid option. secondly, antigen expression can be either transient or stable in plants, but the second is preferred in order to obtain a stable genetic resource. in fact, transgenic seeds represent a constant resource to grow the transgenic plants and to extract proteins. however, stable transformation is time-consuming [ ] . moreover, expression in stable transformed crop plants suffers from low yields, typically less than % of tsp [ ] . on the other hand, transient expression technology using either agrobacterium or viral vectors is robust, quick, and easy to manipulate [ ] . however, this expression is typically unstable [ ] . another important challenge of plant-based oral vaccines is the lack of a proper dosing strategy because low doses may not be able to induce a sufficient immune response and high doses, as previously described, may lead to immune tolerance. to this end, freeze-drying methods were implemented to stabilize plant biomass, concentrate the antigen, and achieve an accurate dosage by quantifying the antigen in terms of dry biomass weight, as mentioned above [ , ] . to date, there are some plant-based vaccines for the hepatitis b virus (hbv), rabies virus, norwalk virus, enterotoxigenic e. coli, and vibrio cholerae in phase clinical trials (table ) . many others are still in preclinical development, including vaccines targeting a variety of pathogens such as avian influenza viruses (hpai h n ) [ ] , helicobacter pylori [ ] , and coronaviruses [ ] . . . algae-based vaccines. green microalgae, such as chlamydomonas reinhardtii, represent another viable option for vaccine production. however, some disadvantages of plantderived vaccines, such as low expression levels and improper glycosylation of antigen proteins, have been described [ ] . thus far, only chloroplast transformation is possible [ ] , and only a single organelle is present, even if it occupies half of the cell volume [ ] . stable transformed lines of green algae are easy to obtain and can lead to increased yield of expressed antigens. in fact, unicellular green algae have all the positive characteristics of plant systems, plus unique advantages over terrestrial plants. biomass accumulation is extremely fast and can be used in its entirety. their growth neither has seasonal constraints nor relies on soil fertility. cross-contamination of nearby crops cannot occur, as algae can be cultured with enclosed bioreactors [ ] . furthermore, in regard to regulatory aspects, green algae, such as c. reinhardtii, are generally recognized as safe (gras) by the fda. finally, algae can be easily lyophilized and, when dried, can be stored at room temperature for up to months without losing antigenic efficacy [ ] . in fact, the algae cell wall assures the bioencapsulation effect, as it was proven to prevent antigen degradation by enzymes of the git [ ] . collectively, these characteristics indicate that algae would be an ideal host for vaccine transport without a cold chain supply. thus, as already described for plant-derived edible vaccines, the low cost and simpler logistic in terms of manufacturing, storage, delivery, and administration of the algae-based technology make it an ideal system in the context of resource-limited settings compared to conventional vaccine formulations. there are no algae-based vaccines currently in clinical trials; however, preclinical formulations against human papillomavirus (hpv), hbv, and foot-and-mouth disease virus (fmdv) are under development [ , , ] to overcome some technical problems, such as a low expression level from the nuclear genome and lack of glycosylation following chloroplast expression [ ] . . . insect cell-based vaccines. insect cell systems have been widely adopted because of their capacity to produce high levels of proteins and to perform cotranslational and posttranslational modifications, including glycosylation, phosphorylation, and protein processing. this expression platform allows for generation of stable transformed cell lines or transient expression driven by recombinant baculovirus infection. the baculovirus-insect cell expression system, referred to as bevs, is one of the most well-known and used systems for large-scale production of complex proteins and, most recently, for the development of subunit vaccines [ ] . to date, there are three commercially available vaccines produced in insect cells for different indications: cervarix (gsk) for cervical cancer, flublok (protein sciences, now owned by sanofi pasteur) for influenza, and provenge (dendreon) for prostate cancer [ ] . importantly, the baculovirus expression system is not limited only to cultured cells. insect larvae or pupae can be used for protein production. in the context of edible vaccines using insect larvae or pupae, silkworm bombyx mori larvae or pupae have been commercially used for the production of recombinant proteins and also as a feasible delivery system for the vaccine [ , ] . as mentioned above, the baculovirus-silkworm expression system is able to perform cotranslational and posttranslational modifications and also able to produce large amount and multiple proteins. moreover, since baculovirus is unable to replicate in vertebral animals, it can be considered a gras. furthermore, the presence of protease inhibitors and biocapsule-like fat in the silkworms may protect recombinant proteins from enzymatic digestion in the git [ , ] . several vaccine prototypes are currently under evaluation, and strong systemic immune protective responses support the use of silkworm as a mucosal vaccine vector, as shown, for example, by high immunogenicity in mice of the urease b subunit of helicobacter pylori produced in silkworm [ , ] . while the data collected so far support the possible use of baculovirus-silkworm vaccines as a promising edible vaccine platform, it is only approved for food ingestion in a few asian countries. . . whole-cell yeast-based vaccines. the industrial usage of yeasts cells for production of heterologous proteins has been well described [ , ] . additionally, the capability of this system to perform posttranslational modifications, the gras status, and the cellular wall that could protect the antigen across the git make engineered yeasts an attractive vaccine delivery system [ , ] . in addition, the major drawback of this system is hyperglycosylation of recombinant proteins, but it has been already addressed by generating defective n-glycosylation strains of yeasts [ , ] . whole-cell yeast-based vaccines have been studied for their ability to elicit an immune response. remarkably, some preclinical studies based on orally administrated saccharomyces cerevisiae and developed for different infectious agents, such as hpv and actinobacillus pleuropneumoniae, showed that this delivery system is able to induce a protective mucosal and a systemic immune response [ ] [ ] [ ] . moreover, the increased immunogenicity of this delivery system could be explained by the adjuvant activity of β-glucans on the yeast cell wall, which demonstrates immunomodulatory and adjuvant effects through binding of innate pathogen receptors on macrophages, dc, and neutrophils [ ] . currently, two clinical trials have been developed: gs- for hbv treatment and gi- for hepatitis c virus (hcv) treatment (table ). regarding the clinical trial for gs- , despite the positive results obtained from phase [ ] , the second phase, conducted in virally suppressed, noncirrhotic patients with chronic hbv infection did not show a clinical benefit. however, other safety and efficacy studies have been conducted on another group of patients (in particular, in treatment-naïve patients with chronic hbv) [ ] . regarding the clinical trial for gi- , phases i and ii reported promising results [ ] . in particular, in this trial, gi- was used also in combination with peg-ifn/ribavirin. however, data on efficacy have not been published yet. lactic acid bacteria (lab) are gram-positive, nonsporulating, and nonpathogenic bacteria that have been used for decades for the production and preservation of food as well as for therapeutic heterologous gene expression, like the production of different anti-human immunodeficiency virus (anti-hiv) antibodies (scfv-m , dab-m , and dab-m . ) by lactobacillus jensenii and the production and functional expression of the antilisterial bacteriocin enta in l. casei [ ] [ ] [ ] . given these and the ability of lab to elicit a specific immune response against recombinant foreign antigens, these bacteria have been considered potential candidates as mucosal vaccine vectors. this delivery system can confer protection against antigen degradation and, thanks to its uptake at the git level, can activate both innate and adaptive immune responses [ , ] . many lab, in particular, lactobacillus spp and bacillus subtilis, were used in preclinical studies against different infectious diseases. different results have been obtained from these studies, but an elicited immune response was observed in all of them. as an example, the production of high levels of specific iga and systemic igg after immunization with bacillus spores expressing toxin a peptide repeat was reported [ ] , while in another paper, l. lactis expressing hev antigen orf vaccine was tested and a specific th -based cellmediated immune response was revealed as well as the production of specific mucosal iga and serum igg [ ] . another study reported a th /th immune response elicited after the immunization with csenolase-expressing bacillus subtilis [ ] . another example is the oral administration of b. subtilis spores expressing urease b of helicobacter pylori that provide protection against helicobacter infection [ ] . an important feature of lab is their natural adjuvanticity and their immunomodulatory effects, although the molecular mechanism of these capabilities is not completely understood [ ] . moreover, other studies reported an effect on dendritic cell maturation and an induction of cytokine secretion [ , ] . despite the promising characteristics of recombinant lab as mucosal vaccine vectors and given the encouraging results from murine studies, some aspects need to be taken into consideration, namely, the fact that vaccine strains cannot be considered avirulent, even if it could be listed as gras, due to potential transfer of antibiotic selection markers among microbes [ , ] . furthermore, other factors are important for the development of lab-based vaccines. as an example, the necessity of a suitable delivery system since different administration routes produce different immune effects. additionally, the role of specific adjuvants and the correct localization (intracellularly or on the bacterial surface) of each expressed antigen need consideration [ ] . overall, additional studies and clinical trials are needed for the development of efficient vaccines based on lab. a different carrier system based on nonrecombinant lactococcus lactis bacteria was recently developed. this system, called gram-positive enhancer matrix (gem), is composed of the rigid peptidoglycan (pgn) cell wall of the bacterium resulting in a nonliving particle that preserves the shape and the size as the original bacterium [ ] . gems are used in two different ways: mixed with vaccine antigens as adjuvants or as antigen protein carriers, with the antigens bound to the surface of gems. regarding the use of gems as adjuvants, because of their nature, gems are safer adjuvants compared to others. moreover, they retain the inflammatory properties of live bacteria and enhanced specific mucosal and systemic immune responses of the influenza subunit vaccine [ ] [ ] [ ] . therefore, the use of gems was further examined in a study investigating the immune response elicited by intranasal delivery of the influenza subunit vaccine coadministrated with gem (flugem). in detail, an influenza-specific memory b cell response and the presence of long-lived antibodysecreting plasma cells were reported. additionally, this immune response was able to confer protection from influenza infections [ ] . these important results obtained in murine studies have led to a phase i clinical trial which confirmed the positive preclinical data. systemic hemagglutination inhibition (hai) titers and local siga responses were reported. further studies will assess if this immune response confers protection against the influenza virus [ ] . gems have also been used as antigen protein carriers. in particular, antigens are bound to gem through the presence of a pgn-binding tag (protan) in the antigen. several works used this vaccination strategy, and the data support the potential of gems as safe vaccine delivery vehicles and their ability to elicit systemic antibodies [ ] [ ] [ ] [ ] . moreover, gems are also able to present several antigens at the same time which could be helpful for the preparation of multivalent vaccines [ ] . furthermore, the delivery of an adjuvant (gems) and an antigen together has been correlated with enhanced vaccine immunogenicity [ ] . lastly, as opposed to a vaccine based on lab, the absence of recombinant dna avoids its dissemination into the environment. however, the inability of gems to colonize any compartment does not allow the reduction of the number of vaccine doses. these promising premises allowed the development of a vaccine against respiratory syncytial virus (rsv). in particular, an intranasal formulation based on the trimeric rsv fusion protein coupled with gems and named syngem was developed. also, in this case, positive results from studies in mice and rats have been obtained, and as for flugem, vaccination with syngem resulted in the induction of a robust systemic and mucosal immune response as well as a balanced cytokine profile. these data supported further study of this vaccine in phase i clinical trial, which is currently ongoing [ ] . in conclusion, gems represent an interesting vaccination strategy either as adjuvant or as antigen protein carriers, but as in the case of vaccine based on lab, further studies are needed. immune response. another vaccine delivery route capable of triggering both systemic and mucosal immunities is the intradermal route, in which the antigen is delivered through the skin using recently developed self-administrable devices. in particular, the application of microneedle technology overcomes the skin permeation barrier imposed by the stratum corneum and facilitates antigen delivery. the efficacy of this new microneedle-based immunization approach is due to the presence of several types of immune cells (such as dcs, t lymphocytes, nk cells, macrophages, and mast cells) in the epithelium [ , ] . in fact, the cells that are responsible for triggering the inflammation cascade in the skin are the langerhans cells (comprising - % of epithelial cells). langerhans cells are a specific dc subset that migrates into the lymph node following antigen capture and aids in the initiation of an adaptive immune response [ ] . these cells are also efficiently stimulated by pathogen-associated molecular patterns (pamps) using an array of germlineencoded pattern recognition receptors (prr), including toll-like receptors (tlr) and langerin (cd ) [ ] . importantly, skin resident mast cells are also key drivers of the innate immune response in the skin through the release of granules containing inflammatory mediators [ ] . . . intradermal vaccination. using conventional syringes for intramuscular or subcutaneous vaccinations, large volumes of vaccine solution can be injected (≥ ml). thus, the choice of the muscle or hypodermis as vaccination targets is mainly based on convenience [ ] . intradermal immunization for vaccine delivery is an upcoming strategy showing significant advantages over conventional vaccination routes. in particular, in the last few years, intradermal vaccination has gained momentum as an alternative and more effective vaccine delivery route, both from a scientific and a commercial point of view (table ) . intradermal vaccination designates the delivery of an antigen directly into the dermis with a syringe, a needle, a microneedle, or a pressure injector. the standard intradermal immunization technique was invented by the french physician charles mantoux in , while he was developing the tuberculin test. this technique allows the injection of - μl of vaccine solution. however, mantoux's technique requires skilled medical personnel to be performed [ ] . recent advancements have led to the development of techniques and instruments that can overcome the difficulties associated with intradermal administration [ ] . in fact, different devices have been developed over the years for intradermal vaccination. among them, solid microneedles, particle injectors, and self-administrable patches with coated microprojections or biodegradable needles have been described [ ] . as previously mentioned, intradermal vaccination can induce mucosal and systemic immunities. these immunological capabilities, coupled with its ease of access, make the intradermal route an attractive vaccination delivery target [ ] . intradermal vaccination has been demonstrated to be very safe. in fact, novel devices involve the use of needles with a smaller size than the usual ( μm and mm) and make it possible to bypass the corneous layer of epidermis by creating transient micropores in the cutaneous tissues. even if some studies have shown that intradermal vaccination can be associated with a higher incidence of local reactogenicity, including primarily mild pain, swelling, and redness, systemic side effects have not been reported. in fact, the intradermal route limits the transfer of vaccine components to the blood circulation (and the risk of septic shock) and the possible toxicity due to hepatic first-pass effect [ ] . typically, when present, local effects resolve quickly, as reported in a study comparing the safety and immunogenicity of a large number of intradermal versus intramuscular influenza vaccines [ ] . another important aspect is the possibility of improving the immunogenicity of various vaccines in immunocompromised hosts as well as during pregnancy via the intradermal route [ , ] . as an example, it has been reported that the hbv vaccine fails to yield seroconversion in - % of recipients. however, a significant improvement was observed following intradermal vaccination [ ] . additionally, it has been demonstrated that in patients on dialysis or in hiv-positive subjects, the intradermal route was more immunogenic than the standard intramuscular route [ ] . from a commercial point of view, intradermal vaccination has been primarily explored for its ability to elicit equivalent antibody responses at lower doses, a phenomenon typically described as "dose sparing" [ ] . in this regard, the advantage of a low dose is most evident in high-surge situations, such as during pandemic and seasonal influenza waves, in which large populations are at an increased risk and large amounts of new antigen preparations are quickly required each year [ ] [ ] [ ] . above all, dose sparing is also important in a large-scale setting and in reducing the production-associated costs, especially in developing countries, where the price of the vaccine limits its use and coverage. in this regard, the u.s. food and drug administration (fda) approved the trivalent inactivated intradermal influenza vaccine for use in adults - years of age for use during the - season, and a quadrivalent formulation was subsequently approved in . however, similar to intramuscular vaccines, the formulation of these approved intradermal vaccines is liquid and thus still dependent on the cold chain and administered through a syringe. for these reasons, novel dried solid microneedle devices, while eliciting comparable immunogenicity to intramuscular-administered vaccines, represent an innovative approach to facilitate self-administration and allow vaccine storage at room temperature [ ] . infectious diseases represent a global concern, and the most effective strategy to reduce them is vaccination. unfortunately, not every disease can currently be prevented through vaccines. however, many strategies have been developed against infectious agents, such as the generation of neutralizing antibodies, antibiotics, and antiviral drugs [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and innovative approaches are currently under development [ ] [ ] [ ] . many vaccines have been developed and approved against various pathogens, and countless studies have been conducted to improve their efficacy by testing new adjuvants and performing the rational identification of the antigen formulations and pathogen contaminations [ ] [ ] [ ] . promising results have been also achieved by changing the delivery strategy. in fact, most of the approved vaccines are administrated by injection with intrinsic limitations like those concerning the immunological aspect. injected vaccines are able to elicit a strong humoral immunity but a weak cellular response. in addition, this type of administration is strongly associated with a systemic immunity but with a lack of mucosal response, which is helpful to block the early stages of infection since most pathogens infect through the mucosal membranes. for these reasons, new vaccination strategies have been proposed. in particular, edible vaccines, triggering the galt, and intradermal approaches, involving langerhans cells, are able to elicit both a mucosal and a systemic immune response. the increased knowledge of these approaches has led to the progression of many preclinical studies and several promising clinical trials (tables , , and ). moreover, these vaccine strategies are considered safe and cost-effective as no extensive antigen processing is needed [ , ] and they are easy to administrate (table ). in fact, due to the opportunity of self-administration and ease of distribution compared to an injection-based approach, these two vaccination systems could improve the overall coverage. there remain a number of obstacles and drawbacks associated with each antigen delivery platform that still need to be addressed (table ). presently there are no fda-approved compounds for edible vaccination, but new emerging technologies like nanoparticles (nps) could help to control antigen bioavailability to avoid mucosal tolerance. nps are particles with a mean size of - nm (up to nm), which can be used as carriers and/or adjuvants in vaccine preparation [ ] [ ] [ ] . moreover, nps can be targeted to specific cell populations. as an example, nps can be coated with antibodies recognizing a surface protein on dendritic cells [ , ] . this approach enabled a more accurate measurement of the quantity of antigen required to elicit an immune response [ ] . finally, a more efficient immunization was demonstrated using np-based approaches combined with an intradermal vaccine delivery [ ] , while oral delivery needed further investigations as they have been tested only in vitro [ , ] . in conclusion, novel approaches eliciting a stronger mucosal response are showing promising results both in preclinical and clinical studies. further studies are needed to implement and improve these delivery systems; however, mucosal delivery is becoming the most preferred mode of vaccination. ten great public health achievements-united states the intangible value of vaccination single mucosal, but not parenteral, immunization with recombinant adenoviralbased vaccine provides potent protection from pulmonary tuberculosis mucosal adjuvants and long-term memory development with special focus on cta -dd and other adp-ribosylating toxins recent progress in mucosal vaccine development: potential and limitations a prime-boost vaccination strategy using attenuated salmonella typhimurium and a replication-deficient recombinant adenovirus vector elicits protective immunity against human respiratory syncytial virus mucosal immunity and vaccines function of mucosa-associated lymphoid tissue in antibody formation experience with registered mucosal vaccines gut-associated lymphoid tissues for the development of oral vaccines current state and challenges in developing oral vaccines keynote review: intestinal peyer's patch m cells and oral vaccine targeting adaptive immune regulation in the gut: t cell-dependent and t cell-independent iga synthesis human iga-and igm-secreting intestinal plasma cells carry heavily mutated vh region genes diversification of memory b cells drives the continuous adaptation of secretory antibodies to gut microbiota antigen-driven evolution of b lymphocytes in coronary atherosclerotic plaques anatomical and cellular basis of immunity and tolerance in the intestine uptake through glycoprotein of fimh + bacteria by m cells initiates mucosal immune response green therapeutic biocapsules: using plant cells to orally deliver biopharmaceuticals transgenic plants: a -year update on oral antipathogen vaccine development plant-based vaccines against viruses preclinical and clinical development of plant-made virus-like particle vaccine against avian h n influenza food-grade organisms as vaccine biofactories and oral delivery vehicles plant-made oral vaccines against human infectious diseases-are we there yet? need of cost-effective vaccines in developing countries: what plant biotechnology can offer? the potential of plants as a system for the development and production of human biologics using storage organelles for the accumulation and encapsulation of recombinant proteins seed-based oral vaccines as allergen-specific immunotherapies the increasing value of plant-made proteins plant-produced vaccines: promise and reality oral delivery of bioencapsulated exendin- expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-tc cells disease prevention: an opportunity to expand edible plant-based vaccines? plastids: the green frontiers for vaccine production exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic the role of heterologous chloroplast sequence elements in transgene integration and expression chloroplast-derived vaccines against human diseases: achievements, challenges and scopes transient expression systems for plant-derived biopharmaceuticals expression of the b subunit of escherichia coli heat-labile enterotoxin as a fusion protein in transgenic tomato a plant-based system for rapid production of influenza vaccine antigens oral immunogenicity and protective efficacy in mice of a carrot-derived vaccine candidate expressing ureb subunit against helicobacter pylori severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato immunogenicity of recombinant lt-b delivered orally to humans in transgenic corn human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes expression in plants and immunogenicity of plant virus-based experimental rabies vaccine a plant-derived edible vaccine against hepatitis b virus immunogenicity in humans of an edible vaccine for hepatitis b a rice-based oral cholera vaccine induces macaque-specific systemic neutralizing antibodies but does not influence pre-existing intestinal immunity induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin b subunit vaccine without plant-associated sugar modification good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants mucoricecholera toxin b-subunit, a rice-based oral cholera vaccine, down-regulates the expression of α-amylase/trypsin inhibitorlikeprotein family as major rice allergens algae-based oral recombinant vaccines recent developments in the production of human therapeutic proteins in eukaryotic algae plant-derived vaccines and other therapeutics produced in contained systems heatstable oral alga-based vaccine protects mice from staphylococcus aureus infection recombination and expression of classical swine fever virus (csfv) structural protein e gene in chlamydomonas reinhardtii chroloplasts insect cell technology is a versatile and robust vaccine manufacturing platform non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules oral administration of a cholera toxin b subunit-insulin fusion protein produced in silkworm protects against autoimmune diabetes expression of ureb and hspa of helicobacter pylori in silkworm pupae and identification of its immunogenicity silkworm expression system as a platform technology in life science canine parvovirus vp protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity recombinant protein production in yeasts heterologously expressed aspergillus aculeatus β-glucosidase in saccharomyces cerevisiae is a cost-effective alternative to commercial supplementation of β-glucosidase in industrial ethanol production using trichoderma reesei cellulases whole pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize plasmodium antigens protease-deficient saccharomyces cerevisiae strains for the synthesis of humancompatible glycoproteins enhanced expression of heterologous proteins in yeast cells via the modification of n-glycosylation sites induction of antigen-specific immune responses by oral vaccination with saccharomyces cerevisiae expressing actinobacillus pleuropneumoniae apxiia whole recombinant pichia pastoris expressing hpv l antigen is superior in inducing protection against tumor growth as compared to killed transgenic leishmania oral immunization with whole yeast producing viral capsid antigen provokes a stronger humoral immune response than purified viral capsid antigen characterization and optimization of the glucan particle-based vaccine platform safety, tolerability and immunogenicity of gs- , a hepatitis b virus-specific therapeutic vaccine, in healthy subjects: a randomized study randomized phase ii study of gs- as a therapeutic vaccine in virally suppressed patients with chronic hepatitis b whole recombinant yeast-based immunotherapy induces potent t cell responses targeting hcv ns and core proteins expression of human immunodeficiency virus type neutralizing antibody fragments using human vaginal lactobacillus cloning strategies for heterologous expression of the bacteriocin enterocin a by lactobacillus sakei lb , lb. plantarum nc and lb. casei cect recombinant protein production in bacterial hosts recent advances in the development of live, attenuated bacterial vectors live-attenuated yersinia pestis vaccines immunization with bacillus spores expressing toxin a peptide repeats protects against infection with clostridium difficile strains producing toxins a and b oral immunization with recombinant hepatitis e virus antigen displayed on the lactococcus lactis surface enhances orf -specific mucosal and systemic immune responses in mice surface display of clonorchis sinensis enolase on bacillus subtilis spores potentializes an oral vaccine candidate expression of helicobacter pylori urease b on the surface of bacillus subtilis spores lactic acid bacteria -promising vaccine vectors: possibilities, limitations, doubts probiotic modulation of dendritic cell function is influenced by ageing immunomodulatory properties of lactobacillus plantarum and its use as a recombinant vaccine against mite allergy characterisation and transferability of antibiotic resistance genes from lactic acid bacteria isolated from irish pork and beef abattoirs lactic acid bacteria as mucosal delivery vehicles: a realistic therapeutic option novel surface display system for proteins on non-genetically modified grampositive bacteria influenza antigen-sparing by immune stimulation with gram-positive enhancer matrix (gem) particles intranasal delivery of influenza subunit vaccine formulated with gem particles as an adjuvant cta : purified and display onto gram-positive enhancer matrix (gem) particles as mucosal adjuvant lactic acid bacteria- years exploring their potential as live vectors for mucosal vaccination lactococcus lactis gem particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization development of lactococcal gem-based pneumococcal vaccines immunogenicity of a malaria parasite antigen displayed by lactococcus lactis in oral immunisations bacterium-like particles for efficient immune stimulation of existing vaccines and new subunit vaccines in mucosal applications lactic acid bacteria as a surface display platform for campylobacter jejuni antigens intradermal, epidermal and transcutaneous vaccination: from immunology to clinical practice disentangling the complexity of the skin dendritic cell network innate control of adaptive immunity: dendritic cells and beyond controversies in experimental dermatology: who is really in control of skin immunity under physiological circumstances -lymphocytes, dendritic cells or keratinocytes? intradermal vaccine delivery: will new delivery systems transform vaccine administration? cutaneous delivery of prophylactic and therapeutic vaccines: historical perspective and future outlook the success of microneedle-mediated vaccine delivery into skin noninvasive vaccination against infectious diseases vaccine allergy and pseudo-allergy a meta-analysis of intradermal versus intramuscular influenza vaccines: immunogenicity and adverse events microneedle patch for immunization of immunocompromised hosts microneedle patch delivery of influenza vaccine 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escherichia coli assessment of acceptability and usability of new delivery prototype device for intradermal vaccination in healthy subjects a phase study of safety and immunogenicity following intradermal administration of a tetravalent dengue vaccine candidate randomized controlled clinical trial of fractional doses of inactivated poliovirus vaccine administered intradermally by needle-free device in cuba priming with a simplified intradermal hiv- dna vaccine regimen followed by boosting with recombinant hiv- mva vaccine is safe and immunogenic: a phase iia randomized clinical trial cloning of the first human anti-jcpyv/vp neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy entry inhibition of hsv- and - protects mice from viral lethal challenge neutralization activity and kinetics of two broad-range human monoclonal igg derived from recombinant fab fragments and directed against hepatitis c virus e glycoprotein monoclonal antibodies isolated from human b cells neutralize a broad range of h subtype influenza a viruses including swine-origin influenza virus (s-oiv) anti-hepatitis c virus e (hcv/e ) glycoprotein monoclonal antibodies and neutralization interference diverging effects of human recombinant anti-hepatitis c virus (hcv) antibody fragments derived from a single patient on the infectivity of a vesicular stomatitis virus/hcv pseudotype a phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments chimeric antigen receptor (car)-redirected t cells: is there a place for them in infectious diseases? chimeric antigen receptor (car)-engineered t cells redirected against hepatitis c virus (hcv) e glycoprotein adoptive t-cell therapy in the treatment of viral and opportunistic fungal infections peptide-based vaccinology: experimental and computational approaches to target hypervariable viruses through the fine characterization of protective epitopes recognized by monoclonal antibodies and the identification of t-cell-activating peptides epitope mapping by epitope excision, hydrogen/deuterium exchange, and peptidepanning techniques combined with in silico analysis elicitation of broadly protective antibodies following infection with influenza viruses expressing h n computationally optimized broadly reactive hemagglutinin antigens microneedle patches as drug and vaccine delivery platform microneedles-based transdermal drug delivery systems: a review non-carrier nanoparticles adjuvant modular protein vaccine in a particle-dependent manner vaccine delivery using nanoparticles antigen delivery via hydrophilic peg-b-page-b-plga nanoparticles boosts vaccination induced t cell immunity protein corona-mediated targeting of nanocarriers to b cells allows redirection of allergic immune responses biomedical nanoparticles: overview of their surface immunecompatibility nanovaccine: a novel approach in immunization intradermal delivery of vaccine nanoparticles using hollow microneedle array generates enhanced and balanced immune response interaction of cruciferinbased nanoparticles with caco- cells and caco- /ht -mtx co-cultures in vitro stability of cucumber mosaic virus nanoparticles carrying a hepatitis c virus-derived epitope under simulated gastrointestinal conditions and in vivo efficacy of an edible vaccine the authors declare that they have no conflicts of interest. e.c. and v.c. contributed equally to this work. key: cord- - o htufh authors: borrow, persephone; shaw, george m. title: cytotoxic t‐lymphocyte escape viral variants: how important are they in viral evasion of immune clearance in vivo? date: - - journal: immunol rev doi: . /j. - x. .tb .x sha: doc_id: cord_uid: o htufh summary: although viral variants which are not recognized by epitope‐specific cytotoxic t lymphocytes (ctl) have been shown lo arise during a number of persistent virus infections, in many cases their significance remains controversial: it has been argued that the immune response is sufficiently plastic to contain their replication. in this review, we describe the mechanisms by which amino acid changes in viral proteins may affect epitope recognition by virus‐specific ctl, and discuss the viral and immunological basis for the emergence of viral variants bearing such amino acid changes during infection. we then consider the impact that viral variation may have on the host ctl response and its ability to contain virus replication. we argue that the emergence of a viral variant demonstrates that it must have an in vivo replicative advantage, and that as such, the variant must tip the balance between virus replication and immune control somewhat in favor of the virus. further, we suggest that although the immune response can evolve to recognize new viral epitopes, the ctl generated following such evolution frequently have a reduced ability to contain virus replication. we conclude that this escape mechanism likely does make a significant contribution to persistence/pathogenesis during a number of different virus infections. following virus infection, a series of complex interactions occur between the virus and the host immune system. the host aims to ehminate the infection and minimize associated pathological consequences, whilst the virus tries to avoid clearance by the host immune response so that it can persist and be disseminated to other hosts over a long time period. viruses have evolved a variety of different strategies for avoiding clearance by the host immune response; a single virus often employs multiple strategies simultaneously to increase its chances of persisting in the face of an adaptive host defense system with an array of different effector mechanisms ( ). as cell-mediated immime responses, in particular the virus-specific cd + cytotoxic t-lymphocyte {ctl) response, frequently play a key role in the ehmination of established virus infections, many viral evasion strategies are targeted to this arm of the immune response. viral immune evasion strategies may broadly be divided into two categories: those that enable the virus to avoid detec-jrrow & shaw • in vivo importance of ctl escape viruses tion by the host immune response, and those that impair the functioning of the host immune system. viral impairment of the functioning of the host immune system may be non-antigen-specific, as exemplified by the production of homologs of host cytokines or rheir receptors that mimic (in the case of immune downregulatory) or block (in the case of immunestimulatory or antiviral effector) the actions of host cytokines ( ), or hy infection of cells of the immune system, causing either impairment of their functions or resulting in their destruction; the latter may occur either directly or hy the cells being rendered targets for immune lysis ( ). however, as too severe a generalized impairment of host immune functions can lead to the death of the host, thus reducing the opportunity for virus spread, a preferable strategy is impairment of the virusspecific immune response. examples of how this may be achieved are by viruses infecting and targeting the destruction of antigen-specific b cells ( ) or inducing exhaustion of highaffmity clones of virus-specific t cells ( ), strategies viruses may employ to avoid detection hy the host immune response include i) the establishment of latent infections during v\'hich viral protein production is minimized, a strategy commonly employed hy herpesviruses and perfected by herpes simplex virus ( ); ii) infection of immune-privileged sites (to which access of the immune system is restricted and where levels of major histocompatibility complex (mhc) antigen expression are iow and immune downregulatory mediators may be present), the best example being the hrain where a surprisingly large number of different viruses persist ( ); iii) virus-induced downregulation of levels of mhc antigens or adhesion molecules on the surface of infected cells so :hat they do not trigger host t-cell recognition ( ); and iv) evolution of antigenic variants whose recognition by the specific immune response is impaired, antigenic variation was initially documented as a means for viral escape from antibody neutrahzation ( ), but more recently has also heen shown to confer viral escape from ctl control ( ). although the above immune evasion mechanisms all have [he potential to promote viral persistence, the contribution that some of these mechanisms in fact make to persistence in vivo, particularly during human virus infections, is not completely dear. for example, although viral variants which are not recognized by epitope-specific ctl bave been found during a number of persistent virus infections ( ), in many cases their significance remains controversial: it has heen argued that the immune response is sufficiently plastic to contain their rephcation ( ), in this review, the mechanisms hy which amino acid changes in viral proteins may affect epitope recognition by virus-specific ctl are described, and the viral and immunoiog-ical basis for the emergence of viral variants bearing such amino acid changes during infection is discussed. the impact that viral variation may have on the antiviral ctl response during the course of virus infections is then considered, and the contrihution that this escape mechanism may make to persistence/pathogenesis in different virus infections is discussed. mechanisms by which amino acid changes may affect epitope recognition by virus-specific ctl for a viral epitope to be recognized by cd -^ t cells, it must be processed and presented on the cell surface in association with mhc class i molecules. amino acid changes may thus affect epitope recognition by specific cd + t cells hy altering any of the steps involved in this pathway, from the initial generation of the epitopic peptide to its formation of a stable complex with mhc, they may of course also affect the interaction of the peptide-mhc complex with the t-cell receptor (tcr), either ablating tcr binding altogether, or altering the signahng which occurs via the tcr, as described below, examples have been found of amino acid changes in viral proteins that act via each of these mechanisms. alteration ofantigen processing/peptide transport the efficiency of processing and presentation of ctl epitopes is determined not only by the sequence of the epitope itself, but also by the residues which surround it in the protein. amino acid mutations in flanking sequences may thus affect the recognition of viral ctl epitopes, as was originally demonstrated in experiments using cytomegalovirus and influenza virus-specific ctl ( , ), the precise mechanism by which antigen processing was affected was not defmed in these studies or a subsequent paper describing mutations in the nef protein of human immunodeficiency virus type (hiv- ) which also affected presentation of a nearby ctl epitope ( ), however, in a more recent report, alteration of proteasome-mediated degradation due to a single amino acid difference within a ctl epitope was shown to form the basis of lack of presentation of this epitope by infected cells ( ). in mice infected with akv/mcf type murine leukemia virus, an epitope in the pi e transmembrane viral envelope protein constitutes the immunodominant sequence recognized by virus-specific ctl, these ctl do not recognize cells infected with friend/moloney/rauscher type murine leukemia virus, where the epitope sequence differs by a single residue ( ); this is because the amino acid difference causes epitope destruction by specific proteasomal cleavage. mice infected with the latter virus thus do not mount a ctl response to this epitope, amino acid changes that alter the processing of ctl epitopes can therefore have a dramatic effect on the in vivo antiviral ctl response. it is unclear how commonly such mutations are selected for during persistent virus infections. that relatively few examples of amino acid changes in viral proteins which affect ctl recognition via this mechanism have been described could be a reflection of the fact that such mutations are not readily picked up by the methods most frequently used to analyze antiviral ctl responses. alternatively, the requirements for antigen processing/peptide transport may be so much less stringent than those for peptide interactions with mhc and the t-cel! receptor that amino acid changes which affect the former arise less often. amino acid mutations within epitopic peptides may ablate peptide binding to mhc altogether, or reduce the affmity of peptide-mhc interaction so that the peptide-mhc complex has an extremely short half-hfe and is unlikely to trigger t-cell activation. viral mutations which alter peptide interactions with mhc class i molecules in each of these ways have been described; examples in hiv were recently reviewed ( ). as mutations which prevent the stable association of peptides with mhc confer escape from recognition by all epitope-specific t cells regardless of their tcr usage, this is an efficient mechanism for viruses to avoid ctl recognition. it is of interest that the human leukocyte antigen (hla)-a epitope loss from epstein-barr virus (ebv) isolates derived from populations where the frequency of hla-al is extremely high is conferred via mutations in anchor residues of the epitope that are important for binding to hla-al ( , ). ebv is a genetically stable dna virus which generates mutants at a relatively low frequency; mutants will thus only come to prevail in the population if they confer advantages for the spread of virus to new hosts, in which very different tcrs may he used to recognize the same peptide-mhc complex. mutations that ablate binding of the immunodominant hla-a ] -restricted ebv ctl epitope to mhc will allow escape from recognition from epitope-specific t cells in any host. alteration of peptide interaction with the tcr amino acid changes in a peptide which do not prevent it from being presented in association with mhc may result in alterations to the surface recognized by the tcr. this surface may be altered by changes in the tcr contact residues, or by changes in other residues in the epitope that cause the peptide to bind to mhc in a distorted conformation ( ). epitopes bearing mutations that cause alterations in the tcr contact sur-face have been termed altered peptide ligands (apl) ( ). epitope-specific t cells bearing distinct tcrs may be affected by apl in different ways. the altered peptide-mhc complex may fail to interact with a particular tcr altogether; or it may be recognized, but the t cell may receive a reduced or even a different type of signal when it recognizes an apl-bearing cell ( - ), the responding t cell may thus be only partly activated (e.g. to proliferate but not induce cell lysis ( )), or even anergized ( ). presentation of certain apl to t cells can therefore inhibit the response to the index peptide not just by competing with it for binding to mhc, but by negatively signaling the responding t-cell population: this phenomenon is known as t-cell antagonism. the abihty of apl to act as ctl antagonists can be tested by measuring the capacity of cells presenting the apl to inhibit lysis of labeled target cells presenting the index peptide ( ), using this technique, mutations which confer antagonistic properties on epitopic sequences have been shown to exist in the persisting virus population in individuals chronically infected with both hepatitis b virus (hbv) ( ) and hiv- ( ), although viral mutations which result in the generation of apl may not provide as efficient a means of simple escape from immune recognition as mutations which abrogate epitope presentation altogether (because the apl may still be immunogenic to a proportion of host t cells which could be expanded following emergence of the mutant), they could potentially have powerful effects on the overall control of virus replication via other mechanisms. if they are able to act as t-cell antagonists, they may not only inhibit the lysis of cells on which the mutant epitope is presented, but also that of cells in which nonmutant virus is replicating, hence conferring protection from immune control on the entire viral quasispecies. further, as some apl are able to stimulate and sustain the growth of ctl despite the fact that they do not induce ctl lysis ( ), they could drive an ineffectual ctl response and hence modulate the ctl repertoire in a detrimental fashion, reducing the overall efficiency of ctl control of virus replication. genetic variation is a strategy viruses exploit to promote their survival not just in the face of the host immune response, but under any environmental conditions they may encounter. they can achieve variation by a number of different mechanisms, including mutation, homologous and non-homologous recombination and (for viruses with a segmented genome) reassortment: different virns families utihze these to different extents. rna viruses (including retroviruses) and dna viruses such as immtinologicd reviews / bepadnaviruses, whose genome replication involves an rna intermediate, have extremely high mutation rates. this is due in large part to the absence or very low efficiency of proof-reading-repair activities associated with rna replicases and transcriptases ( ), and the lack of post-rephcative error correction mechanisms such as those diat normally operate during rephcation of cellular dna. misinsertion errors during rna rephcation and reverse transcription have been estimated to be in the range of ""^ to "^ substitutions per nucleotide per round of copying; for a kb genome, this w(mld result in each progeny rna strand including an average of . to mutations ( ). rna viruses thus exist not as homogeneous populations, but as complex, dynamic mixtures of heterogeneous sequences termed quasispecies ( , ). if a virus is replicating under a constant set of environmental conditions to which it is optimally adapted, although the precise composition of the quasispecies will continually be fluctuating, the average sequence of the viral population will remain unchanged. however if conditions alter, mutations which confer an increase in fitness (the relative ability of the virus to produce infectious progeny) will be selected for and come to predominate in the viral population. the quasispecies nature of rna viruses, conpled with the short replication times and high viral yields they frequently exhibit, favors rapid adaptation to environmental changes. dramatic examples of viral evolution in response to environmental pressure have been provided by the emergence of drug-resistant viral variants in hiv- -infected patients treated with antiretroviral agents (e.g. ( - )), hiv is a retrovirus with an in vivo mutation rate of approximately x "^ nucleotides per rephcation cycle ( ). human infection with this virns is characterized by high levels of persisting virus, much of which is actively replicating and turning over at remarkable rates ( - ). as many as "^ virions may be produced per day ( ), allowing great potential for variation ( ), although not all of these will be infectious, and the effective population size may be much smaller ( ). w^here a single nucleotide change is sufficient to confer a high level of resistance to antiretroviral treatment, resistance may be selected for very rapidly eor example, a single nucleotide change can reduce hiv's susceptibihty to the non-nucleoside reverse transcriptase inhibitor nevirapine by - , -fold. viral variants bearing this mutation have been shown to pre-exist in the plasma hiv rna in untreated patients ( ), and in some patients high level resistance to this drug may be acquired within weeks of starting therapy ( ), in cases where multiple mutations are required to achieve drug resistance, either because a series of mutations needs to occur before a high level of resistance to one antiretrovirai agent is achieved ( , ) or because the patient is being treated simultaneously with a combination of several antiretroviral agents ( , ), it takes longer for resistance to evolve. indeed, in some patients, particularly where virus replication has been reduced to very low levels, it may not do so ( , ). however, these observations illustrate the tremendous potential for rna viruses to overcome controlling forces during the course of an infection via selection for resistance-conferring mutations. under what conditions are ctl escape viral variants selected in vivol the above outline of how mutations are selected for in a virus population allows a number of predictions to be made about the in vivo conditions under which ctl escape virns variants are likely to emerge. firstly, as a mutation will only be selected for if it confers a fitness advantage on the virus hearing it, ctl escape virus variants will only grow out in infections where ctl pressure exerts significant control over virus replication. it is thus perhaps not surprising that the first report of the in vivo emergence of a ctl escape virus variant came from a study performed using lymphocytic choriomeningitis virus (lcmv) ( ), a murine virus: during infection with lcmv the cd + ctl response is well known to be the critical determinant of control of virus rephcation (reviewed in ( )), conversely, demonstration that viral variants bearing mutations which confer escape from cd "^ t cells come to dominate the viral quasispecies during a particular virus infection can be used to provide evidence for the importance of this arm of the immune response in controlling the infection, which in human virus infections may otherwise be difficult to demonstrate convincingly ( ) ( ) ( ) . secondly, as the stronger the control over virus rephcation exerted by the ctl response directed against a particular epitope, the greater the selective pressure it will exert for escape mutations in this epitope to emerge in the viral population, the frequency of ctl directed against a particular epitope and the avidity of their target cell interaction will clearly influence the selection of escape mutations in this epitope. the original demonstration of ctl escape virus selection in the lcmv system was actually made in tcr transgenic mice where cd + t cells specific for the epitope in which escape mutations were observed made up - % of the peripheral t-cell population prior to infection ( ), this was an artificial system; however, due to advances in methods for quantitating epitope-specific t cells, it has recently become apparent that epitope-specific cd "^ t cells can reach extremely high frequencies in natural virus infections in both mice and humans, particularly during the primary immune response ( ) ( ) ( ) . primary infection is thus clearly a setting under which escape-conferring mutations may potentially emerge very rapidly although lower frequencies of epitope-specific ctl will also have the capacity to drive the selection of escape viral variants, this selection will occur more gradually over the course of a greater number of rounds of viral rephcation. the greater the selective advantage conferred hy a particular viral mutation, the faster this mutation will be selected for in the viral quasispecies. thus, not only the strength of the ctl response to a particular epitope hut also the immunodominance of this response will have a great impact on the likelihood that escape mutations will be selected for within the epitope. escape mutations in subdominant or even co-dominant ctl epitopes may confer such a shght selective advantage on the viral variants bearing them (whose replication will still he controlled by the more dominant or co-dominant ctl responses in the host ( , ) ) that they may never emerge in vivo. virus rephcation must clearly be ongoing for viral variants to be generated and selected. particularly where the selective advantage conferred by a certain amino acid mutation is not very great, many cycles of virus replication may need to occur before viruses bearing it become predominant in the viral quasispecies. how dominant the ctl response to a certain viral epitope is in the overall control of virus replication is thus also an important determinant of the emergence of escape mutations in this epitopic sequence from this perspective. if other arms of the immune response or cd + t-cell responses to other epitopes in the virus which are not affected by the mutation concerned are able to reduce virus replication to very low levels or mediate viral clearance, the escape mutation will not have time to hecome fixed in the viral population during the course of infection. ctl escape virus variants will thus be most likely to emerge in the face of a host ctl response which is highly dominated by t cells of a single epitope specificity; even a very strong response to a particular epitope is unhkely to have the chance to select for escape viral variants in the context of strong responses to other epitopes. this is illustrated by observations made in the murine lcmv infection model. the ctl response moanted to lcmv by infected c bl/ mice has a broad epitope specificity: in the viral glycoprotein (gp) and nucleoprotein (ne) alone, at least five different ctl epitopes have heen identified ( , ). although the response to some of these epitopes is extremely strong (t cells recognizing the epitope at ne - constitute up to % of the cd -^ t-cell population, and those recognizing the epitope at ge - up to % at the peak of the antiviral immune response ( )), the infection is rapidly cleared, and ctl escape viral variants do not emerge. if mice are immunized with one of these epitopes in a carrier protein prior to infection, the ctl response mounted upon lcmv infection is then biased in favor of this epitope. viral clearance still occurs; however, if virus is isolated from the mice just prior to clearance and grown up in vitro, it is found to contain escape mutations in the epitope to which the ctl response was biased ( ) , escape viral variants thus arise in the context of this more immunodominant ctl response, but they do not grow out in vivo as the infection is cleared too rapidly the lcmv tcr transgenic mice discussed above represent a situation of epitope immunodominance at the extreme of the spectrum: their t-cell repertoire is so dominated by transgene-expressing cells recognizing the lcmv ge - epitope that ctl responses cannot be effectively mounted to other viral epitopes. thus, when they are infected with lcmv and viral mutants arise which cannot be recognized by the ge - ]-specific ctl, their replication is not contained by ctl of other specificities and they emerge in the context of viral persistence ( ). the ahove predictions that ctl escape viral variants are most likely to. emerge in the context of a strong host ctl response which is highly focused on a single viral epitope are supported by the observation that one of the clearest examples of the emergence of ctl escape virus variants during a human virus infection occurred under just these conditions. both we and others have shown that strong cd + ctl responses are mounted very early following infection with hiv- , prior to seroconversion, and have hypothesized that they play an important role in containing viral replication ( , ), in one patient we studied ( ), the early ctl response ( - days following the onset of symptoms indicative of acute hiv- infection, which occurred days after the homosexual encounter during which he initially contracted the virus) appeared to he strongly directed against a single viral protein, gpl . epitope mapping performed using the gpl sequence of the patient's autologous early hiv- population indicated that this response was in fact extremely focused on a single epitope encompassing gpl amino acids - ( ), recognized in association with hla-b , the frequency of epitope-specific ctl was extremely high: at the earhest timepoint available for study, which may have been shghtly after the peak of the primary immune response, in peripheral blood mononuclear cells (ebmcs) were found to score as virus-specific ctl precursors by limiting dilution analysis, a technique which has recently been shown to greatly underestimate the total numher of epitope-specific t cells ( , ) , as shown in fig, , viral variants bearing mutations in the epitopic sequence which conferred escape from recognition by epitope-specific ctl rapidly appeared in this patienc, and then increased in frequency until / they had cotnpieteiy repiaced the transmitted virai strain. interestingly, the variants which came to predominate in the virai quasispecies aii possessed changes at gpi amino acid i, the position which constituted the major anchor residue for hla-b . as discussed above, mutations which abiate peptide binding to mhc confer escape from recognition by aii epitope-specific t ceiis, and indeed, not oniy individual ctl ciones but aiso bulk ctl derived from this patient were unabie to recognize peptides corresponding to the mutant virus sequences in in vitto assays (fig. ) . thus in both human and murine systetns, there is evidence that mutations which confer resistance to controi by epitopespecific ctl are most iiiieiy to be seiected for in highiy immunodominant epitopes. under conditions of epitope immuno-dominance, a singie mutation which provides escape from recognition by ctl of one specificity wiii confer a greater repiicative advantage on the mutant virus, thus the seiection for it wiii be stronger; pius there wiii be a greater chance that virus replication wiii be abie to continue for iong enough for the mutation to compieteiy repiace the index residue in the virai quasispecies. evolution of the ctl profile in response to the emergence of escape viral variants the virai quasispecies can ciearly evoive in response to host immune pressure: as the host immune response is aiso adaptive, can it in turn evoive in response to the emergence of escape viral variants? the specific immune response is nltimately driven by antigen; the emergence of a ctl escape mutation in a virus population during the conrse of an infection could potentially affect the antigen to wbicb tbe immune system is exposed, and hence the ctl profile, in several ways. firstly, unless the mutation selectively affects ctl stimulation so that only effector functions are diminished, the level of ctl specific for the index epitope will decline as the level ofantigen available to stimulate them decreases. secondly, if the mutant sequence is presented to and can stimnlate novel populations of host ctl, these will increase in frequency. thirdly if the mutation reduces the efficiency with which the epitope is presented, this may allow the level of presentation of other viral epitopes to increase, and hence ctl of different specificities to increase in frequency, or novel populations of host ctl to be stimulated. finally, as tbe mutant virus must bave a rephcative advantage over tbe original virus in order to be selected, the antigen load will increase, which will drive the overall expansion of virusspecific ctl. as discussed earlier, ctl escape mutations are most likely to be selected for in immunodominant ctl epitopes. the end result of the above effects will be that the breadth of tbe ctl response will increase, with the previously dominant t-cell clones declining in frequency, and subdominant or novel responses being stimulated. sucb broadening of ctl specificity in response to tbe emergence of viral variants able to escape recognition by ctl directed against a highly immunodominant epitope was apparent in the hiv patient described in the previous section ( ) . as illustrated in fig. , whereas pbmc derived from tbe patient very early during tbe infection mediated detectable lysis after in vitro restimulation of only target cells expressing the immunodominant gpl - ( ) epitope, as viral variants bearing mutations in this epitope emerged, responses to epitopes elsewhere in gpl and in other viral proteins became apparent. a decline in the frequency ofctl directed against the gpl - ( ) epitope occurred simultaneously ( ) . this patient clearly bad the capacity to make ctl responses to a broad range of epitopes in hiv; why then was his initial response so highly foctised on a single epitope? how epitope immunodominance is dictated dnring an antiviral immune response is not clear: both the level of presentation of different viral epitopes and the available t-cell repertoire impact on this. the former can be affected by epitope processing ( ), peptide transport into tbe endoplasmic reticulum ( , ) , tbe binding affinities of peptides to class i molecules ( ) and the stabihty of peptide-mhc complexes on the cell surface ( ), if ctl epitopes overlap, different mhc molecules may compete for their presentation ( ) . the t-cell repertoire is initially deter-mined by positive and negative selection in tbe tbymus, and subsequently reshaped in tbe periphery during the course of successive immune responses. tbe influence that tbe previous infection history may have on the subsequent immune response to a virus infection bas been illustrated in mnrine arenavirus infections ( ) . in the hiv patient discussed above, both properties of the epitopic sequence and the t-cell repertoire at the time of infection may bave contributed to the extreme immunodominance of the early ctl response, tbe gpl - ( ) peptide may have had advantages over other potential epitopic sequences in its processing and transport: there appear to be differences in the processing of transmembrane and cytoplasmic proteins for presentation with class i whicb may give tbe former a presentation advantage under certain circumstances ( ); further, it is of interest that during the natural processing of gpl , signal peptide cleavage occurs between amino acids and , thus generating the same n terminns as the - ( ) epitope. in addition, this epitope is predicted to have a very high binding affmity to hla-b . t-cell repertoire effects may also bave played a role: it is possible tbat prior unrelated infections in this patient left bim with a population of memory t cells that cross-recognized this epitope, and that these cells, being present at higher frequency and more readily activated than naive t cells, dominated the initial hiv- -speciflc immune response. this cannot be demonstrated conclusively as no samples are available from tbis patient prior to infection; bowever, the fact that epitope-specific ctl were contained in an ohgoclonal population of vp + t cells which underwent a massive expansion very early after infection in this patient ( ) would be consistent with this. another study in the hiv system wbich emphasized the effect that escape mutations in ctl epitopes may bave on ctl specificity focused on the ctl responses of two hla-identical hemophiliac brothers who were exposed to the same batch of contaminated factor viii and became seropositive within weeks of one another ( ) . their ctl responses were very dissimilar: one made strong responses to two hla-a and hla-a -restricted epitopes in gag, whilst the other instead responded to two hla-b -restricted ctl epitopes. mutations in botb immunodominant epitopes of the gag responder were seen in provirai sequences from the non-responder; it was thus very hkely that be had initially made a ctl response to these epitopes, and tbat following the emergence of escape mutant viruses, tbis response had been downregulated and the b restricted responses stimulated. in this individual, the escape mutant viruses did not revert to the index sequence when the ctl response against them diminished, implying tbat the mutations they bore were not detrimental to viral replication. in other cases, however, ctl escape mutations may reduce viral fitness ( ) . if they impair virus replication too severely, tbey will not be selected for in the first place; this may contribute to the fact that in some studies escape variants have not been seen to arise in particular ctl epitopes despite the fact tbat ctl pressure is exerted on them over long periods of time ( . ). if they have a smaller impact on viral fitness, they will emerge when epitope-specific ctl are exerting a strong pressure on viral replication, as overall they will confer an advantage on the virus, but as the frequency of epitope-specific ctl declines, they will tend to be replaced in the viral quasispecies by the index sequence. this in fact occurred in the hiv patient we described, where a strong selection occurred in tbe viral quasispecies for viral variants bearing mutations at the residue w-bich constituted tbe anchor motif for the hla-b -restricted ctl epitope to which a dominant response was made in tbe early phase of the infection. later in tbe infection, wben the frequency of epitope-specific ctl bad declined significandy, clones bearing the index sequence again began to emerge in tbe viral quasispecies (x. wei, p borrow, . epitope immunodominance and mutant virus frequency tbus drive one another, which can lead to complex fluctuations in the makeup of tbe viral quasispecies and ctl specificity over the course of a persistent infection, sucb fluctuations have been observed in patients cbronically infected with hiv- , and mathematical models developed to describe them ( - ), these models predict tbat antigenic variation in immunodominant epitopes can shift responses to weaker epitopes and tbereby reduce immunological control of the virus. that this certainly can occur bas been illustrated in experiments performed in the lcmv model system, ctl clones directed against the three most dominant epitopes recognized during the antiviral ctl response in c bl/ mice were used to select in vitro for lcmv variants bearing escape-conferring mutations in one, two or all three of these epitopes ( ) ( ) ( ) . wben cs bl/ mice were infected with tbe mutant viruses, they mounted ctl responses to subdominant epitopes including epitope(s) in the viral polymerase wbich are not readily apparent during tbe response to wild-type virus, but the responses driven by the in vitro-generated escape viral variants controlled virus replication less efficiently than the response to wild-type virus ( , ) . these experiments indicate tbat although tbe immnne response is extremely plastic, its capacity to evolve to efficiently contain the replication of escape mutant viruses may be limited, particularly in inbred mice where the diversity of mhc alleles is more restricted than in the outbred human population. the evolution of escape viral variants may thus make a significant contribution to viral persistence during some infections by driving a suboptimal immune response. for a ctl escape viral variant to emerge, it must tip the balance between virus replication and the host antiviral immune response at least somewhat in favor of the virus. this replicative advantage can in itself affect the course of a virus infection, as was seen in tbe experiments originally demonstrating the in vivo selection of ctl escape viral variants in lcmv epitope-specific tcr transgenic mice: these animals developed a persistent infection following inocnlation with a dose of virns whicb non-transgenic mice were rapidly able to clear ( ). however, in tbis system, the tcr repertoire was artificially restricted, leaving open the question of whether ctl escape viral variants may also have biologically significant effects on the course of infection under more natural circumstances, where the immune system has the capacity to respond to a mucb broader range of viral epitopes and, as discussed above, the ctl response may co-evolve with the viral quasispecies. mutations which affect epitope-specific ctl lysis in vitro bave been described in a number of different viruses; here, their likely significance in some of these virus infections is discnssed. mouse hepatitis virus-strain jhm (mhv-jhm) is a coronavirus which produces an acute fatal encephalitis in most inbred strains of mice. if c bl/ mice are infected wbilst suckling on dams previously immunized to the virus, tbey are protected from tbe acute encephahtis; a proportion of mice then control the infection, but the majority develop a persistent infection associated with chronic demyelinating encephalomyehtis ( ), tbe virusspecific cd + t-cell response is important in controlling this infection; in c bl/ mice this is directed against two epitopes in the viral surface gp (s), an immunodominant epitope at s - , and a subdominant epitope at s - s ( ), a recent study showed that mutations which cause a loss of recognition are present in tbe immnnodominant epitope sequence in almost all virus sampled from symptomatic mice, but not in other t-cell epitopes ( ) , mutations in this epitope were not detected in mice witb acute encephalitis (whicb die very soon after the antiviral t-cell response is mounted) and were fonnd at only low frequency in the residual viral rna in the central nervous system of animals which had cleared infectious virus and remained asymptomatic at late times after infection. further, when mhv-jhm variants bearing mutations in epitope s - isolated from infected mice were used to infect naive mice, they produced an increased morbidity and mortahty ( ) . this is thus a convincing example of an infection in wbicb ctl escape variant selection does appear to be a key factor influencing virus pathogenesis. this infection may represent a situation where the host's abihty to control virus rephcation is tenuous (cd "^ t-cell control of virus replication in the central nervous system may not be tbat efficient), and the rephcative advantage conferred on the virus by escape mutations may tip the balance firmly in favor of the virus. ebv is a human gamma herpesvirus that is carried by the majority of individuals as a hfelong asymptomatic infection, but has oncogenic potential and is implicated in the pathogenesis of a range of malignancies. the increased risk of development of ebv-positive mahgnancies associated with immune suppression illustrates the importance of immune control in maintaining a non-pathogenic equilibrium between this potentially oncogenic virns and its host; the potent antiviral ctl response is thought to play an important role in controlling virus replication and spread and the development of disease ( , , ) . the abihty of tbis virus to evade clearance from the infected host has been attributed to a number of different mechanisms (reviewed in ( )), but ctl escape variant selection is not thought to play a major role. unlike tbe other viruses discussed in this section, fbv is a genetically stable dna virus; mutations whicb may confer escape from the prevailing host ctl response are tbus hkely to arise too infrequently for escape variant generation and selection to constitute an efficient means ofimmune evasion within a given host. mutations have been documented in the virus population prevaihng in certain areas wbich confer escape from recognition by ctl directed against epitopes whicb are presented by hla alleles possessed by a high proportion of the local populace. tbe best known example involves hla-a -restricted ctl epitopes in the ebna b protein which are conserved in tbe majority of type ebv isolates from caucasian and african populations, where the hla-a allele frequency is low, but are mutated in virtually all isolates from highly all-positive immunologicoj reviews / populations in china and coastal new guinea ( , ) . the majority of these mutations are located within anchor residues and abrogate epitope binding to the hla-al molecule; as discussed earher, such mutations would provide escape from epilope-specific ctl regardless of their tcr usage, suggesting that they may have conferred a selective advantage on the virus in the a -positive populations from which they were isolated. however, fbv isolates from south-east asia differ from caucasian and african isolates at a number of loci, and other polymorphisms have been shown to affect the antigenicity of epitopes that are not present at high frequency in south-east asian populations ( ) , raising the possibility that the changes observed in the al -restricted epitopes may be coincidental. support for the hypothesis of specific epitope loss was not obtained when ebv isolates from a highly hla-b -positive african population were sequenced across a b -restricted epitope-containing region of the ebna a protein ( ), however, the hla-b frequency in this population was only about half that of the hla-a frequency in the south-east asian populations where a epitope mutations were detected; further, the hla-b -restricted ctl response to ebv is not usually as strong as that restricted by hla-a ( ) . whether the ebvspecific ctl response does in fact select for viral variants with a growth advantage within human populations bearing certain hla alleles thns remains an unresolved issue. hbv is a hepadnavirus which, although it has a dna genome, replicates via reverse transcription from a pregenomic rna and thus has a high mutation rate. this virus produces acute and chronic infections in man during which the cd "^ ctl response plays a key role both in virus clearance and in the pathogenesis of the associated liver disease ( ) . during acute hbv infection, most patients develop a strong polyclonal ctl response against multiple epitopes in different viral proteins ( ) . as discussed above, the likelihood of selection of escape mutant viruses under these conditions is prohably low, because a mutation which provides escape from ctl of just a single specificity may not confer a significant selective advantage on the virus bearing it in the presence of strong ctl responses to many other epitopes. in contrast, the ctl response is usually much weaker during chronic hbv infection ( ) . in some patients it may also be more ohgospecific, providing greater opportunity for the selection of epitope-specific escape mutations. indeed, there is one report ( ) of two patients who showed strong hla-a -restricted ctl responses narrowly focused on an epitope in the hbv core protein at amino acids - and failed to respond to any of the other hla-a -restricted ctl epitopes in hbv that are frequently recognized in acutely infected patients. when the persisting virus in these patients was sequenced, the quasispecies was found to be dominated by variant virus carrying mutations within the hbv core - epitope that affected epitope recognition by the patients' ctl and could antagonize the response of certain ctl clones to the index epitope ( ). although conversion from a wild-type to the mutant sequence was not demonstrated in these patients, it is very likely that this does represent an example where escape variants were selected by the strong epitopespecific ctl response. ctl responses restricted by other hla alleles were not analyzed in these patients, and no information is available about how the ctl repertoire may have evolved in response to the emergence of the mutant viruses. it is thus difficult to assess how the escape variant may have impacted on the overall efficiency of ctl-mediated control of virus rephcation in these individuals. other reports suggest that the emergence of virus variants bearing ctl escape mutations may not be a common event during chronic hepatitis b infection: no escape mutations were identified in a stndy where the ctl response against eight different hla-a -restricted epitopes defined in patients with acute hbv infection and the sequence of these regions in the in vivo viral quasispecies were analyzed in parallel in patients chronically infected with hbv ( ) . virus-specific ctl responses were undetectable in eight of these patients and weak in the other four; ctl-mediated pressure may thus have been too low to select for escape variants. whether selection of escape mutations plays a critical role in hbv persistence is thus questionable ( ) ; it is likely that the development of a weak antiviral immune response is ol much greater importance in determining the course of this infection ( ) . hcv is a positive-stranded rna virus, assigned to a new genus in the flaviviridae, which is well adapted for persistence in its human host: at least % of infected individuals develop a chronic infection that is frequently associated with clinical hepatitis ( ) , as in hbv infection, the virus-specific cds-' ctl response is thought to play an important role in limiting virus replication, and simultaneously to contribute to disease pathogenesis. however, unlike the situation in hbv infection, where cell-mediated immune responses to the virus are generally low/undetectable in chronically infected individuals, hcv persistence in the presence of strong virus-specific ctl responses has been observed in both humans and experimentally infected chimpanzees (reviewed in ( ) ). the highly mutable nature of this virus's rna genome raises the possibility that antigenic variation may he one of the mechanisms via which it aciiieves persistence in the face of the antivirai immune response. indeed, there is one report that in a chimpanzee which became persistentiy infected with hcv tiie virai quasispecies underwent compiete repiacement with virai variants hearing a conservative amino acid suhstitution at residue of an epitope in non-structural protein (ns ) recognized hy iiver-derived ctl iines from this animal; this mutation ahrogated recognition of the epitope by the animai's ctl ( ) . the ctl response in this animai recognized epitopes in mtiltipie virai proteins (ioo); whetiier mutations were seiected for in any of the other epitopes, how epitope specificity/immunodominance may iiave ciianged over the course of infection in response to virai variation, and what impact escape variant seiection may have had on the overali efficiency of controi of virus repiication were not determined. however, tiiis study iliustrates that even though the ctl response to hcv is frequentiy poiycionai, escape virai variants can be seiected during hcv infection. as discussed beiow for hiv, where more information ahout the co-evoiution of the virai quasispecies and tiie immune response is avaiiahie, escape mutations iikeiy do compromise immune control of hcv infection, and are probabiy one of the factors which contrihute to the persistence of this virus in its human host ( ) . hiv ehcits strong ctl responses in most infected individuals. the eariy cd + ctl response is temporally coincident with the reduction in acute plasma viremia ( , ); as discussed ahove, virus-specific ctl iikeiy expand to extremeiy iiigh frequencies during the primary immune response ( , ) . the frequency of virus-specific ctl then deciines somewhat; however, high ieveis of activated ctl are commoniy detected in chronicaiiy infected asymptomatic individuais, even in the setting of an extremeiy iow virai ioad ( , i - ). as disease progression occurs, virus-specific ctl frequencies may initialiy increase in response to increasing viral ioad, but generaily faii to imdetectabie ieveis in the end stages of the infection ( ) . a number of hnes of both direct and indirect evidence (discussed in ( ) ) indicate that the cd + ctl response does piay an important role in controiiing virus repiication in hiv- -infected individuais, and a variety of mechanisms have been proposed to contribute to virus persistence in the face of the host immune response ( ) ( ) ( ) ( ) ( ) ( ) ( ) . we focus here just on the role that the selection of ctl escape virus variants may piay during this infection. the high virai turnover during hiv- infection, rapid mutation rate of this virus and strong virus-specific ctl responses provide conditions under which ctl escape mutants wouid be iikeiy to emerge. indeed, as indicated earher. there have been numerous reports of variant viruses within the quasispecies in different patients w-hich are able to escape recognition by epitope-specific ctl. escape virus variants may be selected at different stages of infection and correspondingly impact on the virus-host baiance in different ways. during primary hiv- infection, when the virus undergoes an intensive hurst of rephcation and ctl directed against dominant epitopes may reach extremeiy high frequencies, escape variant seiection may be particuiariy favored. we have observed repiacement of the virai quasispecies hy variants with mutations in an epitope targeted by tiie primary ctl response in the eariy stages of infection in two patients anaiyzed ( ) , and there has aiso been another study documenting ctl escape variant emergence in a seroconverter ( ) . interestingly, the two patients in whom we observed eariy seiection of ctl escape variant viruses botii underwent rapid disease progression: studies of larger numbers of patients are required to reveai how frequentiy eariy seiection of mutations in epitopes recognized during the primary ctl response in fact occurs, and whether this phenomenon is aiways associated with rapid progression to aids. the emergence of virai variants with mutations that confer resistance to controi by dominant ctl responses in the eariy stages of infection may potentialiy impact on the suhsequent course of infection in two ways. first, as a virai variant wiii oniy be seiected for if it has a rephcative advantage, the emergence of ctl escape variants must he associated with an increase in eariy virus rephcation and spread. consequences of this may inciude increased ioss of cd + t cells and estabhshment of a lareo er pooi of iatentiy infected ceiis or a higiier setpoint ievei of persisting virus: the iatter has been demonstrated to be a good predictor of the subsequent rate of disease progression ( ) . second, because (as discussed above) mutations in immunodominant ctl epitopes may promote evoiution of the immune response to recognize subdominant epitopes, the host may he left after tiie acute phase of the infection not oniy with a higher ievei of persisting virus, but aiso with a ctl response that is iess adequate to contain it. in mid-hiv infection, mutations which affect recognition by epitope-specific ctl have been demonstrated in the persisting virai quasispecies in a numher of studies (e.g. ( . , )); however, clear examples of such variants being selected for and fixed in the virai quasispecies over time are difficuit to find. this is hkeiy hecause ctl responses to muitipie virai epitopes are frequendy present and, as discussed earher, under such conditions a complex cycie of fluctuations in the domi-nance of ctl responses directed against different viral epitopes and corresponding shifts in the epitopic composition of the viral quasispecies may occur ( , ) , there are also examples of strong ctl responses being exhibited over long periods of time in the absence of appearance of mutations in the epitopes to which they are directed { , ). ctl epitope mutation during chronic hiv infection tbus does not seem to be essential for the continued persistence of virus; however, when it does occnr it may allow higher levels of virus replication, and bence promote disease progression. in the later stages of the infection, when the immune system is failing, the impact of viral variation on immune control may become more pronounced. the capacity of the immune response to evolve to focus on tmmutated epitopes as escape variants are selected may be more limited at this time. simultaneously, the rehance of the host on ctl lysis as a means of controlling virus replication and spread may be increasing, particularly if syncytium-inducing viruses resistant to control by chemokines such as rantes, miela and mip p appear. ctl escape variants may thus grow out and contribute to tbe escalating viremia. a recent paper described two examples of patients where mutations conferring evasion from a previously stable immunodominant ctl response became fixed in the viral quasispecies in the late stages of the infection ( ), providing a clear illustration that tbis can occur ctl escape viral variants may thus influence the immune system's abihty to control virus replication at all stages of hiv infection, and likely do bave an important impact on the overall disease course in at least a proportion of infected individuals. the preceding discussion illustrates tbat viral variants whicb are able to escape recognition by host ctl emerge during a number of different virus infections, and clearly have a biologically significant impact on tbe balance between virus replication and its control by the immune response in at least some of these cases. the latter include several infections that are of clinical significance in humans; it is thus important that immunebased propbylactic and therapeutic strategies to combat these infections should be designed to minimize the likelihood of ctl escape variant selection. there are examples in the literature of both prophylactic ( ) and therapeutic strategies ( ) wbich have led to the selection of viral variants able to escape recognition by the ctl response wbich was intended to mediate a beneficial effect. as described in this review, studies in both human virus infections and murine model systems have illustrated the conditions under which ctl escape viral variants are likely to emerge: this information should be considered in future vaccine/therapy design to prevent a similar outcome from occurring. given that escape-conferring mutations will not be selected for if they have detrimental effects on virus replication, and that escape mutations emerge most rapidly wben ctl pressure is predominantly focused onto a single bighly immunodominant epitope, vaccines should aim to induce broad immune responses that recognize multiple co-dominant viral epitopes, at least some of which should lie in conserved regions of viral proteins on which there are stringent functional constraints. eeptide or minigene-based vaccination strategies are probably not the optimal choice for use in such infections: even if they include a carefully chosen cocktail of epitopes that can be recognized in association with multiple hla alleles, there is a bigh probability that they will induce mono-or oligospecific immune responses in individuals of certain hla types. particularly if the infecting virus shows sequence variation compared to the antigens used for vaccination, there will be a danger that the vaccine might prime for an immune response that could be more detrimental than the immune response that may have been ehcited naturally following infection. vaccines that inclnde multiple viral antigens are preferential immunogens for the induction of beneficial multispecific antiviral immune responses. viral pathogenesis. philadelphia: lippincott-raven publishers one-step ahead of the gameviral immunomodulatory molecules viral pathogenesis. philadelphia: lippincott-raven publishers; i specific cytotoxic t cells eliminate cells producing neutralizing antibodies virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector t cells overview of herpesvirus latency viral pathogenesis. philadelphia: lippincott-raven publishers viruses use stealth technology lo escape from the host immune system nevirapine-resist ant human immunodeficiency virus: kinetics of replication and estimated prevalance in untreated patients ordered accumulation of mutations in hiv protease confers resistance to ritonavir combination therapy with zidovudine and didanosine selects for drugresistant human immunodeficiency virus type- strains with unique patterns of poi gene-mutations emergence of human immunodeficiency virus type- variants with resistance to multiple dideoxynucleosides in patients receiving 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non-dominant anchor residues the hfe span of major histocompatibility complex-peptide complexes influences the efficiency of presentation and immunogenicity of two class i-restricted cytotoxic t lymphocyte epitopes in the epstein barr virus nuclear antigen different mhc class i alleles compete for presentation of overlapping viral epitopes siliciano re, soloski mj, mhc class irestricted processing of transmembrane proteins -mechanism and biological significance major expansion of cd -i-t cells with a predominant vb usage during the primary immune response to hiv patterns of immunodominance in hiv- -specific cytotoxic t lymphocyte responses in two human histocompaiibilicy leukocyte antigens (hla)-identical siblings with hla-a* are influenced by epitope mutation sequence constraints and recognition by ctl of an hla-b -restricted hiv- gag epitope in vivo persistence of a hiv- -encoded hla-b -restricted cytotoxic t lymphocyte epitope despite specific in vitro reactivity cytotoxic t lymphocytes in asymptomatic long-term non progressing hiv- infection antigenic oscillations and shifting immunodominance in hiv- infections late escape from an immunodominant cytotoxic t-lymphocyte response associated with progression to aids in vitro selection of lymphocytic choriomeningitis virus escape mutants by cytotoxic t lymphocytes ctl escape viral variants, i. generation and molecular characterization discriminated selection among viral peptides with the appropriate anchor residues: implications for the size of the cytotoxic t-lymphocyte repertoire and control of viral infection late onset, symptomatic, demyelinating encephalomyelitis in mice infected with mhv-jhm in the presence of maternal antibody cd + t cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity cytotoxic t cell-resistant variants are selected in a virus-induced demyelinating disease ctl escape mutants cause increased mortality and morbidity ln mice infected with mouse hepatitis virus, strain jhm. keystone symposium on molecular aspects of viral immunity cytotoxic t lymphocyte responses to epstein-bair virus human cytotoxic t lymphocyte responses to epstein-barr virus infection boftow & shaw • in vivo importance of ctl escape viruses t cell responses and virus evolution: lossof hla al -restricted ctl epitopes in epstein-barr virus isolates from highly al -positive populations by selective mutation of anchor residues unusuaey high frequency of epstein-barr virus genetic variants iji papua new guinea that can escape cytotoxic t-cell recognition: implications for virus evolution epstein-barr virus isolates with the major hla b . -restricted cytotoxic t lymphocyte epitope are prevalent in a liighly b . -positive african population hepatitis b virus immunopathogenesis cytotoxic t lymphocyte response to a wild-type hepatitis b virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope chisari fv hepatitis b virus (hbv) sequence variation in cytotoxic t lymphocyte epitopes is not common in patients with chronic hbv infection chisari fv is antigenic variability a strategy adopted by hepatitits b virus to escape cytotoxic t-lymphocyte surveillance? hepatitis c viruses. in: fields bn, ed. fields virology cytotoxic t-lymphocyte responses to the hepatitis c virus in humans and chimpanzees persistent hepatitis c virus infection in a chimpanzee is associated with emergence of a cytotoxic t lymphocyte escape variant hepatitis c virus-specific ctl responses in the liver of chimpanzees with acute and chronic hepatitis c hiv specific cytotoxic t lymphocytes in seropositive individuals aids virus specific cytotoxic t lymphocytes in lung disorders high levels of anti-human immunodeficiency virus type (hiv- ) memory cytotoxic t-lymphocyte activity and low viral load are associated with lack of disease in hiv- -infected long-term nonprogressors kinetics of gag-specific cytotoxic t lymphocyte responses during the clinical course of hiv- infection: a longitudinal analysis of rapid progressors and long-term asymptomatics why can't cytotoxic t cells handle hiv? hiv-!-specific cytotoxic t lymphocytes and the control of hiv- reph cation evidence for rapid disappearance of initially expanded hivspecific cd -(-t cell clones during primary hiv infection vigorous hiv- -specific cd -i-t cell responses associated with control of viremia hiv versus cytotoxic t lymphocytes -the war being lost what can we learn about human immunodeficiency virus infection from a study of lymphocytic choriomeningitis virus? immunoirev hiv- nef protein protects infected primary cells against killing by cytotoxic t lymphocytes prognosis in hiv- infection predicted by the quantity of virus iji plasma human immunodeficiency virus genetic variation that can escape cytotoxic t cell recognition transfer of hiv- -specific cytotoxic t lymphocytes to an aids patient leads to selection for mutant hiv variants and subsequent disease progression bourgault-villada l selection of virus variants and emergence of virus escape mutants after immunization with an epitope vaccine the authors are supported by grant numhers ro i ai - (pb)and uo ai (pb and gms) from the nih and by core funding from the edward jenner institute for vaccine research key: cord- - yo rfml authors: bortolin, michelangelo; ciottone, gregory r. title: disaster medicine date: - - journal: trauma team dynamics doi: . / - - - - _ sha: doc_id: cord_uid: yo rfml millions of people every day face disasters, for example, typhoons, terrorist attacks, earthquakes, famine, civil wars, explosions, and tornadoes. disaster is defined as every event that causes serious disruption which exceeds the ability of the affected community or society to cope using its own resources. disasters are usually categorized as natural or man-made and are described using a series of steps called the disaster cycle, defined in four phases: mitigation and prevention, preparedness and planning, response, and recovery. disaster medicine is an emerging specialty that integrates the medical response to disaster with the systems of disaster management. devastating events such as natural disasters like the typhoon in the philippines ( ) and the earthquake in haiti ( ), intentional events like the terrorist attack in new york ( ) , and the sarin attack in tokyo's subways ( ) demonstrate that disasters are both unpredictable and ubiquitous. disaster is defi ned as any event that causes "a serious disruption of the functioning of a community or a society involving widespread human, material, economic or environmental losses and impacts, which exceeds the ability of the affected community or society to cope using its own resources" [ ] . considering that any number of different events could hit a population at any time, there is no place on earth completely immune to disasters. therefore, disaster medicine was created as a broad specialty grounded in emergency medicine, but utilizing the skill sets of other surgical and medical specialties, and only able to become operational in combination with the systems supported by disaster management. for example, during an earthquake, several specialties are involved in the response and immediate care of the victims: emergency physicians, surgeons, anesthesiologists, and orthopedics; however, other subspecialties are also required in the ongoing care of victims. these include nephrologists to treat acute renal failure related to crush syndrome, both during and following the event, and psychosocial and rehabilitation specialties for continued care. these medical and surgical specialists are only able to perform their roles under the umbrella of disaster management. without being enabled by the logistics and operations capabilities seen in a large-scale disaster response, these specialists would not be functional. outbreaks and human immunodefi ciency virus (hiv) are just two examples of natural disasters that for several reasons can spread quickly, ravaging entire communities. the spanish fl u in the early s caused more than million deaths, and severe acute respiratory syndrome, (sars) that arose from china in , resulted in thousands of death in that part of the world and concern for a global pandemic. another virus that affl icts and kills millions of people, particularly in the poor areas of africa and developing countries, is hiv. it is estimated that from the s, hiv has caused more that million deaths around the world. an estimated % of natural disaster-related deaths occur in developing countries (world bank, - [ ] . man-made disaster is defi ned as any event that is caused by the activity of human beings. explosions, building collapse, civil wars, and nuclear accidents are some examples. the collapse of the hyatt regency hotel in kansas city in is one such example. investigations found that the cause of the collapse was due to an engineering problem. there were more than deaths and more than casualties from that disaster. the transportation industry is commonly involved in incidents that cause large numbers of injured and dead. in in eschede, germany, a high-speed train derailed causing fatalities. other industrial sectors, such as the chemical industry, have also been involved in man-made catastrophes. in december in bhopal, india, more than half a million people were exposed to methyl isocyanate. in the immediate phase after the leak, almost , people died. the indian government has calculated that this event has caused over the years almost , casualties due to lingering effects of the chemical exposures [ , ]. the / terrorist attack in new york and the oklahoma city bombing ( ) are also man-made disasters. it is important to recognize and underline that disasters also result from war, the conduct of repressive regimes, the use of sanctions, as well as economic and social policies, particularly in developing countries [ ] . the syrian civil war arose in the and has caused more than , deaths and more than . million refugees since [ ] . disasters and the response to them follow a pattern called the disaster cycle, which is defi ned in four phases: mitigation and prevention, preparedness and planning, response and recovery. mitigation and prevention involve measures designed either to prevent hazards from occurring or to lessen the effects of the disasters [ ] . these measures involve multiple different agencies and commissions, for example, policymakers introducing regulations regarding the storage, transportation, and disposal of chemical substances. another example of mitigation is to empower a public health system to monitor and conduct surveillance for infection diseases and at the same time introduce rules regarding health screening at the borders. the importance of the mitigation phase is to avoid disaster or to reduce the impact on the population. it is clear if we compare the earthquake in haiti, with a magnitude of . , and similar earthquakes in japan where despite the same magnitude the number of dead and injured were more limited, that we see the effect of a disaster is often dependent on the underlying condition of the area affected. for decades, japan has introduced strict building codes that follow seismic regulations. nevertheless, it is not possible to fully mitigate against all disaster events. for instance, the earthquake in the pacifi c ocean produced a tsunami that hit the east coast of japan and caused severe damage, in particular a failure of the nuclear plant in fukushima, with release of radiation that affected the local community. the preparedness and planning phase includes all activities conducted on an ongoing basis, in advance of any potential incident. preparedness involves an integrated combination of assessment; planning; procedures and protocols; training and exercises; personnel qualifi cations, licensure, and certifi cation; equipment certifi cation; and evaluation and revision [ ] . the fi rst step of preparedness is defi ning what events are more likely to hit a community. the hazard and vulnerabilities assessment (hva) is a way to objectively risk-stratify those hazards that are more likely to strike a given community. the hva takes into account different events: natural, man-made, and cbrn (chemical, biologic, radiological, and nuclear). the output from an hva prioritizes the risks to which a population is most susceptible and should therefore be prepared for. after the hva, it is possible to then establish standard operating procedures (sop) and the emergency operations plan (eop) for the community or hospital. it is a good rule that the eop adopts an allhazards approach to preparedness, with annexes and appendices specifi c for every type of probable event [ ] . an important part of the preparedness phase is training. in particular, every healthcare professional must be trained when to activate a disaster response and their own specifi c roles and responsibilities within the framework of the response. the typical drills commonly used are tabletop and full-scale exercises. they are important also to identify shortfalls, bottlenecks, and gaps in the eop. the staff should take part in the training, and the eop should be tested, reviewed, and updated at least once per year. response is the phase in which agencies and sections with responsibility to deploy to disasters activate their emergency response plan as a result of specifi c threats or situations and can incorporate local, regional, and federal response agencies [ ] . the response is conducted through the intervention of several agencies and must be fl exible and adaptable for any type of event. it is important to immediately establish a response framework with a unifi ed command structure that establishes a chain of command, control, and coordinate the resources, in terms of staff, stuff, and structure. the incident command system (ics) provides a structure to enable agencies with different legal, jurisdictional, and functional responsibilities to coordinate, plan, and interact effectively on scene [ ] . the medical response is provided at the scene by the emergency medical services (ems), with triage, treatment, and transport to the hospitals. ems plays a crucial role in the immediate phase of the response to disaster. the response must be quick and effective on the scene as well as in the determination of hospital destinations for every patient, to guarantee an appropriate standard of care and to avoid bottlenecks and congestion at hospitals. rarely, ems is able to triage, treat, and transport every single patient from the scene. often some casualties reach the closest hospital on their own, giving rise to disruption in the chain of triage and to the hospital. the ability of a healthcare system to suddenly expand its capacity beyond normal services to meet the increased demand for qualifi ed medical staff and services during a large-scale event is defi ned as "surge capacity" [ ] . the surge capacity depends on the features of the healthcare system, but also by an effective eop and training of the staff. the post-impact period revolves around disaster recovery in which the goal is to eliminate impairment caused by a disaster and rebuild communities and infrastructures [ ] . also this phase involves several agencies and may be long lasting, ranging from weeks to years. people affected by a tremendous disaster must face a long recovery phase. survivors from the september , , terrorist attacks on the twin towers not only had immediate treatment in the fi eld and in the hospitals that day, but their treatment has continued for years. the majority of people exposed to disasters do well; however, some individuals develop psychiatric disorders, distress, or risky behaviors such as an increase in alcohol or tobacco use [ ] . the department of health and human services spent months and years following the / attacks and has gone on to provide health care, both physical and mental, to those who were, and continue to be, affected and in need [ ] . the recovery phase often also involves rescue workers as their exposure to the traumatic event has a severe impact on their mental health. studies confi rm that rescue workers are prone to have diseases or documented behavioral health disturbances following events. for example, several articles describe how the acute and prolonged exposures were both associated with a large burden of asthma and posttraumatic stress (pts) symptoms years after the / attack. during an incident, the response must be effective and efficient. to achieve this, and thereby ensure that the best care possible is rendered to victims, it is fundamental to have a well-prepared and organized system. the incident command system (ics) is a standardized, on-scene, all-hazards incident management concept and allows its users to adopt an integrated organizational structure to match the complexities and demands of single or multiple incidents without being hindered by jurisdictional boundaries [ ] . the ics was developed in the s in california to manage, command, and control fi re brigades during their operations to extinguish wild fi res. shortly after that, it was adopted by ems and other agencies, as well as endorsed by the u.s. department homeland security as a fundamental element of incident management. the ics is used for all events and is modifi ed depending on the size of the incident. its goal is to manage and to resolve the incident with an effi cient use of resources while protecting all persons involved. the ics is a modular and fl exible organizational system that can be standardized for multiple uses. the ics is modifi ed according to the size and complexity of the incident, specifi cs of the hazard, environment effected by the incident, the incident planning process and incident objectives (ics expansion and contraction) [ ] . the ics establishes an incident commander (ic), who is in charge of all the activities regarding the incident, a chain of command, and unifi ed command between the agencies. the priorities of the ic are three: the safety of the casualties and the rescue team, incident stabilization, and property preservation. every incident must have an incident action plan (iap) that establishes incident goals, operational period objectives, set activities, and the response strategy defi ned by the ic during response planning [ ] . the ic manages and carries out his responsibilities with three features of command that are important for every role within the framework of the ics: the chain of command, the unity of command, and the span of control. the chain of command is a key part of the ics and is defi ned as a structure with a clear line of authority. the unity of command infers that every responder knows without question who his/her supervisor is. span of control describes the typically - people a supervisor directly leads. during a disaster, it is extremely important to establish a unifi ed command, because it enables all responsible agencies to manage and coordinate an incident together by establishing a common approach and a single iap. it permits the integration of staffi ng and shared facilities, with everyone having the same objectives and not replicating efforts [ ] . the iap describes activities, responsibilities, and the communication procedures. this system is fundamental to avoid confusion and lack of communications. adequate and redundant communication systems are very important during the response to disaster. it is essential that the ics use common terminology and integrated communications among agencies and establish precise ways of communications. the communication systems should be: interoperable between agencies; reliable to function in the context of any kind of emergency; portable, built on standardized radio technologies, protocols, and frequencies; scalable as the needs of the incident dictate; resilient to perform despite damaged or lost infrastructure; and redundant to enable the use of alternate communications methods when primary systems go out [ ] . the ics is supported by a command staff that includes a safety manager, a liaison offi cer, and a public information offi cer. it is organized into four sections, the general staff, which supports the ics: operation, planning, logistics, and fi nance/administration. the operation section is in charge of managing all the tactical operations on the scene; tactical operations include fi re brigades, ems, and every agency required for the incident. the planning section is responsible to draft the iap; to get, receive, elaborate, and share information; and to track all the resources. the logistic section provides the supplies, needs, and the facilities and supports the personnel with food, water, and fi rst aid. the fi nance/administration section is in charge of tracking all the costs and to negotiate and supervise contracts. these sections, like the ics, are modular organizations and can be further expanded into: units (the organizational element with functional responsibility for a specifi c incident planning, logistics, or fi nance/administration activity), divisions (only for operation section, used to divide an incident geographically), groups (only for operation section, established to divide the incident management structure into functional areas), and branches (used when the number of divisions or groups exceeds the span of control and can be either geographical or functional for major aspects of incident operations) [ ] . triage in a disaster event places casualties in four classes: black (or expectant), red (or immediate, priority ), yellow (or delayed, priority ), or green (or minor, priority ) in agreement with the severity of the injuries. when sorting casualties, it is important to give immediate medical care to critical patients that have a chance of survival with prompt, advanced treatment. in minor patients and patients who are so severely injured that they have very little chance of survival treatment is delayed. the goal is to provide the greatest good for the greatest number of patients, forcing the triage offi cer to decide whether the chance of a patient surviving is so low in comparison to the burden such care would place on the medical system that the patient must be consigned to the "expectant" category (dying; little or no treatment) [ ] . the concept of triage must be seen in a wider context and is composed of the following elements: rapid evaluation of all disaster victims, assessment of the nature and severity of the injuries and its consequences on the vital functions of the casualties, and categorization of the casualties, resuscitation, stabilization, and conditioning for transport, distribution, and evacuation of the casualties [ ] . triage is a quick and dynamic process. this means that it must be repeated often and at every moment in which a new healthcare professional takes control of a patient, for example, during transport, at arrival at the hospital, or if there is the suspicion that the state of the patient has changed. triage will be discussed further in the chap. . the "second hit" is a classic tactic and pattern seen in terrorist attacks. it is defi ned as second incident caused by the terrorists, following the fi rst event, with the goal of striking the fi rst responders that are on scene. typically, it is a second explosion close to the scene and often more powerful than the fi rst detonation. this is because the intention is to create casualties during the fi rst blast and to attract people to the scene and then striking them with a larger detonation. this achieves the goal of a terrorist attack: to cause additional chaos thereby delaying the response and causing great physical and psychological impact on the populations and on the rescuers. an example of this was the terrorist attack in in bali. two bombs detonated within a short period of time. the fi rst was concealed in a vest worn by a suicide bomber. the second charge was in a minivan about m away when the fi rst explosion happened. the force of the car bomb was enormous [ ] . terrorist attacks are very challenging and diffi cult to manage, because they are designed to create loss of life and property damage, disruption of the agencies involved in the response, and fear and harm to the population. the israel response system is very seasoned to terrorist attack and has specifi c guidelines and protocols in case of attacks to avoid damage from a "second hit." traditionally, medical teams do not enter the scene of the explosion until it is deemed safe by police or army personnel. with many of the terrorist attacks in israel, a secondary explosion or bomb is set off timed to cause additional injuries to the emergency personnel and bystanders responding to the primary event. however, because time is critical, often ems does not wait for such security clearance and attempts to rapidly remove the casualties from the immediate vicinity of the initial event. the only medical care given before this initial evacuation is external hemorrhage control [ ] . they apply the "scoop and run" approach on the scene (minimal resuscitation on the scene and immediate transportation to the trauma center) to clear the casualties from the area of the event but in the meantime minimize the risk for the rescuers. therefore, in case of a terrorist attack, it is imperative to maintain the role of the incident commander, the coordination between the agencies, and training of the rescuers to guarantee the safety of all the workers involved in the response. the united nations offi ce for disaster risk reduction, terminology world bank seminar on the role of local governments in reducing the risk of disasters man-made disaster and development-the case of iraq united nations high commissioner for refugees, stories from syrian refugees community emergency preparedness: a manual for managers and policy-makers guide for allhazard emergency operations planning public health and disasters, chapter department of homeland security national response. national response framework (nrf) das konzept von "surge capacity" im katastrophenfall notfall rettungsmed koenig and schultz's disaster medicine: comprehensive principles and practices individual and community responses to disasters testimony before the committee on oversight and government subcommittee on government management, organization and procurement united states house of representatives, on / health effects: hhs's monitoring and treatment of responders federal emergency management agency "ics- . a: introduction to ics emi course number: is ". student manual version ics- : single resources and initial action incidents" emi course number: is instructor guide version offi ce of the assitant secretary for preparedness and response. what is an incident action plan? ics- intermediate ics for expanding" student manual is- .a: national incident management system, an introduction federal emergency management agency. introduction to the incident command system (ics ) student manual mostly dead: can science help with disaster triage? triage unit for emergency preparedness, committee for disaster medicine studies. the terror attack on bali medical lessons from terror attacks in israel • disaster is defi ned as any event that causes serious disruption of society which exceeds the ability of the affected community to cope using its own resources. • disasters are usually categorized as natural or manmade, and response to them follows the same pattern, called the disaster cycle, defi ned in four phases: mitigation and prevention, preparedness and planning, response, and recovery. • the incident command system (ics) is a standardized and modular organization, on-scene, and allhazards incident management concept and allows its users to adopt an integrated organizational structure to match the complexities and demands of single or multiple incidents. • the word triage means "to categorize, to sort." the purpose of triage in a disaster event is to catalogue the casualties in agreement with the severity of injuries. sorting casualties is important to give immediate medical care among critical patients that have a chance of survival. the goal is to provide the greatest good for the greatest number of patients. • the "second hit" phenomenon is a classic tactic and pattern of terrorist attacks. it is defi ned as a second incident caused by the terrorists, a little bit later than the fi rst event that is geared to injure the fi rst responders that are on the scene. key: cord- -pztn authors: roach, jeff; kemish, ian title: bali bombings: a whole of government response date: - - journal: tourism in turbulent times doi: . /b - - - - . - sha: doc_id: cord_uid: pztn nan which provide services to the community. local government and voluntary organisations also play important roles, as both groups have close links with the community. while no legislation requires the australian government to act in emergencies, it has always accepted a responsibility to assist the states and territories where their resources are insufficient or inappropriate for the situation. a number of australian government disaster response plans are administered by emergency management australia (ema). the most important for the purposes of this chapter are the commonwealth government disaster response plan (comdisplan) for physical assistance to the australian states and territories, and the australian government overseas disaster assistance plan (ausassist-plan) for assistance overseas. in discussing the australian government's responses to crises, it is useful to note the political context. with agencies taking their cues from ministers, the whole of government response will be conditioned by the strength of political decisiveness and unanimity among ministers. the response to the tragic terrorist attack in bali on october underscores this observation. from the outset, the prime minister's instructions to senior officials were decisive: the government's response needed to be comprehensive and effective. issues concerning resources could not be allowed to constrain the policy response -these matters could be addressed later. there was strong bipartisan support for the government's approach. reflecting these decisive political instructions, the government established explicit and appropriate chains of command. given its responsibility for consular services to australians in distress overseas, the department of foreign affairs and trade (dfat) was tasked to coordinate the whole of government response to international aspects of the crisis. domestically, the department of family and community services (facs) coordinated government policy and delivery of assistance to australians and their families affected by the crisis. a clear lesson from bali was the extent to which overseas events can resonate at the local community level, underlining the importance of domestic and state/territory agencies being activated early in response to a major overseas crisis. the decisive establishment of clear roles and chains of command in this case contrasts with comments by mcconnell and stark ( ) concerning the response by the uk ministry of agriculture, fisheries and food to the outbreak of foot and mouth disease (fmd) in february . the authors describe the response as "suffering from an institutional malaise and a fragmentary civil service, incapable (at least in the early stages) of providing a 'joined-up' response to match the scale of the crisis"(p. ). in the case of bali, two 'hub and spokes' models were used to coordinate the whole of the government response. dfat took on the 'hub' role in coordinating the interagency 'spokes' response to the international aspects of the crisis. at the same time, facs took on another 'hub' role, coordinating the interagency 'spokes' response to domestic aspects of the recovery. the two clusters of 'hub and spokes' worked alongside each other, attending each other's meetings where necessary, to provide a comprehensive overall response. the 'hub and spokes' arrangement worked well to draw together key agencies and players to share information and coordinate policy responses. this approach represented the combination of two interdepartmental committees, each chaired by a line agency. these arrangements provided the context for effective consultation, rapid decision making, close attention to the implementation of decisions and action to address new or unforeseen difficulties. within each committee, clear directives identified the roles and responsibilities of respective agencies, thereby ensuring that mandated issues were resolved early. figures . and . illustrate these institutional arrangements. while a 'hub and spokes' structure could be used in a range of crisis scenarios where there is a distinction between international and domestic issues or another clear thematic division, ultimately portfolio departments should ensure that their own crisis plans consider the appropriate linkages and structure for different scenarios relevant to their particular portfolio or industry structure. a number of observations can be made about this approach. first, within each 'hub and spoke' there was a clear division and respect for the different mandates of respective agencies. rather than normal bureaucratic rules being abandoned, there was a strong appreciation figure . : international aspects: crisis phase -interagency emergency taskforce. that the simplest and most direct means of achieving goals was to use the appropriate agency and established channels. efforts at short-cutting, even where motivated by a noble desire to expedite an outcome, were ultimately more likely to result in delays and confusion. the use of traditional channels of liaison and coordination means that new relationships do not need to be established in the tumult of a crisis situation. this provides a higher degree of comfort for downstream organisations, such as state authorities, which are involved in delivering a specific response. coordination by dfat for international issues and facs for domestic issues reflected the overall responsibilities of the two departments: dfat has responsibility for consular services overseas, assisting in responding to the deaths of about australians overseas each year (dfat, ) ; facs supports australian families in need (see http://www. familyassist.gov.au/). both dfat and facs held daily (twice daily in the initial aftermath of the attacks) interagency taskforce meetings, which drew major stakeholders together to share information and coordinate policy responses. australian public service (aps) employees attending these meetings were at a senior level, meaning they had the authority to make on-the-spot decisions on behalf of their agency. the bali response brought together a diverse range of agencies, many with little prior experience in working with each other. however, participants worked cohesively and collaboratively throughout the period. they were hindered little by the different departmental cultures and work practices which can be found in the aps. while it can be argued that organisational culture is the key to the whole of government success (this issue is explored further in "culture and capability", in management advisory committee, connecting government: whole of government responses to australia's priority challenges, ), a crisis situation elicits a high level of goodwill which appears to eliminate cultural barriers. one of the key lessons from the bali attack was the decision to establish an overarching national plan, to be coordinated by ema, to provide a framework for coordination to: • clarify the roles of agencies and non-government organisations in crisis responses; • review links between australian government and state disaster plans; and • identify and rectify any gaps in interagency coordination arrangements. the development of the national response plan for overseas mass casualty events will provide the blueprint for federal and state contingency plans to be examined further and tested against a variety of different scenarios. at the time of publication, the preparation of this plan was well advanced. while the government's response to bali was pursued through the parallel processes of the 'hub and spokes' models, there was no competition or mandate clash between the two structures. in the days after the bali attack, as the evacuation of injured australians was completed, remains stabilised and positive identification of the deceased began, the transition from crisis phase to recovery phase was well advanced. in fact, it was important that activities associated with the recovery phase were conducted in tandem with activities associated with the crisis response. while there was no clear distinction in the transition from one phase to the next, the emphasis of policy making increasingly shifted from international to domestic aspects. in driving response from crisis to the recovery phase, a high degree of discipline is required to ensure that decision making is followed through by rigorous implementation and follow-up. coordinated whole of government policy making and implementation is integral in driving towards the recovery phase. within the facs bali response taskforce, core issues were addressed in each daily meeting, with recent progress and forward planning reported against each. the core issues were: • public communication; • financial support; • domestic health services; • disability issues; • counselling; • return of effects of deceased victims to next-of-kin; • community harmony; • community support; • rural issues; • intergovernmental welfare issues; • role of airlines; • insurance coverage; • domestic economic issues; • international issues; and • interaction with other disasters. the daily review of these issues ensured that policy outcomes were closely monitored and driven forward. similarly important is the need for crisis managers to rise above the maelstrom of the moment and obtain a more strategic view of the overall policy response. one way to do this is to create a regular opportunity for key decision makers to briefly canvass what might be the policy and media issues of the day. this discipline assists in ensuring that decision making continues to strike a balance between the proactive and the reactive, looking beyond the issues of the moment. in the case of bali, these approaches meant that within days of the attack the focus of government decision making had moved smoothly from the international to the domestic, from crisis to recovery phase. effective crisis management is founded on good preparation. this should include: the negotiation of protocols with likely key participants and stakeholders; the maintenance of the crisis infrastructure to ensure that it is ready to use at any time (this includes fundamentals such as after-hours contact lists of key employees from other agencies); and the appropriate training and development to ensure that people can fulfil key roles, whatever the dimensions of the crisis. human resource issues also need careful attention -a sustained crisis has the potential to burn out key people. one of the lessons of the australian government's fmd simulation, exercise minotaur (see http://www.affa.gov.au/exerciseminotaur), was the need for agencies to look at human resource capacity in a number of key areas, particularly that of skilled and trained technical employees. experience indicates that the long-term nature of individual and community recovery will also place significant strain on human resources. a further recurring issue is the need for a compatible communications system that allows information to be shared quickly between agencies without the need for special handling. the bali response, for example, showed that two agencies (dfat and facs) that had rarely communicated with each other before found electronic communications difficult. it is critical that agencies prepare for a whole of government crisis during normal business operations. as part of this, agencies need to develop -and test -contingency plans that map out how the agency will respond in a range of different scenarios. such plans require regular review, monitoring and testing to ensure that they can deliver against their stated goal. desktop and trial exercises are important, both within agencies and across the whole of government. furthermore, after a crisis, it is critical that agencies undertake a formal review and, if required, further reform of response plans subsequent to a crisis. this needs to be undertaken on a whole of government basis and it is critical that agencies come together to pool lessons learnt and negotiate reforms to their own departmental processes. the australian government comprehensively tested its response systems in through exercise minotaur. the breadth of the simulation was impressive, with the scenario testing diverse issues such as animal health responses, trade advocacy skills and even consular dimensions (see http://www.affa.gov.au/exerciseminotaur). the simulation was conducted over days in september , after months of planning. more than people from a range of government and industry agencies were formally involved, with the simulation overseen by a panel of evaluators and observers. another part of forward planning is the development of financial management protocols to give aps employees the discretion to authorise action. once a crisis has begun, financial systems must be able to deliver appropriate resources to enable decision makers to quickly meet policy priorities, while also satisfying australian government financial guidelines. this lesson is illustrated by the issuing of ex gratia payments by the australian government that are covered neither by legislation or regulation. such payments require written authority, normally from the prime minister. however, australian government agencies need to understand how these payments will be handled where a decision and announcement has been made at the ministerial level, but corresponding authorisations are not yet available. while the lag may be only hours, announcements about government assistance will trigger an immediate response from the community. departmental secretaries may need to develop a consistent approach to flexible bridging arrangements to ensure that financial assistance can be provided quickly in times of crisis. some private sector corporations have taken testing one step further by using 'internal assassins' -well-versed employees who devise worst-case business disruption crises to test management systems (see klinger, ) . testing such as this on a whole of government basis would mean crisis management responses could be reviewed to ensure that existing protocols and practices keep pace with changes in the threat environment, technology and political imperatives. exercise minotaur enabled a thorough testing of the fmd coordination arrangements. this simulation also highlighted the importance of response protocols that include 'fire drills' to make sure that all systems are working well, including a managed approach to public communication. an approach like this moves beyond frameworks and standards, and puts in place specific action pathways with which all players can become familiar. the period between crises also provides an opportunity to review possible jurisdictional barriers to an effective crisis management. there are a number of possible sources for such difficulties. the first lies in the balance of powers between the australian government and state/territory governments, with divisions of responsibility established by the constitution. protocols are required to ensure that the whole of government response at the australian government level is matched by seamless coordination at the state/territory level. experience has shown that e-mail should be used with some caution for priority communication during crisis management. systems need to be robust enough to cope with the increased demands of a crisis and employees need to regularly monitor e-mail to ensure that responses are not delayed. the potential for information management issues to arise during a crisis also needs to be considered. state and australian government agencies collect an array of data about individuals. however an agency's obligations under the privacy act (see http://www. privacy.gov.au/) will impact on what information can be shared among agencies. the privacy act can allow pragmatic decisions in times of national disaster. however, the need for personal data to be protected means it is difficult to collect information from different authorities to support ongoing whole of government work during a recovery period. agencies may find it useful to develop a common approach to understanding the way in which the act applies to their operations in a crisis. without this common understanding, different interpretations of the act can lead to inconsistent policy formulation and agency responses during the crisis. approaches to whole of government crisis management in other countries can also provide learning opportunities for australia. the uk government has established rapid deployment crisis teams which can move within hours to lead the government's response to an overseas crisis (see response of the secretary of state for foreign and commonwealth affairs to the twelfth report of the foreign affairs committee session session - session , . the exact composition of the teams, possibly drawn from a range of agencies including foreign affairs, law enforcement and aid delivery, would depend on the nature of the crisis, from a natural disaster to a terrorist attack. the capacity of the teams to work together is likely to hinge on joint training opportunities. the pre-crisis development of a solid understanding between team members of each other's responsibilities and portfolio mandate would be vital. lessons learnt through crisis management can also be applied to other whole of government work. all agencies address business continuity issues and lessons learnt here can be disseminated throughout the aps using reports such as this one. times of crisis provide a litmus test of a government's capacity to work cohesively to convey information, extend medical, financial and counselling support and provide reassurance and leadership to its citizens. community expectations are influenced by a government's record in responding to previous crises, as well as by media commentary, which closely shadows every government statement and action. the media impact on driving community expectations and turning public opinion cannot be underestimated by crisis managers (beirman, ; wilks & moore, ) . as the crisis management response unfolds in the context of community expectations, government messages can be roughly divided into two categories: • educational messages that seek to reshape community expectations in cases where expectations exceed the power and authority of the government. • reassuring messages that confirm that the government response will be generous and equitable. getting the balance right in blending these messages requires sophisticated and wellintegrated public affairs management. the importance of public affairs management in educating and shaping community expectations was underscored in the response to the bali bombings. in the aftermath of the attack, there was considerable public anguish about the disaster victim identification process. the indonesian government implemented a positive identification process, in line with international norms and protocols. although undertaken swiftly, the collection of information about victims from australia meant that the identification process could not be undertaken immediately. this generated anxiety within australia. calls were made for the australian government to assume responsibility in bali, thereby overriding indonesian sovereign responsibility for its coronial processes. others suggested that australia should encourage the indonesians to set aside international norms, thereby running the risk that a less-rigorous identification process might lead to a serious and tragic error. given the intensity of media coverage of the bali attacks, the educational message was difficult to advance. however, primary agencies, such as the federal and state police services, dfat and coroners, worked to send a single and simple message: that the positive identification process, which was being properly implemented by the indonesian government, was the only appropriate course. media commentary and community expectations shifted fairly quickly towards a clearer understanding of the issue. reassuring messages to the community were delivered swiftly in the bali example. given a clear mandate to support australians affected by the bombing, facs established a taskforce that arrived quickly to assist the community on a range of issues: emergency medical treatment, assistance for family members to visit loved ones at interstate hospitals, the establishment of family liaison officers to work one-on-one with those affected and long-term packages of support. experience has shown that a case management approach can provide effective liaison and support for the affected families. this should be based on clearly defined agency roles and responsibilities and be provided by appropriately trained employees. determination of the level of support provided to victims and their families required careful judgement. issues of precedence were considered as governments frequently offer emergency assistance packages in response to natural and other disasters. there was also a need to be clear about the extent of flexibility around the application of the guidelines which provided assistance. this was important to ensure that the government's approach was not criticised for being either too strict or too lax. clearly, this balance was struck, as media treatment and the community's response to assistance measures were uniformly positive. it is important to carefully consider the full range of community needs in the aftermath of a crisis. as well as financial assistance, people affected need information and will naturally turn to media or other sources to meet this need. the bali experience showed also that people directly affected crave information over emotional support, at first. over time, as the implications of the crisis become clearer, community need will, however, turn to counselling and social support. nevertheless, this switch from information to support needs to be judged carefully and timed correctly to ensure that the government's communication efforts are appropriately formulated. in the aftermath of bali, facs established a newsletter for affected families. the newsletter conveyed the government's key messages and ensured that the messages were tuned to the emotional and information needs of families at different times. a major challenge in informing the community is to make sure every agency is giving out the same message. this is difficult in any whole of government task and more so in the fast-moving environment of crisis response. while web-based information is an excellent way of providing a suite of information with links to partner agencies and other relevant sites which can be accessed at all hours, some caution is needed as some areas of australia have difficulty opening and downloading some sites. caution also needs to be used to ensure that websites are not the only source for information. in the bali response, a number of different agencies needed to contact families to provide information on different elements of the government's response. dfat consulted families about whether their loved ones had told their family that they were safe , while police were in touch with families to seek material for the victim identification process, and centrelink employees provided information about government assistance packages. while each agency provided contact details about where cross-portfolio questions could be directed, none was initially able to answer queries from a whole of government perspective. however, a common set of questions and answers was quickly developed to meet this need. one option to ensure that messages are conveyed consistently and to avoid multiple agencies contacting families in times of already high stress is to delegate authority to a single agency to represent the australian government. in planning for an approach like this, agencies would benefit from regular contact with each other during normal business operations (e.g. prior to bali, contact between dfat and centrelink occurred only rarely). this would assist people to understand other corporate cultures and the agency's core priorities, roles and responsibilities. the handling of media briefings in times of crisis is also important. in some cases, a media briefing might be more appropriately handled by departmental employees, given technical or other specific knowledge required on a subject. an example of this was the decision during the severe acute respiratory syndrome (sars) outbreak to use the commonwealth chief medical officer to lead media briefings. given the technical complexity of many potential crisis triggers (e.g. animal disease) there may be value in establishing a protocol to guide when departmental employees, rather than ministers, should lead public communications. work to integrate the whole of government public affairs management has been taken forward by the attorney general's department under national counter-terrorism committee jeff roach and ian kemish following the attacks, the department of foreign affairs and trade pursued almost whereabouts inquiries triggered by families registering concern that their loved ones may have been in bali at the time of the attack arrangements. this is aimed at ensuring that if there is a domestic terrorist crisis, careful public affairs management will reduce the scope for both rumour to replace information and for multiple or contradictory statements by different agencies. these measures rest on five key principles: (i) the community has a right to be informed and information should only be withheld if its release would be to the detriment of the national interest, including operational security. (ii) public information management and media liaison can play a key role in national security operations, and therefore must be strategic, accurate and undergo all necessary clearances. (iii) agencies must not comment on another agency's area of responsibility without first seeking appropriate approval from the agency in question. (iv) all agencies have a responsibility to ensure that they have a single point of coordination contact, as well as appropriately trained media liaison employees and resources to respond to any national security incident. (v) it is the responsibility of all agencies to ensure that they have clear coordination processes within their own agencies, with their ministerial offices and across agencies. in order to embed these principles in organisational behaviour, training workshops are being run which bring together public affairs employees from various governments. these workshops will not only improve skills but also build links between media staff to ensure that a collaborative approach is taken during a crisis. the community has high expectations about the substance of the australian government's actions during a crisis. it can be valuable to use strategic partnerships that agencies have developed in normal day-to-day business operations to assist in this response. in the bali response, a company with long-standing experience in mass casualty incidents, kenyons international, was contracted within hours of the attacks to manage the repatriation of all deceased australians on behalf of the australian government. qantas also agreed to put on additional flights to repatriate the many hundreds of australians who wished to leave bali immediately. from the non-government field, the red cross agreed to coordinate all voluntary requests for assistance from the community, drawing on its long record in the field of international humanitarian issues. the red cross was also a member of the domestic taskforce and provided important support to individuals such as non-australian citizens who were affected by the bombing but were unable to access assistance provided by facs. volunteers too may need to be integrated into the overall crisis response effort. whether planning for crises at home or overseas, agencies need to include the capacity and desire of australians on the ground to play a constructive role in the crisis response. clearly, with the degree of volunteer support unknown until the crisis hits, this role needs to be carefully scoped and defined. the likelihood of unaffiliated volunteers appearing should also be addressed. if there is no role for volunteers, this willingness to assist could translate into understandable frustration -most likely vocalised -about the australian government's handling of the crisis. the department of agriculture, fisheries and forestry has recognised in developing its fmd plans the need to harness local communities, given their pivotal role in providing additional, appropriately qualified human resources to any fmd emergency. ongoing consultation with the community is also important. once a crisis has begun, it is also important that the affected community is involved in, and has a sense of ownership of, their own recovery. consultation should also occur before a crisis, as part of an agency's contingency planning arrangements. there are some useful examples of how this consultation can occur. over recent years, the government and livestock industries have reached a comprehensive agreement on the sharing of costs in dealing with outbreaks of animal diseases (see department of agriculture, fisheries and forestry, ) . the agreement was a landmark as it established a positive partnership of responsibility and decision making involving industry. in conclusion, the following lessons can be learned from the bali bombings for a whole of government crisis management response: • plan early and test the plan; • establish clear leadership; • define roles of all players early; • use formal chains of command; and • ensure strong public affairs management. restoring tourism destinations in crisis: a strategic marketing approach. sydney: allen & unwin. department of agriculture, fisheries and forestry leadership and terror connecting government: whole of government responses to australia's priority challenges foot and mouth : the politics of crisis management response of the secretary of state for foreign and commonwealth affairs to the twelfth report of the foreign affairs committee session risk management for tourism in the asia pacific region. southport: cooperative research centre for sustainable tourism ian kemish was head of consular branch at the australian department of foreign affairs and trade at the time of the bali attack. he was responsible for the overall management of the commonwealth's response to the attacks for which he was inaugurated as a member of the order of australia (am). he is currently the head of the international division in the department of the prime minister and cabinet. jeff roach was director of the consular information and crisis management section at the dfat at the time of the bali bombing. he was responsible for coordinating the activities of australian government agencies in response to the attacks, and was awarded an order of australia medal (oam) for his work. jeff is currently posted with the australian embassy in paris. key: cord- -ztp w yh authors: land, walter gottlieb title: cell-autonomous (cell-intrinsic) stress responses date: - - journal: damage-associated molecular patterns in human diseases doi: . / - - - - _ sha: doc_id: cord_uid: ztp w yh in this chapter, the role of cell-intrinsic stress responses is examined which include autophagic processes, the oxidative stress response, the heat shock response, the unfolded proteins response, and the dna damage response. autophagy (macroautophagy, microautophagy, and chaperone-mediated autophagy) is a self-digestive process in response to environmental stress to eukaryotic cells, by which cytoplasmic components are delivered to the lysosome for recycling and degradation. the oxidative stress response is directed against any oxidative stress and is mediated by antioxidative defense systems including antioxidant enzymes such as superoxide dismutase, detoxifying enzymes such as glutathione peroxidase, and energy-dependent efflux pumps. the heat shock response is induced upon exposure of cells to any stress condition and characterized by emission of heat shock proteins which operate as damps to maintain and restore homeostasis. the unfolded protein response is induced by any stress of the endoplasmic reticulum that is perceived by three sensor molecules. under remediable endoplasmic reticulum stress conditions, the sensors trigger signalling pathways to resolve this stress. however, in severe irremediable endoplasmic reticulum stress, the unfolded protein response may lead to pro-inflammatory and pro-apoptotic responses resulting in regulated cell death. finally, the dna damage response is induced by any dna damage that occurs in a variety of exogenous and endogenous conditions. when successful, this stress response leads to dna repair and is associated with the emission of various damps which contribute to restoration of homeostasis. when unsuccessful, the dna damage response, like the unsuccessful unfolded protein response, can result in regulated cell death, either in form of apoptosis or necrosis. together, the ultimate goal of all the stress responses is to maintain cellular homeostasis and ensure cell integrity. when they fail, the incidence of regulated cell death is frequently observed. as comprehensively described in part ii, prms are specifically involved in the recognition of mamps and damps. as will be discussed in part vi, each of these recognition receptors can trigger distinct signalling cascades in innate immune cells that modify their gene expression to create and execute efferent innate immune responses that involve ( ) production of inflammatory mediator substances such as cytokines and chemokines, ( ) phagocytosis, and ( ) cytotoxicity, as well as, as described in part viii, may elicit and shape antigen-specific adaptive immune responses. beyond this well-characterized mamp/damp engagement of prms leading to a variety of downstream efferent cellular and humoral responses, the innate immune defense program also depends on cell-autonomous, that is, cell-intrinsic, responses which counteract any stressful insult [ ] . constitutive cell-autonomous immunity mobilizes pre-existing molecules and processes in order to primarily and quickly defend the cell and the host against infectious and sterile injury. hence it can be considered as the very first line of innate immune defense. here, the role of constitutive cell-autonomous responses will be examined, whose involvement in the innate immune defense to stress and injury has only been appreciated within the last few years. the focus of this brief overview will be mainly directed toward cellular stress responses. the term autophagy comes from the greek words "phagy" meaning eat and "auto" meaning self. autophagy is an evolutionarily highly conserved self-digestive process in response to environmental stress to eukaryotic cells, by which cytoplasmic components such as defective/damaged or redundant organelles or protein aggregates are delivered to the lysosome for recycling and degradation. there is convincing evidence indicating that activation of the autophagic process is promoted by mamps and/or damps [ , ] . in more simple words, autophagy is a classical cellprotective and cell-autonomous process of the innate immune system aimed at maintaining and restoring homeostasis at both the cellular (cell-intrinsic) and organismal (cell-extrinsic) level [ ] . although autophagy was initially identified in mammals, a significant breakthrough in our understanding of how autophagy is controlled came from the analysis in the genetically tractable yeast system. pioneering work from ohsumi's group showed that the morphology of autophagy in yeast was similar to that documented in mammals [ ] . (as known, ohsumi received the nobel prize in physiology or medicine .) in fact, the discovery of the autophagyrelated genes in yeast has significantly advanced the understanding of the molecular mechanisms participating in autophagy and the genes involved in regulating the autophagic pathway. many yeast genes have mammalian homologues, confirming that the basic machinery for autophagy has been evolutionarily conserved along the eukaryotic phylum [ ] [ ] [ ] [ ] . notably, a panel of leading experts in the field of autophagy has recently published a new definition of several autophagy-related terms based on specific biochemical features [ ] . accordingly, in the following, three types of autophagy are briefly sketched including macroautophagy, microautophagy, and, in mammals, chaperone-mediated autophagy. each of them fulfils very specific tasks in intracellular degradation. there is general agreement on two main features that characterize bona fide, functional autophagic responses, irrespective of type: ( ) they involve cytoplasmic material; and ( ) they culminate with (and strictly depend on) lysosomal degradation [ ] . thus, although autophagy substrates can be endogenous such as damaged cellular organelles or exogenous such as viruses or bacteria escaping phagosomes, autophagy acts on entities that are freely accessible to cytosolic proteins. this property is essential in order to distinguish between autophagic responses and branches of vesicular trafficking that originate at the plasma membrane, which also culminates in lysosomal degradation. such endocytic processes include phagocytosis, receptor-mediated endocytosis, and macropinocytosis, that is, processes which will be dealt with in part vi, sect. . . of note, however, some forms of autophagy and the endocytic pathway interact at multiple levels, and the molecular machinery responsible for the fusion of late endosomes (also known as mvbs) or autophagosomes with lysosomes is essentially the same [ ] . as stressed [ ] , the strict dependency of autophagic responses on lysosomal activity is necessary to discriminate them from other catabolic pathways that also involve cytoplasmic material, such as proteasomal degradation [ ] . thus, the s proteasome (box . ) degrades a large number of misfolded cytoplasmic proteins that have been ubiquitinated (for (poly)ubiquitination, see box . ) as well as properly folded proteins that expose specific degradation signals, such as the socalled n-degrons [ ] . on the other hand, the proteasome system shares some substrates with different forms of autophagy whereby these two catabolic pathways differ drastically in their final products. thus, proteasomal degradation results in short peptides that are not necessarily degraded further but may flow into additional processes including but not limited to antigen presentation/cross-presentation at the plasma membrane, thereby generating mhc-ii and mhc-i epitopes (compare part viii, chap. ). by contrast, lysosomal proteases fully catabolize polypeptides to their constituting amino acids which eventually become available for metabolic reactions or repair processes. together, as summarized [ ] , bona fide functional autophagic responses navigate cytoplasmic material of endogenous or exogenous origin to degradation within lysosomes (or late endosomes, in specific cases). the binding of many ubiquitin molecules to the same target protein. in its simplest form, ubiquitin can be attached to the target protein as a single moiety resulting in monoubiquitination. ubiquitin itself can be ubiquitinated, resulting in the formation of ubiquitin chains attached to the target protein: polyubiquitination. polyubiquitination of proteins is the triggering signal that leads to subsequent degradation of the protein in the proteasome. ligases play a central role in polyubiquitination. ligases are enzymes that catalyze the synthesis of polyubiquitin chains. ubiquitin conjugation requires typically box the proteasome is a common complex for all living cells, needed to recycle and eliminate unwanted proteins. in analogy, it resembles a chaff-cutter. this molecular machine provides a pathway that is involved in many cellular levels such as protein degradation, antigen processing, cell cycle, apoptosis, and dna repair. the s proteasome that is present in the cytoplasm and nucleus is usually formed by one s proteasome complex and two s proteasome complexes, which are composed of proteases and structural units. the s proteasome is a giant protease responsible for the regulated degradation of polyubiquitylated proteins (see box . ) . it consists of at least distinct subunits and is arranged into two modules: core particle containing catalytic sites and regulatory particles. the cylinder-shaped proteolytic core is the s core particle, which is capped at one or both ends by s regulatory particles. further reading: wehmer m, sakata e. recent advances in the structural biology of the s proteasome. int j biochem cell biol ; : - . basically, the term macroautophagy is often used when describing autophagy in general. the phenomenon is characterized by its typical morphological features which involve dedicated vesicles that can occupy a considerable part of the cytoplasm. typically, macroautophagy is one type of autophagic processes in which the substrates are sequestered within cytosolic double-membrane vesicles termed autophagosomes. the substrates of macroautophagy include superfluous and damaged organelles, cytosolic proteins, and invasive microbes. mechanism of formation and regulation of macroautophagy are very complex and complicated processes that are outlined here in a considerably simplified way. macroautophagy involves the sequestration of cytoplasm via a double-membrane intermediate structure termed the phagophore which matures into an autophagosome; the latter compartment fuses with a lysosome allowing degradation and recycling of the cargo [ ] . in more detail, the process begins with the formation of a membrane of unknown origin, the initial phagophore or isolation membrane. the phagophore then expands, surrounds proteins or organelles, sequesters cytoplasm, and, on completion, develops into a large double-membrane transport vesicle, the autophagosome. subsequently, the autophagosome fuses with a lysosome containing acid hydrolases and releases its contents into the lytic acid hydrolases-containing compartment as part of single-membrane vesicles, termed autophagic bodies. the fused compartment where the autophagic body and its contents are degraded is called an autophagolysosome or autolysosome ( fig. . ) . notably, the process of phagophore expansion three classes of enzymes. e (ubiquitin-activating enzyme) hydrolyzes atp and forms a thioester-linked complex between itself and ubiquitin. e (ubiquitin-conjugating enzyme) receives ubiquitin from e and forms a similar thioester-linked intermediate with ubiquitin. e (ubiquitin ligase) finally binds both the e and a substrate and catalyzes the transfer of ubiquitin to the substrate. ubiquitin itself is often a substrate for further ubiquitylation, which results in the formation of so-called polyubiquitin chains. ubiquitin has seven lysine residues, and depending on the lysine residue used for ubiquitin-ubiquitin chain formation, the polyubiquitin chain can signal different functions. proteins modified by lysine- (k )-or lysine- (k )-linked chains are usually degraded by the proteasome. in contrast, modification by k -linked chains or by a single ubiquitin moiety (monoubiquitylation) seems to trigger other functions, e.g., protein sorting, gene expression, and dna repair. further reading: callis j. the ubiquitination machinery of the ubiquitin system. arabidopsis book ; :e . provides tremendous flexibility and capacity with regard to cargo, allowing entire organelles to be deleted via autophagy; however, this flexibility also means that autophagy must be tightly controlled in order to prevent inappropriate degradation, which could lead to cell death (for relevant papers, see [ ] [ ] [ ] [ ] [ ] [ ] [ ] . intensive studies have been carried out in the past two decades to understand the mechanism and regulation of autophagy. the biogenesis of autophagosomes needs the ordered intervention of autophagy-regulated (atg) proteins that act on different modules. thus, more than atg genes have been identified in human that orchestrate the complex membrane dynamics involved in autophagic sequestration. these atg proteins act sequentially in three macromolecular complexes involved in the three successive stages of autophagy. initiation of autophagy requires the unc- like kinase (ulk )-atg -fip (also known as rb -inducible coiled-coil ) complex, whereby the kinase activity of ulk is controlled by the kinase mammalian target of rapamycin (mtor) in mtor complex (mtorc ), which is sensitive to rapamycin [ ] . the next process, membrane nucleation, requires the beclin / class iii pi k complex, which also plays a major role in membrane trafficking and restructuring involved in autophagy [ , ] ; the final process refers to the elongation, expansion, and closure of the phagophore membrane/autophagosome which mainly relies on atg /microtubule-associated protein light chain (lc ) lipidation. in fact, atg /lc lipidation is regarded as a hallmark of autophagy and is established by a covalent linkage of cytosolic lc to the lipid phosphatidylethanolamine on the surface of the autophagosome [ , ] . of note, in addition to the cytoplasmic ptm of various atg proteins, recent studies have explored the transcriptional and epigenetic control of autophagy [ ] . notably, in human cells, tfeb (for transcription factor eb) and zkscan (for zinc finger with krab and scan domains ) were shown to be implicated in playing a crucial role in autophagy regulation [ , ] . also, there is growing evidence in support of the notion that histone modification/dna methylation acts as an alternative approach for long-term autophagy control [ ] (for histone modification, see part vi, sect. . . ) . also recently, a new ampk→skp →carm (for: ampactivated protein kinase; s-phase kinase-associated protein (p ); coactivatorassociated arginine methyltransferase ) regulatory axis was reported that incorporated cellular nutrient sensing with transcriptional as well as epigenetic control of autophagy [ ] . as concluded by xu and klionsky [ ] , "…this ampk-skp -carm signalling axis integrates the various levels of autophagy regulation including cell signalling, and transcriptional regulation as well as epigenetic modification. epigenetic and transcriptional regulation provides an energy-saving approach for control and also create an enduring memory in preparation for future adverse events. thus, this study has deepened our understanding of how autophagy can be controlled in a holistic manner by pathways linking a multitude of regulation mechanisms. given the extensive involvement of autophagy in human diseases, this work also presents potential directions for novel therapeutic intervention." indeed, besides its beneficial function in controlling cellular homeostasis, macroautophagic pathways when disrupted can have severe consequences leading to major diseases such as cancer, metabolic and neurodegenerative disorders, and cardiovascular and pulmonary diseases [ ] . of note, macroautophagy can be divided into two subtypes depending on the organelle that is targeted for autophagic degradation; thus, the process of mitophagy corresponds to autophagy of mitochondria, whereas the term er-phagy refers to autophagy of the endoplasmic reticulum (er). both processes deserve a few more words in the following subsection. the term mitophagy corresponds to cargo-specific autophagy of mitochondria, a process which mediates the selective removal of mitochondria [ , ] . the aim of mitophagy is to eliminate mitochondria, either to regulate their number to adjust to metabolic demand or to explicitly remove those that are damaged in terms of a quality control. mechanistically, mitochondria are selectively recruited into isolation membranes, which seal and then fuse with lysosomes to eliminate the trapped mitochondria. as discussed [ ] , mitophagy is preceded by so-called mitochondrial fission that divides elongated mitochondria into pieces of manageable size for encapsulation and also controls segregation of damaged mitochondrial material for selective removal by mitophagy. the term er-phagy (also called micro-er-phagy) refers to a process of distinct selective degradation of er membranes and proteins in the lysosome under stress, and this is independent of the core autophagy machinery [ ] [ ] [ ] (for er stress, see sect. . ) . studies on yeast showed that er-phagy is characterized by the fact that stress-induced er whorls are selectively taken up into the vacuole, the yeast lysosome. import into the vacuole was found not to involve autophagosomes but occurs through invagination of the vacuolar membrane, indicating that er-phagy is topologically equivalent to microautophagy [ ] . recent studies on yeast provide evidence suggesting that the atg proteins atg and atg are specific receptors for this pathway of er-phagy [ ] . at this point, it also appears worthwhile to mention that a more recent study on yeast revealed a novel er quality-control pathway, namely, the so-called macro-er-phagy. first results from this study suggest that this pathway delivers an excess of integral-membrane proteins from the er to the lysosome for degradation and, typically, requires the core autophagy machinery [ ] . the brief overview about macroautophagy provides another typical example of innate immune responses which, when controlled, operate in a beneficial homeostatic way but, when uncontrolled, may lead to severe pathologies. for other forms of phagocytic responses, this phenomenon has not been investigated sufficiently. clearly, mitophagy also plays a key homeostatic role in mitochondrial quality control. upregulation of mitophagy has been shown to mitigate excessive mitochondrial accumulation and toxicity to safeguard mitochondrial fitness. hence, mitophagy is a viable target to promote longevity and prevent age-related pathologies [ ] . concerning the two types of er-phagy (micro-and macro-er-phagy), one has to state that research in this exciting field has just begun. several questions remain to be addressed, for example, what is the purpose of er-phagy and what are the underlying mechanisms. future studies will probably provide a clue to elucidating the molecular mechanisms and physiologic roles of er-phagy in other organisms. of note, besides mitophagy and er-phagy, other specific forms of phagocytic pathways have been described. they include -pexophagy as a macroautophagic response preferentially targeting peroxisomes -nucleophagy as an autophagic response selectively targeting portions of the nucleus -ribophagy as a specific autophagic response targeting ribosomes -aggrephagy as an autophagic response specific for protein aggregates -lipophagy in terms of selective autophagic degradation of neutral lipid droplets -bacterial xenophagy as a macroautophagic removal of cytoplasmic bacteria which have escaped the phagosomal compartment upon phagocytosis -viral xenophagy as a macroautophagic response targeting fully formed cytoplasmic virions or components thereof -proteaphagy in terms of macroautophagic responses specific for inactive proteasomes -lysophagy as a specific macroautophagic disposal of damaged lysosomes in mammalian cells for details of these specific forms of phagocytic responses, the reader is referred to the excellent comprehensive review article of galluzzi et al. [ ] . in addition to macroautophagy, two other types of autophagy have been described called microautophagy and chaperone-mediated autophagy (cma). microautophagy together with macroautophagy plays, for example, a role in nutrient recycling under starvation. on the other hand, cma is known to contribute to the maintenance of cellular homeostasis by facilitating recycling of amino acids of the degraded proteins and by eliminating abnormal or damaged proteins, thereby exerting major regulatory functions in different pathophysiological scenarios such as metabolic regulation. here, a few aspects of these two types of autophagic responses are skimpily touched. by contrast to macroautophagy, the process of microautophagy is much less defined in mammals since most studies have been performed in yeast and plants. according to current models, the term refers to a collection of diverse processes. unlike autophagy, microautophagy does not involve the autophagosome-dependent degradation of cytoplasmic components but rather and characteristically relies on the direct engulfment of small portions of cytoplasm into lysosomes or late endosomes by invagination and inward budding of the lysosomal/endosomal membrane, a process that leads to their degradation [ ] [ ] [ ] . though microautophagy is the least studied form of autophagy, a molecular signature of the process has begun to emerge and has led to the definition of microautophagy as a type of autophagy in which the cargo is directly internalized in small vesicles that form at the surface of the lysosome/vacuole or late endosomes (multivesicular bodies), respectively [ ] . in addition to macroautophagy and microautophagy, there is another type of autophagy experiencing increased attention, the cma. characteristically, in cma, cargo delivery also occurs directly at lysosomes, but it does not require formation of vesicles nor membrane invagination. instead, the substrate proteins for this autophagic pathway cross the lysosomal membrane through a protein-translocation complex, that is, a process that requires protein interaction with the chaperone hspa (also known as hsc ) and association of hspa with a specific splicing isoform of lamp- , that is, the lysosomal protein lamp- a. thus, chaperone-bound autophagy substrates bind lamp- a monomers on the cytosolic side of the lysosome, which stimulate the formation of an oligomeric lamp- a translocation complex [ , [ ] [ ] [ ] . essential functions that cma fulfils in cells include a contribution to amino acid recycling during prolonged starvation as well as quality control, directly linked to the ability of this pathway to selectively remove single proteins from the cytosol. for example, cma is up-regulated during oxidative stress where it contributes to the degradation of oxidized proteins (reviewed in [ ] ) (see next sect. . ). of note, growing evidence demonstrates that malfunction of cma plays a vital role in the pathogenesis of severe human disorders. often, the mechanisms underlying the alterations of cma in these pathologies involve perturbations in the functioning of the cma translocation complex. both diminished and enhanced cma activities have been shown to associate with diseases, an observation that emphasizes the importance of a tight regulation of cma activity (highlighted in [ ] ). as argued above, current knowledge about mechanism and physiological relevance of microautophagy in mammalian cells is hard to judge since most findings derive from studies in yeast. however, future studies aimed at identifying proteins controlling microautophagy-related vacuolar membrane changes in yeast will probably allow to search for homologues in mammals and then investigate their contribution to mammalian microautophagy. by contrast, research on cma has already made substantial progress. for example, the recent identification of a plethora of new cma substrates and deficiencies in cma associated with diverse human pathologies has expanded our understanding of the importance of cma in multiple cellular functions. in fact, the growing number of connections between cma and human diseases has already generated interest in modulating cma activity for therapeutic purposes. there is a close relationship between autophagy and mamps and/or damps in the cellular response to injury. in fact, autophagy cannot be restricted to an innate immune mechanism that controls intracellular homeostasis alone but has to be extended in terms of "immunological autophagy" to a process that is committed to control and regulate efferent innate and potential adaptive immune responses. the "medium" for achieving this goal is the mamps and/or damps which operate as a link between intracellular and extracellular events. this scenario is briefly touched in the following. growing evidence indicates that autophagy regulates release and degradation of damps-here in terms of inducible damps-including hmgb , atp, and dna in several cell types [ ] . for example, autophagic mechanisms reportedly promote and regulate the release and secretion of hmgb in a ros-dependent manner in fibroblasts, macrophages, and cancer as well as net-mediated release of hmgb in neutrophils [ , ] . moreover, autophagy has been shown to be required for the liberation/active secretion of atp by dying cancer cells [ , ] . in addition, autophagy was found to contribute to the regulation of the ddr at multiple levels, that is, a process associated with the emission of damps [ , ] (for ddr, see sect. . ). via emission of damps, eventually, together with mamps, autophagy can amplify or even instigate mamp/damp-prm signalling leading to efferent innate immune responses. on the other hand, autophagy can inhibit pro-inflammatory signalling cascades. for example, the atg -atg complex, a key regulator of the autophagic process, was shown to negatively regulate rlr signalling by direct binding to card domains of rig-i and interferon promoter-stimulating factor- (ips- ) [ ] (compare part vi, sect. . . ). moreover, as reviewed elsewhere [ ] , autophagy has been found to inhibit both nlrp and aim inflammasome activation and subsequent production of pro-inflammatory cytokines il- β and il- (for inflammasomes, see part vi, sects. . . and . . ). as a possible mechanism, the authors propose that inflammasome components and pro-il- β are subjected to ubiquitination and subsequent degradation by autophagy, thereby leading to functional inactivation of inflammasomes. conversely, an increasing number of studies suggest that damps, including hmgb , atp, and dna, are powerful stimuli and regulators to elicit autophagic responses [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, hmgb was demonstrated to be an important regulator of autophagy in various types of cancer cells and keratinocytes. mechanistically, the reduced form of the hmgb protein was proposed to be responsible for the promotion of autophagy in an ager/rage-dependent fashion [ , ] . also, and of high interest, in a clinical study on patients with chronic hepatitis b, hmgb -induced autophagy was found to maintain treg function during chronic viral infection [ ] . moreover, in studies on a mini pig lung iri model, evidence was provided indicating that autophagy, when triggered by damps such as hmgb and hsp during iri, amplifies the inflammatory response through enhancing k -linked ubiquitination of traf and activation of the downstream mapk and nf-κb signalling (for traf , mapk, and nf-κb signalling, see part moreover, there is already first evidence suggesting a role of atp in the regulation of autophagy [ ] . in addition, there are accumulating data indicating that cytosolic dna, dislocated as a result of dna damage, may contribute to the regulation of autophagy whereby the dna damage-regulated autophagy modulator (dram ) appears to play a mechanistically crucial role [ ] [ ] [ ] . the mechanisms involved in mamp/damp-activated autophagic responses have only partially been elucidated. in fact, there is convincing evidence suggesting that many mamp/damp-recognizing prms including tlrs (in particular endosomal tlrs), nlrs, and anti-dna receptors can activate autophagic responses by triggering specific signalling pathways (reviewed or discussed in [ , , , ] ). the crosstalk between autophagic responses and damps represents a powerful instrument of the innate immune system to integrate and unify various tools for the promotion and regulation of injury-induced inflammation and, in the presence of nonself-or altered self-antigens, injury-induced adaptive immunity. thus, on the one hand, autophagy is known to promote and regulate the release of damps (though the exact mechanisms are still elusive); subsequently, damps via prmtriggered pathways participate in the regulation of inflammation. on the other hand, activation of prms by mamps and/or damps promotes autophagy activation through a mechanism that has been partially elucidated. in fact, an increasing number of findings suggest that this activation process is triggered by prms following recognition of mamps and/or damps. nevertheless, the precise molecular mechanisms by which prms modulate autophagy remain largely unknown. there is increasing evidence in support of the notion that mamp/damp-activated autophagic responses promote emission of damps which in turn support cellular homeostasis in the course of adaptive stress responses in healthy cells. notably, this cellular homeostatic effect may spread out and affect the whole organism via emission of autophagy-dependent damps. in other words, via damps, autophagy as a cell-intrinsic stress response can fortify its defending capability by providing a link to promotion and regulation of cell-extrinsic efferent innate immune and eventually subsequent adaptive immune responses. however, despite the fact that autophagy is one of the best-known cell-autonomous responses in innate immunity and has clearly been shown to counteract dangerous infectious and sterile cell stress, much is left unclear. one such issue concerns the definition of autophagy-dependent cell death. as discussed and summarized [ ] , autophagy-dependent cell death can be defined as a form of rcd (see next chapter) that can be retarded by pharmacological or genetic inhibition of macroautophagy. in this context, as stressed by galluzzi et al. [ ] , it is important to note that ( ) specificity issues affect most, if not all, pharmacological agents employed so far for suppressing macroautophagic responses and ( ) multiple components of the macroautophagy machinery have autophagy-independent functions. in view of these facts and findings, these authors recommend to favor genetic approaches and to test the involvement of at least two different proteins of the macroautophagy apparatus in a specific instance of rcd before etiologically attributing it to macroautophagy. other unclear issues refer to the specific modulation of autophagy by mamps and/or damps, the precise interaction of autophagy with innate immune signalling cascades, and the cooperation between autophagy and other physiologic cell-intrinsic and cell-extrinsic processes during scenarios of cell stress and tissue injury. efforts to solve these problems are of utmost importance in view of the fact that autophagy-when induced by excessive, chronic, or acute-repetitive emission of damps-can contribute to the pathogenesis of many human diseases, that is, acute and chronic, infectious, or sterile inflammatory disorders. at the respective places, they will often be mentioned in the following chapters as well as in volume . oxidative cell stress and tissue injury reflect most potent and omnipresent threats an organism is exposed to. though there is a robust defense response continuously operating, this kind of injury is known to contribute to the pathogenesis of many human diseases. how can this be? oxidative stress is caused by an imbalance between the production of oxidants such as ros on one side and the biological antioxidative defense system's ability on the other side to counter the oxidant levels with antioxidants, that is, to readily detoxify the toxic reactive species or easily repair the resulting damage. thus, it is the excessive production of ros-overriding the antioxidative capacities-that is pathophysiological and contributes to dysfunction, damage, and even death of cells. by contrast, generation of ros in physiological low/ moderate concentrations-operating as second messenger molecules and causing so-called oxidative "eustress" [ ] -assists in intracellular signalling pathways and, thus, is essential for optimal cell functions and homeostasis of an organism. in other words, the biological effects of ros-beneficial or deleterious-considerably depend on the amounts of ros present and, in action, a phenomenon that is in agreement with the idea that cellular ros generation has characteristics of hormesis implying a dose-response phenomenon that is characterized by beneficial effects at low doses and deleterious effectivity at high toxic doses [ ] . to guarantee this homeostatic function of ros, to keep these molecules within physiological limits, and to prevent their deleterious effects, that is, to maintain hormesis, a smooth running of the oxidative stress response is of utmost importance. accordingly, a few aspects of this critical stress response are addressed in the following. reactive oxygen species are produced from molecular oxygen as a result of normal cellular metabolism. to understand any discussion on a role of ros in host defense or human diseases, one should define free radicals. according to halliwell and gutteridge [ ] , "a free radical is any species capable of independent existence that contains or more unpaired electrons." an unpaired electron is one that occupies an atomic or molecular orbital by itself. radicals can be formed by the loss of a single electron from a non-radical, or by the gain of a single electron by a non-radical. in this sense, superoxide anions (o ·− ), hydroxyl radicals ( · oh), peroxyl radicals (ro · ), and alkoxyl radicals (ro˙) are oxygen radicals. of note, ros is a collective term often used by scientists to include not only the oxygen radicals but also some non-radical derivatives of oxygen such as h o , hypochlorous acid (hocl), ozone (o ), and singlet oxygen ( o ). nitrogen-containing oxidants, such as no · are called rns. generation of ros is generally a cascade of reactions that starts with the production of superoxide anions. superoxide rapidly dismutases to h o either spontaneously (especially at the low ph) or catalyzed by sod. other elements in the cascade of ros generation include the reaction of superoxide with no to form the very toxic peroxynitrite, the peroxidase-catalyzed formation of hocl from h o , and the iron-catalyzed fenton reaction, leading to the generation of hydroxyl radical. the oxidants are produced endogenously as by-products or metabolites of various metabolic processes. multiple enzyme systems produce superoxide radicals and their derivatives including xanthine oxidoreductase (xor), the reduced form of nadph oxidases (noxes), and mitochondrial electron transport chain (etc)associated molecular complexes ( fig. [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] mitochondrial etc is believed to be the main source of ros. in the following, some aspects of these three systems (other systems not mentioned here) are briefly touched, exemplified by and focused on their role as vascular sources of ros production as investigated on models of iri. xanthine oxidoreductase (xor), as a housekeeping enzyme, is probably expressed in all cells but primarily in surface epithelia such as capillary endothelial tissue of various organs. xanthine oxidoreductase, a complex molybdoflavoprotein, is the ratelimiting step in the catabolism of purines, where it catalyzes the last steps of purine metabolism: the conversion of hypoxanthine to xanthine and of xanthine to uric acid, with superoxide/h o generated as by-products. there is a definite role of xor in reperfusion of tissue and organs [ , ] . for example, experiments performed in isolated rat hearts have demonstrated that radical generation and functional injury are decreased by inhibition of xor with oxypurinol. similarly, in human aortic or venous ecs, xor-mediated ros generation has been shown to be a central mechanism of oxygen radical generation upon postischemic reoxygenation [ , ] . the nadph oxidases were initially considered as enzymes expressed only in phagocytic cells involved in host defense and innate immunity; however, recent evidence indicates that there is an entire family of noxes based on the discovery of gp phox homologues. the family comprises seven members, including nox , nox (formerly termed gp phox), nox , nox , nox , duox , and duox [ ] . three members out of the enzyme family are important sources of ros in the vasculature, namely, nox , nox , and nox [ ] [ ] [ ] . today, noxes are perhaps the best-studied enzymes involved in ros production in the blood vessels. remarkably, different members of the nox/duox family engaged in iri are localized in various cells, that is, in vascular cells and phagocytes. this may lead to the notion that noxes in vascular cells are responsible for the first wave of ros production because vascular cells are first confronted with reintroduced molecular oxygen. as generally believed, there is, in fact, no vascular specific nox isoform but rather a complex expression of various nox isoforms in different cells and regions of the vascular system. nevertheless, in arteries from humans and animals, nox , nox , and a shallow level of nox have been consistently found to be present both as messenger rna and as protein [ ] . altogether, the findings and data briefly described here make clear that vascular cells are equipped with efficient machinery able to efficiently produce ros. regarding the different enzymatic sources, superoxide radicals appear to be predominantly generated compared, for example, to hydroxyl radicals. mitochondria have been implicated as potential oxygen sensors by increasing the generation of ros which regulate a variety of hypoxic responses [ ] [ ] [ ] [ ] . in fact, mitochondria are increasingly recognized as lynchpins in the evolution of tissue injury during postischemic reperfusion. it is generally acknowledged that the majority of intracellular ros production is generated in the mitochondrial etc and its associated metabolic enzymes. however, very little is known about which mitochondrial sites are involved in physiological or pathological ros production under native conditions. of note, using inhibitors to manipulate the redox states of particular sites and prevent superoxide generation from others, at least ten different locations of superoxide/h o production in the etc and associated enzymes (krebs cycle, β-oxidation, etc.) have been identified in mammalian mitochondria. in fact, the relative and absolute contributions of specific sites to the production of ros in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely valid in cells and in vivo [ ] . for example, superoxide formation occurs on the outer mitochondrial membrane, in the matrix, and on both sides of the inner mitochondrial membrane (fig. . ) . complex i (nadh-ubiquinone oxidoreductase) accepts electrons from nadh; these electrons are carried to complex ii (the succinate dehydrogenase-coq oxidoreductase), where they are used to oxidize succinate to fumarate. afterward, electrons continue to travel down their electrochemical gradient to complex iii (the cytochrome bc complex (ubiquinol-cytochrome c oxidoreductase)), and subsequently to complex iv (cytochrome c oxidase); finally, the electrons are used to reduce molecular oxygen to water. thus, complex i and complex ii oxidize the energy-rich molecules nadh and flavin adenine dinucleotide h , respectively, and then transfer the resulting electrons to ubiquinol that carries it up to complex iii (for competent articles, see [ , ] notably, complexes i and ii generate superoxide within the mitochondrial matrix, whereas complex iii produces superoxide at the qo site, resulting in the release of superoxide into either the intermembrane space or the matrix. regarding complex i, it was recently demonstrated that inhibition of nd , a subunit of complex i, suppresses the activity of this complex and thus ros production [ ] . furthermore, data from another set of studies on complex i showed that stable down-modulation of its subunits grim- and ndufs decreased complex i activity that was associated with a significant reduction in the overall nadh oxidation rate but with an increased production of ros by the target cells [ ] . similar results have been found in studies on complex ii: there is evidence suggesting that inhibition of complex ii on the level of subunits even leads to an increase in ros production. the phenomena can be explained by assuming that, if electrons provided in the course of the etc cannot efficiently be transferred to the next complex, they would leak out from the inhibited complex and generate ros [ ] . the complex iii subunits rieske iron-sulfur protein (risp) encoded by ubiquinol-cytochrome c reductase, rieske iron-sulfur polypeptide (uqcrfs ), and ubiquinol-cytochrome c reductase binding (uqcrb) protein appear to play a crucial role in hypoxia-triggered mitochondrial ros generation (for rieske, see box . ). thus, it was shown that risp promotes the hypoxic stabilization of the transcription factor hif- α protein [ ] and uqcrb was found to mediate hypoxia-induced tumor angiogenesis via mitochondrial ros-mediated signalling [ , ] . also, and of note, a mouse model to permit conditional deletion of the nuclear-encoded risp gene was recently developed to assess its role in hypoxiainduced ros signalling in the pulmonary circulation [ ] . it was found that depletion of risp abolishes the ros response to hypoxia in isolated pulmonary the rieske protein is an iron-sulfur protein (isp) component of the cytochrome bc complex that was first discovered and isolated by john s. rieske and coworkers in . the rieske iron-sulfur protein is an essential subunit of mitochondrial cytochrome bc complexes and, like the majority of mitochondrial proteins, is encoded by a nuclear gene and synthesized on cytoplasmic ribosomes as a precursor with a -residue amino-terminal extension. the iron-sulfur protein is then post-translationally imported into the mitochondria where it is inserted into the bc complex in the inner mitochondrial membrane. at first, the precursor is translocated via translocation contact sites into the matrix. there, cleavage to an intermediate containing an -residue extension occurs. the intermediate is then redirected across the inner membrane, processed to the mature subunit, and assembled into complex iii. arterial smcs and isolated pulmonary artery segments. further, in this article, it was discussed that mitochondria are not the only source of ros during hypoxia. thus, studies using a genetic knockout of p phox suggested that cytosolic nadph oxidase systems may also contribute to a hypoxic pulmonary vasoconstriction response during acute hypoxia [ , ] . according to the authors' conclusion, the blockade of hypoxia-induced ros responses (in these studies observed with depletion of risp) suggests that the mitochondria may act as the initiators of ros production, which could be amplified by engagement of nadph oxidase systems elsewhere in the cell. such "ros-induced ros release" might permit small ros signals generated by mitochondria to activate ros signalling throughout the cell, thereby avoiding mitochondrial damage that might arise if the entire cellular oxidant signal originated from that organelle [ ] (or even leading to excessive ros production?). indeed, the substantial advances in oxidative stress research of recent times, in particular, the specification of hypoxia-sensing ros-producing enzyme systems, will contribute to new therapeutic strategies to be applied in acute and chronic human diseases known to be influenced by oxidative stress. for example, discrimination of oxidative eustress, a fundamental process in maintaining health, from oxidative damage will improve clarity in developing "redox medicine" [ ] . when the redox equilibrium of a cell is upset by pro-oxidant environmental stimuli, that is, when oxidative stress exists, an adaptive stress response takes place which can result in upregulation of antioxidant proteins and detoxification enzymes. these antioxidative defense molecules comprise the following [ ] : ( ) agents that catalytically remove free oxygen radicals and other reactive species, for example, sod, catalase, peroxidase, and thiol-specific antioxidants; ( ) proteins that minimize the availability of pro-oxidants such as iron ions, copper ions, and heme, for example, transferrins, haptoglobins, hemopexin, and metallothionein; ( ) proteins that protect biomolecules against damage (including oxidative damage) by other mechanisms, for example, hsps; and ( ) low-molecular-mass agents that scavenge ros and rns, for example, glutathione, α-tocopherol, and (possibly) bilirubin and uric acid. in the past, it was fashionable to divide the oxidative stress response into three main tiers: ( ) antioxidant enzymes including sod, catalase, glutathione peroxidase, and glutathione; ( ) detoxifying enzymes such as glutathione peroxidase, glutathione s-transferase, aldo-keto reductase, and aldehyde dehydrogenase; and ( ) energy-dependent efflux pumps. as a fourth defense system, the antioxidant nutrients such as vitamins e and c as well as carotenoids were appreciated [ ] . it was generally accepted that the first line of enzymes is of enormous importance in limiting ros-mediated damage to intracellular macromolecules. for example, among the most important regulators of ros levels were the sod enzymes: cu/ znsod in the cytoplasm and outer mitochondrial space and mnsod exclusively in the inner mitochondrial space. mechanistically, superoxide is converted to h o and oxygen (o ·− + o ·− + h + → h o + o ) by sod. peroxiredoxins and abundant catalase enzyme then scavenge h o , converting it to molecular oxygen and water. another example of a first-line defense molecule is trx. thioredoxin contains two adjacent -sh groups in its reduced form which are converted to a disulfide in oxidized trx. notably, it can undergo redox reactions with multiple proteins using the reaction trx however, it then turned out that these antioxidative principles were clearly not % effective at performing this task, as under normal physiological conditions, lipid and dna oxidation products can be detected in blood and urine. because certain compounds of the chemicals generated after an interaction of ros with macromolecules are highly reactive, there must be an equal necessity to detoxify these secondary oxidation products to prevent them from also damaging dna, proteins, and lipids. without the adequate detoxification of such products, an extended chain reaction will occur resulting in the degradation of cellular components and the ultimate death of the cell. this second line of defense against ros is provided by those detoxifying enzymes. finally, detoxified metabolites produced by these enzymes are eliminated from the cell by energy-dependent efflux pumps such as the glutathione s-conjugate transporter, also called the multidrug resistance-associated protein (mrp) [ ] . today, one must state that these previous notions are incomplete. at first, it became apparent that members of the so-called cnc (for cap 'n' collar)-basic region-leucine zipper (bzip) family of transcription factors are principal mediators of defense responses to redox stress. in mammals, the cnc family members nrf and nrf were shown to be involved in the transcriptional upregulation of cytoprotective genes encoding a large number of diverse detoxification, antioxidant, and anti-inflammatory proteins (e.g., glutamate cysteine ligase, nadph-quinone oxidoreductase, glutathione s-transferases, and aldo-keto reductases) as well as enzymes with essential roles in cell metabolism [ ] . more recent studies then revealed that these transcription factors, notably nrf , are activated by keap as the primary negative regulator of nrf , that is, a molecule that simultaneously operates as a sensor protein able to perceive dyshomeostatic subclass iic- damps, for example, in terms of redox changes reflecting electrophilic stress. it is worth to add here that six critical domains have been defined in nfr (neh -neh ), and it is neh , located at the n terminus of nrf that acts as the regulatory domain for the cellular stress response. actually, neh interacts with the cytoplasmic protein keap [ ] . in the following, this very important damp-induced and gene-based antioxidative and cytoprotective system is addressed a bit more in detail. increasing evidence indicates that several redox-regulated gene products serve to protect cells from ros damage. the antioxidant response element (are), a cis-acting dna regulatory element or enhancer sequence is known to be activated by oxidative stress and to be responsible for the transcriptional regulation of several redox-regulated gene products. both nrf and bind to are and regulate are-mediated gene expression and induction. the molecule nrf is more potent than nrf in activation of are-regulated gene expression and is regarded as the principal transcription factor that binds to the are. this transcription factor is ubiquitously expressed and present in various organs and tissues including the kidney, muscle, lung, heart, liver, and brain (for reviews, see [ ] [ ] [ ] [ ] [ ] ). as touched in part ii, sect. . . and part iv, sect. . . , the nrf -triggered antioxidant response is initiated by activation of keap that functions as a substrate adaptor protein for the degradation of nrf and serves as an intracellular sensor for redox changes reflecting the presence of subclass iic- damps (for reviews see [ ] [ ] [ ] ). earlier studies had already shown that nrf is a bzip transcription factor that translocates to the nucleus after liberation under oxidative stress conditions from its cytosolic inhibitor keap [ ] . in the nucleus, nrf was found to form dimers with the proteins maf, jun, fos, activating transcription factors (atf ), and/or creb binding protein (cbp) and, in addition, regulates transcription by binding to the are upstream of a variety of cytoprotective and detoxification target genes to combat the oxidative stress [ ] . thus, established nrf -regulated genes reportedly included cu/zn sod, catalase, trx, trx reductase, glutathione reductase (gr), glutathione peroxidase (gpx), and ferritin l (ftl) [ ] (ftl and ferritin h (fth) subunits are responsible for intracellular iron storage). all of these genes are involved in the response to oxidative stress. there are several other genes also known to be engaged in the response to oxidative stress that are not described here. recently, the molecular signalling mechanism involved in the keap ↔ nrf pathway has been further elucidated and specified. the core can be seen in an axis consisting of redox change (subclass iic- damps)-initiated → keap -induced → nrf -triggered → are-driven expression of antioxidant and detoxifying genes ( fig. . ) (discussed in [ - , , ] ). the complex and complicated sequelae of the pathway are simplified in the following text. under homeostatic and stress-free conditions, binding of keap to the nrf molecule leads to its polyubiquitination and subsequent degradation by the proteasomal pathway, thereby maintaining a consistent generation of nrf and retaining its very low levels in the cytoplasm. in this scenario, keap homodimer binds to a single nrf protein via a high-affinity so-called "etge" motif and low-affinity so-called "dlg" motif. the twosite recognition of nrf by the keap dimer is essential for polyubiquitination of nrf (also see part ii, sect. . . ) (for polyubiquitination, see box . ) . in this sense, the keap ↔ nrf system can be regarded as a vital part of regulating cells under a homeostatic environment. however, in case of dangerous and threatening oxidative (and xenobiotic) stress, the system instigates a stress response that is characterized by a rapid and dramatic cessation of the keap -dependent polyubiquitination process resulting in a rapid increase of nrf abundance. in fact, exposure to ros-mediated stress (or electrophilic stress) is thought to modify the reactive "iic- damps-sensing" cysteine residues in keap , which is associated with a conformational change of the protein resulting in a loss of keap ubiquitination activity (fig. . ) . notably, as a cysteine-rich protein, the human keap possesses cysteine residues, and they are all reactive to stress to varying degrees. among these sensor cysteines of keap , c is best characterized. evidence from a number of studies has suggested that c is the most reactive and critical to the keap ↔ nrf stress-sensing response. even, there is already evidence from a first atomic-level view suggesting that the unique environment of cys (besides cys , cys , and cys ) appears to be the critical residue of keap responsible for detecting increased levels of oxidative stress [ , , ] . oxidative modification of cysteine sensors of keap leads to a loss of its polyubiquitination and degradation activity thereby stabilizing nrf . consequently, fig. . the oxidative stress-induced keap ↔ nrf pathway. under non-oxidative homeostatic conditions, the sensor keap is bound to the nrf molecule resulting in its polyubiquitination and subsequent degradation via the proteasomal pathway. oxidative stress modifies the ros-sensing cysteine residues of keap leading to loss of its polyubiquitination and degradation activity, dissociation of nrf that becomes stabilized and accumulates. then, nrf translocates to the nucleus and forms a heterodimer with the smaf transcription factor. the nrf /smaf heterodimer binds to are and induces transcription of numerous cytoprotective antioxidant and detoxification genes. are antioxidant response element, keap kelch-like erythroid cell-derived protein with cnc homology (ech)-associated protein , nrf nuclear factor-erythroid p -related factors , smaf small musculoaponeurotic fibrosarcoma, ub-ub-ub poly-ubiquitin chain. sources: refs. [ - , , ] stabilized nrf accumulates in the cytoplasm, translocates into the nucleus, and forms a heterodimer with a smaf transcription factor (smaf, small musculoaponeurotic fibrosarcoma). thus, this accumulation of nrf in response to ros (and electrophiles) cannot be regarded as an induction in a strict sense but instead is a mechanism referred to as derepression, that is, from the rapid degradation-based repression. as highlighted and discussed [ ] , there are two models of how to explain the cytoplasmic accumulation process of nrf . the "hinge-and-latch" model holds that the modification of the sensing cysteine residues of keap reduces its affinity for nrf but does not result in release. instead, newly synthesized nrf is translocated to the nucleus to trigger the transcription of nrf -dependent genes. the other model denoted as the "conformation cycling" model claims that keap uses a cyclic mechanism to target nrf for polyubiquitination and proteasomal degradation. an important feature of this cyclic mechanism is that it ensures regeneration of keap which allows the cycle to proceed. modification of specific reactive cysteine residues of keap may block the cycle of keap -dependent nrf degradation allowing de novo synthesized nrf to accumulate. the subsequent transcriptional process in the nucleus has been specified as well. as partially mentioned above, the nrf -smaf heterodimer binds to are or electrophile-responsive element (epre) and induces transcription of numerous cytoprotective genes. of note, recently, an extensive genome-wide analysis of the nrf -smaf-binding sequence, that is, the are/epre, and the maf homodimerbinding sequence (the so-called maf responsive element or "mare") was conducted, and the differences between these elements were clarified. as a result, it was proposed that are, epre, and the nf-e binding sequence be collectively named cnc-smaf-binding elements (csmbe) [ ] . interestingly, prms appear to be co-players in this scenario. thus, tlrs have been observed to induce nrf activation. remarkably, in a recent study, tlr agonists were shown to activate nrf signalling via reduction of keap [ ] . the authors could demonstrate that tlr signalling-induced keap reduction promotes nrf translocation from the cytoplasm to the nucleus, where it activated transcription of its target genes. further, tlr agonists were found to modulate keap at the protein post-translation level through autophagy. in fact, tlr signalling increased the expression of autophagy protein p and lc -ii and induced their association with keap in the autophagosome-like structures. the keap ↔ nrf system as briefly described here is a robust oxidative stress response. its regulatory mechanisms, for example, stress-sensing mechanism, proteasome-based regulation of nrf activity, and selection of target genes have been elucidated mainly in mammals (for proteasome, see box . ). nevertheless, the pathway is now regarded as an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress across the tree of life. thus, the keap ↔ nrf system has been found to be also present in zebrafish, fruit fly, and caenorhabditis elegans indicating that its roles in cellular defense are conserved throughout evolution among vertebrates and suggesting that analogous defense systems are widely conserved throughout the animal kingdom [ , ] . as briefly demonstrated in this subchapter, aerobic organisms have integrated antioxidant systems, which include damp-promoted generation of enzymatic and nonenzymatic antioxidants that are usually effective in blocking harmful effects of ros. however, when ros is produced in excess causing pathological conditions, the stress response against oxidative damage can be overridden. thus, oxidative stress is known to contribute to many pathological conditions, including cancer, neurological disorders, atherosclerosis, hypertension, ards, and chronic obstructive pulmonary disease, just to mention a few of them. plausibly, these disorders are motivation enough to search for new effective therapeutic options by harnessing the new insights into mechanisms of the keap ↔ nrf system. certainly, intense research is essential for a detailed understanding of the precise consequences of targeting keap for disease prevention and treatment. all the more so as the oxidative stress response is integrated into other forms of innate stress responses that will be further outlined in the following subchapters. the heat shock response-one of the most ancient and evolutionarily conserved cytoprotective mechanisms found in nature-is induced upon exposure of living cells to acute, subacute, or chronic stress conditions. this defense response is characterized by the expression of a group of phylogenetically conserved intracellular hsps, which possess the capacity to recognize structures commonly found in the interior of proteins and to bind such structures. thanks to this property, they form a chaperone network involved in correct protein folding, trafficking, and complex assembly (for reviews, see [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). in order to be released from engagement with proteins after folding and take part in further rounds of activity, hsp and other chaperones utilize an intrinsic atpase domain to hydrolyze atp and assume a free conformation [ ] . once released, hsps mediate protective cellular defense mechanisms including regulation of apoptosis, the aim being to maintain and restore cellular protein homeostasis (proteostasis). exposure of almost any cell to heat shock most often leads to the rapid transcription, translation, and accumulation of a variety of hsps that increase to quite considerable levels when the stress is pronounced. in fact, these molecules are induced by various environmental insults that can cause protein denaturation and unfolding within the cells, leading to the formation of nonnative proteins and protein aggregates, thereby emitting dyshomeostatic damps. of note, besides their protective role in different intracellular compartments, hsps in terms of inducible damps can translocate to the cell surface to get exposed or are actively secreted via non-canonical pathways. in case of necrotic cell death, they are passively released in large amounts into the extracellular space to act as constitutively expressed damps (compare part iv, sect. . . and sects . . . and . . . ). once emitted, hsps are sensed by classical recognition receptors and/or non-classical receptors (e.g., cd ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . interestingly, recent knowledge about similarities between allograft and tumor rejection has visualized that the processes of both iri to allografts [ , , ] and therapy-mediated injury to tumors [ ] [ ] [ ] [ ] are characteristically associated with emission of hsps. in all eukaryotes, the hsr is primarily regulated and controlled by the hsfs, in particular, hsf , a sequence-specific factor that binds upstream to heat shock elements in the promoters of target genes [ ] (fig. . ). for example, hsf is activated by environmental stress including oxidative and tumor-associated stress [ , ] . notably, in all eukaryotes, hsf responds to such stress conditions by undergoing a monomer to trimer transition and becomes massively phosphorylated, leading to its acquiring ability to bind to dna rapidly and activate transcription [ ] . there is at least preliminary evidence suggesting that intracellular perturbations reflecting dyshomeostatic damps may activate hsfs [ ] [ ] [ ] [ ] . such molecular alterations reportedly include changes in cytosolic ca + concentration, for example, caused by increase of fluidity in specific membrane domains [ , ] . interestingly, a recent study in support of these earlier findings provided evidence of the existence of a plasma membrane-dependent mechanism of hsf activation in animal cells, which is initiated by specific membrane-dependent trpv calcium channel-like receptors [ ] . these findings lend support to the notion that heat sensing and signalling in mammalian cells are dependent on trpv channels, suggesting that these receptors may act as a major hsr sensor in different epithelial non-cancerous and cancerous cells, capable of triggering the cellular hsr [ ] . in another line of experiments, trpv was demonstrated to mediate the effects of transient heat shock on endocytosis of human monocyte-derived dcs, suggesting a central role of trpv in mediating the cellular action of heat shock on these important cells of the innate immune system [ ] (for trpv channel receptors, also compare part ii, sect. . . ). induction of an hsr is not only mediated by sterile stress conditions but is also believed to be promoted by cell-invading viruses or bacteria. consequent emission of hsps in their role as damps may reflect a mechanism by which pathogens may contribute to sterile inflammation. for example, exacerbation of hepatitis b virus (hbv)-associated liver injury is reportedly characterized by an abnormal immune response that not only mobilizes specific antiviral effects but also poses a potentially lethal non-specific sterile inflammation to the host [ ] . heat shock proteins may be the most extensively studied damps in the context of hbv infection. a number of hsps such as hsp and hsp have been reported to be supportive factors in the process of hbv replication, and selective inhibition of these hsps was proposed to be host-based anti-hbv strategies [ ] [ ] [ ] . thus, as argued [ ] , both infective and sterile inflammation may synergistically contribute to the exaggeration of chronic hepatitis, if the hbv cannot be cleared entirely. likewise, bacterial infections were also reported to promote induction of the hsr [ , ] . for example, in studies on h. pylori and e. coli infection models, the initiation of an hsp stress response could be demonstrated [ , ] . the hsr is recognized and accepted as a classical stress response in nearly all species across the tree of life. it reflects the desperate efforts of a cell to restore homeostasis and survive upon both infectious and sterile insults. its products, the hsps, operate as damps in commission of the innate immune system to reach this goal. notably, via this mechanism, an hsr, induced by infectious damage to a cell, can promote sterile inflammation. whereas a successful hsr upon stress leads to restoration of cellular homeostasis and cell survival, an unsuccessful hsr may result in rcd such as apoptosis [ ] . this phenomenon will be resumed in the following subchapters. the er is a continuous membrane system that forms a series of flattened sacs within the cytoplasm of eukaryotic cells. as a subcellular organelle in the control of proteostasis, it is responsible for calcium storage and lipid biosynthesis as well as the synthesis, correct folding, processing, and maturation of proteins as well as for the orchestration of their transport along the classical/conventional secretory pathway. the er delivers these components to their destination compartments which include the er itself, the golgi apparatus, the plasma membrane, and the extracellular milieu or the endocytic and autophagic pathways. plausibly, the multifunctional nature of this organelle requires a myriad of proteins, unique physical structures, and coordination with and response to perturbations in the intracellular environment. a series of chaperones, folding enzymes, glucosidases, and carbohydrate transferases support and execute these processes. perturbation of er-associated functions such as accumulation of unfolded/misfolded proteins, excessive ros production, hypoxia, calcium and glucose depletion, or viral and bacterial infections reflect stress of the organelle and result in activation of an er stress-coping response, the evolutionary conserved upr [ ] [ ] [ ] [ ] . for example, the processes of both iri-mediated cell damage/cell death [ ] [ ] [ ] and induction of the icd of cancer cells [ ] [ ] [ ] are characterized by demonstration of er stress that is almost always associated with oxidative stress and vice versa [ ] . to cope with er stress, cells have the unique possibility to activate the upr, a dynamic signalling network that orchestrates the recovery of homeostasis or triggers rcd modalities, depending on the level of damage. perception of any perturbation of the er is provided by three sensor molecules of the upr embedded in the er membrane: the perk, ire- , and atf . the upr-mediated recovery of er homeostasis mainly occurs through the perk-eif α-mediated temporary shutdown of protein translation and the activation of a complex genetic program that aims to improve er quality control and adaptive responses [ ] [ ] [ ] [ ] (fig. . ) . accordingly, perk, devoted to perceiving dyshomeostatic damp-emitting er perturbations (so far, no clear evidence for ire- and atf in this respect), may be regarded as a new family of non-classical prms, at least in a broader sense. in nonstressed conditions, the three sensors of the er homeostasis are kept in an inactive state by the er-luminal binding immunoglobulin protein (bip). the protein bip, also known as glucose regulated protein (grp ), is a member of the hsp family of proteins (specifically hspa ). it functions as a chaperone to selectively bind unfolded proteins in the er lumen by interacting with exposed hydrophobic . simplified scenario model illustrating the er stress-induced three arms of the unfolded protein response. perception of any perturbation of the er is provided by three sensor molecules of the unfolded protein response embedded in the er membrane: the perk, ire- , and atf . perk perceives dyshomeostatic damp (subclass iic- damp)-emitting er perturbations (ire and atf still questionable). perk phosphorylates eif a to up-regulate transcription factor atf that induces the expression of transcription factor chop. ire a signals through its rnase via the splicing of xbp mrna. the active transcription factor xbp s translocates to the nucleus. atf is exported from the er to the golgi complex to enter the nucleus as a potent transcription factor. together, these transcription factors of the unfolded protein response determine the cell fate by the regulation of distinct subsets of target genes toward recovery of er homeostasis and cell survival or the induction of regulated cell death in form of apoptosis and ferroptosis. in addition, the transcription factor atf is differentially translated, up-regulating genes participating in autophagy and other homeostatic pathways. atf activating transcription factor, chop cytidine-cytidine-adenosine-adenosine thymidine-enhancer-binding homologous protein, eif α eukaryotic translational initiation factor α, er endoplasmic reticulum, ire α inositol-requiring transmembrane kinase/endoribonuclease α, perk protein kinase-like eukaryotic initiation factor α kinase, upr unfolded protein response, xbp x-box binding protein . xbp s, x-box binding protein whereby the "s" stands for the spliced form of xbp . sources: refs. [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] residues on nascent peptides [ ] . however, in conditions of er stress, bip is detached from these sensors allowing their activation to trigger pathways, collectively included in the term of upr. this stress response acts as a corrective path, capable of both increasing the er folding capacity and decreasing the incoming polypeptide load. of note, this downstream pathway of each of the three upr sensors appears to have an innate preference for a particular type of er stress. moreover, as reviewed [ ] , upon dissociating from bip, each of the three sensors modifies the er to mitigate stress in its own unique way. for example, atf is often the first sensor to respond to er stress. once atf dissociates from bip, it is translocated to the golgi apparatus for cleavage. the cytosolic domain of atf is then free to move to the nucleus, where it moderates increased expression of several proteins involved in lipid biosynthesis and chaperones. this allows an increase in the volume of the er and provides more chaperone proteins to aid in folding, thus relieving some of the er stress. the other two sensors, ire- and perk, remain as integral er proteins but oligomerize and autophosphorylate following bip disassociation (autophosphorylation, a type of post-translational modification of proteins (see part vi, sect. . ); typical for this biochemical process, a phosphate is added to a protein kinase by itself). under remediable er stress conditions, the three sensors trigger signalling pathways to resolve er stress aiming at maintaining cellular integrity (fig. . ) . they are briefly touched in the following (for reviews and original articles, see refs [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] .). for example, misfolded proteins are dislocated in the cytosol where degradation processes such as the er-associated degradation (erad) and autophagy will clear them, thereby reducing their potential toxicity. characteristically, erad is a protein quality control mechanism conserved in all eukaryotic cells and represents a critical arm of the upr, necessary to alleviate er stress. the erad mechanism results in the selective dislocation of unfolded and misfolded proteins from the er to the cytosol via specific membrane machinery. the erad targets are subsequently degraded by the cytosolic ubiquitin proteasome system (ups). the whole process includes transcriptional activation of a variety of er-associated chaperones and folding enzymes which include but are not limited to bip and the lectins calr, calmodulin (cam), and calnexin (cnx). a pathway that represents the most conserved branch of the upr is mediated by ire- , a multifunctional protein that possesses kinase and endonuclease activities. upon activation, ire- aggregates and autophosphorylates, thereby activating its endonuclease activity to catalyze the unconventional splicing of x-box binding protein (xbp ) via removal of a -nucleotide intron. this processing event changes the open reading frame of the mrna, resulting in the translation of the transcription factor now termed xbp s ("s" stands for the spliced form of xbp ) (for splicing, see box . ) . production of xbp s leads to upregulation of several genes involved in the upr's adaptive phase, for example, expression of er-resident molecular chaperones and protein folding enzymes. activation of perk leads to the phosphorylation of eif α that is required for the initiation of translation. this factor inhibits global protein synthesis by inhibiting the assembly of the s ribosome, thereby reducing er load and promoting cellular survival. at the same time and under these conditions, the transcription factor atf is differentially translated, up-regulating genes participating in protein folding, amino acid metabolism and transport, autophagy, and oxidative stress resistance/redox homeostasis. under conditions of prolonged or severe er stress that the upr cannot resolve (see below), atf also contributes to apoptosis through the induction of the transcription factor chop (for cytidine-cytidine-adenosine-adenosine-thymidine-enhancer-binding homologous protein) and by enhancing oxidative stress and protein synthesis. finally, the third branch of the upr is initiated by atf . the sensor atf is retained at the er under homeostatic conditions but translocates to the golgi apparatus under er stress where it is cleaved by the golgi-resident proteases site- protease (sp ) and sp . this event leads to the release of atf n-terminal fragment, a potent transcription factor that moves to the nucleus, where it binds the er stress response element upstream of a subset of upr genes to activate their transcription. together with xbp , this fragment regulates the expression of several genes with functions in protein folding, protein transport, and lipid biosynthesis, that is, genes involved in re-establishing er homeostasis. strikingly, there is a considerable interconnectedness of the er stress-promoted upr with other innate immune processes. for example, an increasing number of studies support the view that oxidative stress has a strong connection with er stress. during the protein folding process, ros are produced as by-products, leading to impaired redox balance conferring oxidative stress. as the protein in molecular biology, splicing is a modification of an rna after transcription in which introns are removed and exons are joined. thus, an intron is any noncoding nucleotide sequence within a gene that is removed by rna splicing during maturation of the final rna product; an exon is any part of a gene that encodes a part of the final mature rna produced by that gene after introns have been removed by rna splicing. this process is needed for the typical eukaryotic messenger rna before it can be used to produce a correct protein through translation. for many eukaryotic introns, splicing is done in a series of reactions catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snrnps), but there are also self-splicing introns. folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and facilitate the production of misfolded proteins, causing further er stress [ ] . moreover, this stress response functions as a productive source of damps. typically, hsps and calr, like other er chaperones, can translocate to the cytosol and eventually to the surface of cells. once exposed, these molecules can operate as subclass ib- damps to facilitate engulfment of antigens as mostly described in the context of induction of icd [ , [ ] [ ] [ ] [ ] [ ] . via these mechanisms and by crosstalk with other molecular machines of the innate immune system including nlrp inflammasome activation via txnip (see part iv, sect. . . . and part vi, sect. . . . ) , the upr may contribute to sterile inflammation and immunity (for articles, see refs [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). also, several signalling cascades triggered by the three sensors are apparently potent inducers of autophagy at a cellwide level that-as mentioned above-normally has an adaptive/protective function and consists of the three major types: chaperone-mediated, macro-and microautophagy. interestingly, recent evidence has indicated that upr-induced autophagic processes are capable of alleviating the upr pointing to a crosstalk between these two innate immune defense mechanisms [ , ] . strikingly, a complex relationship reportedly exists between autophagy and damps in cellular adaption to stress and injury and cell death characterized by a crosstalk between autophagy induction and secretion or release of damps. in fact, growing evidence indicates that autophagic mechanisms are involved in regulating release and degradation of damps including calr, hmgb , atp, and dna in several cell types [ , , ] . this scenario may contribute to the observation that autophagy is also able to shape a supportive cellular immune response [ , ] . this kind of innate immune interrelationships is, for example, involved in mechanisms of both reperfusion-mediated cell injury and icd of cancer cells (see refs [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). overall, depending on the duration and intensity of the stress, the upr engages different cellular pathways to restore and maintain cell survival, on the one hand, or trigger apoptosis, on the other hand. in cases of severe irremediable er stress, however, the balance is tipped in favor of pro-death signalling; that is, the upr, now mediated by different biochemical pathways, may lead to pro-inflammatory and pro-apoptotic responses resulting in catastrophic rcd (fig. . ) . while the precise pathways of apoptosis induced by er stress are not known, the up-regulated perk → eif α → atf → chop pathway plays an essential role by reversing translational arrest, increasing generation of ros, and promoting calcium efflux from the er. together, these signals lead to cytochrome c release from mitochondria and loss of membrane potential, resulting in apoptosis [ ] . in this context, it is interesting that one pathway of rn (ferroptosis; see sect. . . ) apparently shares a partially overlapping machinery with er stress, suggesting a molecular interconnectivity between these two events [ ] . the issue of er-stress-promoted upr is a parade example of an injury-induced innate immune response that can decide besides life and death of a cell. placed at the very beginning of defense processes of multicellular organisms upon injury, this stress response nicely reflects a hierarchy of damp emission (see part iv, chap. ) . striking is also the existence of an inter-organelle communication, for example, between upr, inflammasome activation, and autophagic pathways which emerge as a homeostatic network determining the switch from adaptive life-saving programs to cell death under stress conditions, where specialized sentinels are localized at organelle membranes to induce the core apoptosis pathway. as briefly sketched in the next section, this innate immune defense response is not only directed against sterile stress but also against pathogen-mediated stress. in the previous section, induction of a upr was mainly exemplified by referring to reperfusion-mediated cell damage and icd of cancer cells, that is, instances of sterile stress. however, growing evidence is coming to light clearly indicating that viruses and bacteria also induce er stress, thereby activating a robust upr. in fact, the constitution of cellular stress responses is meanwhile regarded as the first line of defense against both viral and bacterial infection. however, the outcome is not always in favor of the host; the pathogen may also profit from this stress response, at least under certain circumstances. an increasing number of reports have recently been published on this emerging topic (such as refs [ ] [ ] [ ] [ ] [ ] [ ] ), the quintessence briefly being addressed here. there are several mechanisms described of how a virus can induce er stress. the central mechanisms of perturbation of the er during virus infection can be seen in the production of large amounts of viral proteins by the virus concerned. such accumulation of viral proteins in the er implies a challenge to the protein folding machinery which may cause er stress and, in turn, activate the upr resulting in restoration of the er homeostasis or apoptosis. so far, at least viruses have been found to be able to induce er stress and activate the three upr stress signalling pathways [ ] . moreover, er stress can be caused by viruses via other mechanisms, for example, as a result of er membrane exploitation, imbalance of calcium concentration, or sabotage/depletion of the er membrane during virion release. viral infections may activate these pathways resulting in the inhibition or promotion of viral replication. for example, the perk-mediated global translation shutdown is a very efficient antiviral mechanism, and a similar shutdown by pkr has been used in the interferon pathway to defend against viral infection [ ] . also, the virus-related upr was found to trigger host inflammatory signalling cascade through innate immune signalling pathways that activate nf-κb and ap- transcription factors as a result of chronic er stress. in fact, overexpression of viral proteins in the er has long been known to activate these transcription factors which induce expression of pro-inflammatory cytokines such as il- [ , ] . increasing evidence supports the notion that the upr signalling synergistically interacts with virus-induced signalling to produce inflammatory cytokines and type i ifns. in addition, other lines of studies have shed light on a role of nod and nod receptors in transducing virus-related er stress signals to elicit inflammation [ ] . so far, however, a possible contribution of damps to the promotion of these signalling pathways has not been investigated. importantly, however, the effects of virus-induced upr have been observed not only to inhibit but also to potentiate viral infection. in fact, manipulation of the upr response has become an asset for many viruses to promote their translation, thereby leading to chronic er stress. in other words, during infection, viruses are capable of hijacking the host translational machinery and fill the er with viral proteins. for example, this is the case for many positive-strand rna viruses, which house the virus replication machinery in the protective er-membrane. in fact, viruses need host er to produce increased quantities of viral proteins to continue replication. intriguingly, as discussed [ ] , many viruses have evolved strategies aimed at continuing the replication cycle. thus, viruses were shown to manipulate the host upr in various ways to stimulate protein synthesis capacity and to improve cell survival by inhibiting cellular apoptosis. in particular, the link between the upr and autophagy are intensely discussed to be involved in this scenario. these two systems may act dependently, or the induction of one system may interfere with the other [ ] . thus, experimental studies could demonstrate that different viruses modulate these mechanisms to allow them to circumvent and bypass the host immune response or, worse, to exploit the host's defense to their advantage. according to current knowledge, rna viruses including influenza virus, poliovirus, coxsackievirus, enterovirus, japanese encephalitis virus, hcv, and dengue virus were shown to regulate these processes. for example, recent studies on hcv-infected hepatocytes confirmed the evidence that virus-associated er stress and upr are linked to cellular autophagy. thus, induction of the cellular autophagic response is reportedly required to improve survival of infected cells by inhibition of cellular apoptosis. moreover, the autophagic response was demonstrated to inhibit the cellular innate antiviral program that usually inhibits virus replication. nevertheless, as argued by the authors [ ] , though hcv induces er stress and autophagy, their cause-effect relationship is not clear. notably, the upr is not only modulated by viruses! recent evidence indicates that this stress response plays multiple roles during bacterial infections as well. thus, as comprehensively reviewed by celli and tsolis [ ] , the upr has been shown to be induced in murine lungs by m. tuberculosis (associated with apoptotic events) and also be correlated with helicobacter-induced gastric carcinogenesis. similarly, in vitro infectious models have revealed upr induction in macrophages and epithelial cells infected with either brucella melitensis and b. abortus or listeria monocytogenes. on the other hand, there is evidence suggesting that bacteria can subvert the upr for their own advantage. nevertheless, indications that bacteria can modulate this response are still somewhat sparse. one example refers to l. pneumophila that recruits components of the erad on its vacuole to mediate turnover of bacterial effectors on the vacuolar surface and uses the proteasome to generate amino acids necessary for its intracellular growth. interestingly, like with virus-induced upr, a bacteria-initiated upr may turn out to be of advantage for the host or in favor of the bacteria, but it is not clear in each case whether this response benefits the host or the pathogen. thus, as argued by celli and tsolis [ ] , "modulation of er function during infection by intracellular bacteria can promote bacterial infection by providing a replicative niche, but at the same time the resulting disruption of the secretory pathway can provide a pattern of pathogenesis that aids the innate immune system in recognizing intracellular infection and in mounting an appropriate defence. however, considering the more rapid evolution of bacterial pathogens compared to their hosts, it is likely that bacteria have evolved to modulate the upr to their advantage during infection." the function of the er stress-induced upr appears to provide another impressive evidence in support of the notion that immunity is induced by both sterile and infectious stressful injury and not primarily by invading nonself. in fact, this stress response is at the forefront of any stressful injury and is dedicated to initiating involvement of further damp-promoted defense mechanisms. however, like all the other innate immune processes, the upr may become uncontrolled. together, this may be reason enough that this stress response is involved in the pathogenesis of many human systemic and organ-specific disorders. in fact, the list of human diseases that are pathogenetically associated with er stress and activation of the upr is steadily growing [ ] [ ] [ ] . they include neurodegenerative and metabolic diseases, autoimmune disorders, and atherosclerosis. given such an ever-increasing list of diseases, it is not a surprise that chemical compounds and inhibitors targeting the upr signalling pathways with a high degree of specificity have been and will be further developed [ ] [ ] [ ] . maintaining genome integrity and transmission of intact genomes is a condition sine qua non for cellular, organismal, and species survival [ ] . this homeostatic integrity is threatened by dna damage that occurs in a variety of conditions including but not limited to ionizing radiation, chemical reactions, and viral infections, two of the most dominant conditions being oxidative stress and replication stress. these exogenous and endogenous factors induce diverse lesions in the dna such as nucleotide alterations (substitution, deletion, and insertion), bulky adducts, collapsed dna replication forks, ssbs, and dsbs (see refs [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). in response to dna damage, cells initiate and activate a complex network of cellular signalling cascades that cooperate to sense and repair lesions in dna, denoted as the ddr. this stress response plays an important role in fighting against detrimental effects of cell stress and injury. it orchestrates many processes, including not only dna repair but also regulation of cell-cycle checkpoints, transcription of ddr genes, and autophagy ( fig. . ). the ddr is controlled by three pi k-related kinases (pikks): the atm, the dna-pk, and the atr kinases (mostly nuclear proteins). all three pikks are enormous polypeptides with similar domain organizations and various common structural features. equipped with the capability to autophosphorylate (see above however, at the beginning of this dance, sensors have recently been identified which, as prms in a wider sense or even as a new group of recognition receptors, are capable of detecting dna damage-as manifested by the toxic dsbs or generation of ssdna to activate these three pikks. as already briefly touched in part ii, sect. . . . , two highly conserved multiprotein complexes, mrn and ku, are considered the primary sensors of dsbs to subsequently activate atm and dna-pk [ , , ] . in addition, ssdna is sensed by the recognition molecule rpa that then, analogous to ku, acts as a signalling and repair platform for downstream factors such as the prp , an e ubiquitin ligase involved in pre-mrna splicing, and atr kinase [ ] . other lines of studies have provided evidence suggesting that the protein prp itself may act as the primary sensor of rpa-ssdna to subsequently activate atr [ ] (fig. . ) . in brief, upon dna damage, dsbs are recognized by the mrn complex. following recognition, mrn recruits atm to this dna lesion where it binds to the c-terminus of nbs as a component of mrn [ ] . following binding, atm kinase is activated. however, the exact mechanism whereby mrn activates atm is still not fully understood (discussed in [ ] ). recognition of dsbs is also realized by ku / heterodimer that has been shown to bind broken dsdna ends preferentially [ ] . it is the ku / then that recruits the catalytic subunit of dna-pk (dna-pkcs) at the site of dsbs to form the dna-pk holoenzyme. the major role of activated dna-pk is to promote a peculiar dna repair mechanism called non-homologous end joining (nhej), a pathway that repairs double-strand breaks in dna [ ] . in fact, nhej repairs most dsbs in mammalian cells. as its name implies, nhej involves ligation of two broken dna ends without needing a repair template [ ] . in contrast to atm and dna-pkcs which respond primarily to dsbs, atr is activated by a much wider range of genotoxic stresses, for example, reflected by exposure of increasing amounts of ssdna as a consequence of compromised activity of replication proteins or nucleolytic processing of various forms of damaged dna. in fact, first evidence suggests that ssdna is initially sensed by rpa which then recruits and activates prp [ ] . in turn, prp facilitates the accumulation of atr-interacting protein (atrip, the regulatory partner of the atr kinase) at dna damage sites, thereby activating atr [ , ] . plausibly, the recent discovery of dna damage-sensing molecules such as mrn, ku / , and rpa calls for the definition of those molecules they recognize, that is, broken dna ends at the site of dsbs and ssdna. as outlined in part iv, sect. . . , they have been tentatively sorted into a subclass of cell-intrinsically emitted damps (subclass iic- ). future studies will have to assign their exact place in the world of damps. apart from those damps emitted in the nucleus, other lines of studies lend support to the suggestion that dna damage or dna replication stress, in case of unsuccessful dna repair, promotes release of aberrant dna structures into the cytosol in the form of ssdna and dsdna breaks/fragments which operate as cell-intrinsically emitted dislocated damps. they are sensed by the dna receptor cgas and probably other as-yet-not-identified dna receptors to activate sting-dependent pathways to promote defense pathways [ , ] (compare part ii, sect. . . ; part iv, sect. . . ; as well as part vi, sect. . . ). as discussed by the authors [ ] , it is conceivable that cytosolic dna is released by dysfunctional mitochondria upon dna damage or generated during repair of damaged genomic dna. moreover, as shown in studies on tumor models, the ddr-through the activation of sting-mediated pathways-induces the expression of another class of constitutive damps which are exposed at the cell surface, namely, the mics and different ulbps [ ] [ ] [ ] [ ] [ ] (compare part iv, sect. . . ). of note, however, the ddr does not always result in a happy end. in fact, when unsuccessful in repairing dna damage, the ddr can lead to cellular senescence or-like a upr in case of irremediable er stress-ultimately induces an rcd, most often in the form of apoptosis, less frequently in the form of necrosis. such subroutines of rcd are presumably aimed at mitigating the propagation of potentially mutated cells leading to cancer or other age-related pathologies (fig. . ) [ , , ]. the dna damage response must be regarded as an efficient cell-intrinsic defense process, which is sophistically connected with other stress responses such as autophagy that also plays a significant role in maintaining genome stability [ ] and the er stress-induced upr [ ] . also, of particular interest is the observation that three tiers of damps are involved in this pathway, starting with broken dna ends at the site of dsbs and ssdna in the nucleus immediately generated upon dna damage; followed by dna fragments dislocated in the cytosol and operating as class iic- damps to promote intracellular innate immune signalling; and finishing with the exposure of subclass ib- damps (e.g., mics) able to activate nk, nkt, and γδ t cells (compare part vii, sects. . . , . . and . . ). this phenomenon may again reflect a particular hierarchy in the work of damps to maintain homeostasis. in fact, genomic integrity is of utmost importance and key to human health, and this is retained by the ddr. this stress response, however, may fail in maintaining and restoring homeostasis. accordingly, dna damage has been observed to play a causal role in numerous human pathologies associated with genome instability or aberrant pikk function such as cancer, leukemia, premature ageing, and certain chronic inflammatory conditions. furthermore, there is growing evidence suggesting that uncontrolled ddr signalling is associated with various neurodegenerative diseases, as documented by recent work showing that atm inhibition alleviates pathologies in models of huntington's disease [ ] . this is reason enough that ddr pathways have been and are being explored therapeutically to induce freedom from these diseases. in particular, small molecule inhibitors of atm, dna-pk, and atr are regarded as potential therapeutic agents, for example, as innovative drugs in cancer treatment [ , ] . an increasing amount of publications in the international literature provides convincing evidence that cell-intrinsic, 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with a senescent phenotype biphasic ros production, p and bik dictate the mode of cell death in response to dna damage in colon cancer cells autophagy regulates dna repair through sqstm /p endoplasmic reticulum stress, genome damage, and cancer targeting atm ameliorates mutant huntingtin toxicity in cell and animal models of huntington's disease phosphatidylinositol -kinase (pi k) and phosphatidylinositol -kinase-related kinase (pikk) inhibitors: importance of the morpholine ring targeting atr in cancer medicine the mechanism of mitochondrial superoxide production by the cytochrome bc complex key: cord- -fcno z authors: nan title: molecular aspects of viral immunity date: - - journal: j cell biochem doi: . /jcb. sha: doc_id: cord_uid: fcno z nan mechanisms of t-cell mediated clearance of viruses from the central nervous system are poorly understood, but likely to differ from those employed in the periphery because the cns lacks lymphatic drainage and constitutive expression of mhc class i antigen, and the unique structure of the cns vasculature imposes constraints on access by leukocytes and soluble immune mediators. to study the mechanism by which viruses are cleared from neurons in the central nervous system, we have developed a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (oblv ). grew preferentially in the olfactory bulbs of balbk mice. using in situ hybridization, we found viral rna localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. virus was cleared rapidly from the olfactory bulb between and days. athymic nude mice failed to eliminate the virus demonstrating a requirement for t lymphocytes. immunosuppression of normal mice with cyclophosphamide also prevented clearance. both cd + and cd + t-cell subsets were important as depletion of either of these subsets delayed viral clearance. gliosis and infiltrates of cd + and cd + cells were detected by immunohistochemistry at days. the role of cytokines in clearance was investigated using an rnase protection assay for il-la, il-lp, il- , il- , il- , il- , il- , tnfa, tnfp and ifny. in immunocompetent mice there was upregulation of rna for il-la, il-lp, il- , tnfa and ifny at the time of clearance. nude mice had comparable increases in these cytokine messages with the exception of ifny. induction of mhc-i molecules on cells in infected brains was demonstrated by immunohistochemistry in normal and nude mice, suggesting that ifny may not be necessary for induction of mhc-i on neural cells in vivo. luca g. guidotti, kazuki ando, tetsuya ishikawa, lisa tsui and francis v. chisari. the scripps research institute, la jolla, ca although cytotoxic t lymphocytes (ctl) are known to clear viral infections by killing infected cells, recent studies suggest that they can also suppress the replication of certain viruses by noncytolytic mechanisms. we have examined this area by monitoring the immunopathological and antiviral consequences of antigen recognition by hepatitis b virus (hbv) specific ctl in hbv transgenic mice that express the viral gene products in their hepatocytes. we have shown that intravenously injected ctl rapidly trigger their target hepatocytes to undergo apoptosis, but that the direct cytopathic effect of the ctl is minimal in comparison with the cytopathic effects of the antigen-nonspecific intrahepatic inflammatoly response that they activate. in addition to killing the hepatocyte, the same ctl also downregulate hbv gene expression and completely abolish hbv replication in the hepatocytes that they don't destroy. this noncytolytic antiviral ctl effect is mediated by at least two distinct processes in these animals. first, the ctl cause a quantitative reduction in the steady state content of all hbv mrna species in the hepatocyte, and this is followed by disappearance of all of the corresponding viral proteins in the liver and serum. the ctl initiate this process by secreting ifny and tnfa when they are activated by antigen recognition. since the regulatory effect of the ctl can he prevented completely by prior administration of the corresponding antibodies. nuclear run-on experiments reveal that viral mrna transcription is unaffected despite the profound reduction in hbv mrna content in the liver, suggesting that the ctl-derived cytokines accelerate viral mrna degradation in the hepatocyte. a second noncytolytic antiviral pathway is also activated by the ctl. we have recently shown that hbv nucleocapsid particles, and the replicative hbv dna intermediates that they contain, disappear from the transgenic mouse liver following either ctl administration or partial hepatectomy. the latter of which triggers hepatocellular regeneration without any change in hepatocellular hbv mrna content. these results suggest that preformed hbv nucleocapsid particles may be actively degraded during hepatocyte turnover, and they raise the possibility that similar events might also occur in nondividing hepatocytes that are activated by noncytolytic signals delivered by the ctl. we propose that, in addition to their pathogenetic effect, the comhined effects of the ctl response at die hbv mrna. nucleocapsid and rcplicative dna levels may represent a curative antiviral stimulus during hbv infection. since the virus must contain molecular elements that iespond to these ctl-induced antiviral signals. inactivating mutations at these loci could be very efficiently selected by immune pressure, because a single mutation could abrogate the antiviral effect of a wide spectrum of t cell responses, irrespectrve of epitope specificity. identification of these viral response elements and the intracellular pathways that interact with them may lead to the development of new strategies for antiviral drug design. human fibroblasts infected with hsv are resistant to lysis by cd + cytotoxic t lymphocytes (ctl), yet human b cell lines can be efficiently lysed by these ctl. the effect on human fibroblasts is rapid (within hr of infection of cells), occurring before synthesis of mhc class i is altered by virus infection. a recombinant hsv, f-usbmhc, which expresses mouse mhc class i proteins does not render human fibroblasts sensitive to lysis by mouse ctl. mhc class i molecules are retained in the er of hsv-infected fibroblasts i n a misfolded, unstable form and stability of the mhc complex can be restored by addition of exogenous peptides. using a panel of hsv mutants and ad expression vectors we demonstrated that the hsv ie protein icp was both necessary and s f i c i e n t to cause retention of class i and icp expression in fibroblasts caused the cells to resist lysis by cd + t lymphocytes. icp is a soluble, cytosolic protein and we have found no evidence of membrane association. therefore, it appears that icp inhibits cytosolic stages of the antigen presentation pathway so that antigenic peptides do not reach the er. to date, polyclonal and monoclonal antibodies directed to icp have not specifically precipitated any of the previously described components of the antigen presentation pathway and we have not found icp associated with tap transporter proteins or proteosomes i n these experiments. the effects of icp are being assessed in proteosome and tap transporter assays. gst-icp fusion proteins tightly bind a . kda cytosolic cellular protein which is found in a number of adherent human cell lines but not lymphocytes. the protein has been purified and sequencing is in progress. in addition, radiolabelled icp binds to a single cellular protein of = kda on ligand blots. these proteins are good candidates as cellular targets of icp and as novel components of the antigen presentation pathway. preliminary experiments support the hypothesis that icp is very effective i n blocking cd + t lymphocyte responses in vivo, perhaps explaining the predominance of cd + vs. cd + anti-hsv ctl i n vivo. we expect that icp may be very useful, not only to elucidate antigen presentation pathways, but also to prevent immune recognition of gene transfer vectors and a s a immunosuppressive agent. susceftibility to polyoma virus-induced tumors is conferred by an endogenous mmtv superantigen. aron e. lukacherl, yupo ma , john p. carroll , sara r. abromson-leeman , joseph c. laning , martin e. dorf , and thomas l. benjamin . idepartment of pathology, emory university school of medicine, atlanta, ga , and department of pathology, harvard medical school, boston, ma . susceptibility to tumors induced by mouse polyoma virus varies among inbred mouse strains. we have previously shown that polyoma tumor susceptibility is controlled by products of mhc as well as non-mhc genes. in crosses between mhc-nonidentical strains differing in tumor susceptibility, resistance correlates with dominantkodominant inheritance of the resistant h- haplotype. we have observed the opposite pattern of inheritance of susceptibility in crosses between mhc-identical strains. in crosses between the highly susceptible c wbida mouse and the highly resistant but mhc-identical (h- k) c bwcd.i mouse, polyoma tumor susceptibility is conferred by a single autosomal dominant gene, which we have designated pyvs. pyj does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody responses, or affect intracellular events essential for productive infection or cell transformation by the virus. whole-body irradiation renders cs bwcd.i mice fully susceptible to polyoma-induced tumors, indicating an immunological basis for this strain's resistance. we hypothesized that p y j encodes an mtv superantigen (sag) that confers susceptibility to c wbida mice by deleting precursors of polyoma-specific t cells. we found that tumor susceptibility in (c wbida x c bwcd.i) x c bwcdj backcross mice cosegregated with mtv- . inheritance of mtv- showed perfect concordance with absence of peripheral vp + t cells. genotyping of backcrossmice using markers of simple sequence repeat polymorphisms flanking mtv- showed no evidence of recombination between pyvs and mtv- . strongly biased usage of vp by (a) polyoma-specific cd + ctl from virus-infected c bwcdj mice and by @) cd + t cells infiltrating a polyoma tumor in a virus-immune c bwcd.i host provide further evidence that t cells bearing this mtv- sag-reactive vp domain are critical anti-polyoma tumor effector cells. these results indicate identity between p y j and mtv- sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's t cell repertoire. infection of mice with lymphocytic choriomeningitis virus (lcmv) causes a transient to longlasting immunosuppression dependent upon virus-isolate dose of virus and age, h- , non h- , level of cd + t cells, of cd + t cells and kinetics of neutralizing antibodies of the host. the immunohistological analysis suggests that cd + t cell dependent disappearence of marginal zone macrophages of follicular dendritic cells and of virus infected cells in general correlates with immunosuppression. the details of mechanisms responsible for these findings are now being analysed. a role of this cd + t cell dependent immunosuppression in the establishment of a lcmv carrier state in immunocompetent mice is suggested by the following experiments: the otherwise slow and low neutralizing antibody response agamst lcmv is accelerated and enhanced by cd + t cell depletion at the time of infection, suggesting virus-specific immunopathology being responsible at least partially. the elisa antibody response is not significantly altered under the same conditions but is abrogated if lcmv-specific t cell receptor transgenic mice are infected with high doses of lcmv, indicating, that suppression of the specific antibody response depends upon the relative kinetics of ctl versus antibody responses. whether exhaustion of specific ctl responses is enhanced by similar mechanisms remains to be tested. the role of interleukins of the relative distribution of virus in the mouse and in the various aspects of immunosuppression are now being studied. immunosuppression, caused by cd + t cell-dependent immunopathology, may also be operational in hiv infection in humans. such a pathogenesis of hiv-triggered aids could explain several aspects of the disease process not readily fitting the (unproven) conventional idea that hiv is causing immunodeficiency via direct viral pathogenicity. the cellular immunity against two dna tumor viruses (i.e. human adenovirus type (ad ) and human papillomavirus type (hpv )) was studied with respect to possible immune escape mechanisms and to the development of ctl epitope based peptide vaccines. after identifying an immunorelevant ctl epitope in the ad e a protein to which ctl clones were directed that could eradicate ad e induced tumors in nude mice, an amino acid replacement study of this epitope revealed a point mutation that totally eliminated the possibility to recognize the mutant peptide by the ctl clones directed against the wild-type peptide sequence. new viral constructs were made that contained this point mutation and used to transform mouse embryo cells. however, these mutant tumor cells were still immunogenic and ctl clones specific for these mutant tumor cells were shown to react with a peptide derived from the ad e b protein. these ad eib specific ctl clones, however, were as effective as the ad e a specific ctl clones in the eradication of ad e induced tumors in nude animals, indicating that a choice can be made of immunorelevant epitopes to which an immunization strategy could be developed. in addition, we discovered that by supertransfection of ad e induced tumor cells with the activated ras oncogen the possibility of ad e b specific ctl to recognize the ad e induced tumors was eliminated whereas the ad e a specific ctl could still kill these tumor cells. this might indicate a new mechanism of tumors to escape ctl. in an hpv induced mouse tumor model an immunosubdominant ctl epitope was identified in the e protein that could, upon immunization with that peptide,protect mice against a subsequent challenge with hpv induced tumor cells. by changing the anchor residues in that peptide an even more immunoprotective peptide could be generated. combined, these data indicated a successful use of a ctl epitope based peptide vaccine in the prevention of hpv induced tumors in mice. subsequently this led us to identify relevant ctl epitopes of hpv , that is highly associated with cervical carcinoma in humans, for the major hla-a alleles (i.e. hla-a * , a * , a" , a* and a * ). together these alleles cover a majority of all humans. ctl epitopes were identified through peptide-mhc binding assays followed by in v i m peptide immunizations with high affinity binding peptides to induce primary ctl responses and immunogenicity studies in hla-a transgenic mice. thereafter, memory ctl responses were measured in cervical cancer patients against selected peptides. combined, these data led us to develop a ctl epitope based peptide vaccine that could be of use in hpv induced cervical cancer patients. a clinical trial for this disease is scheduled to start in the fall of . class ii presentation of an endogenously synthesized glycoprotein. carol s. re is^'.'.^, shirley m. bartido', miriam stein', and stephanie diment . , biology department', and center in contrast to class i presentation which is well characterized to use peptide fragements of proteins sythesized in the cytoplasm, exogenously administered experimental antigens enter the class ii mhc pathway through endocytosis. we have been studying the recognition of the glycoprotein of vesicular stomatitis virus (vsv) which can enter either the exogenous or endogenous pathways for presentation to cd + t cells. investigations of the intracellular sites involved, the proteolytic processes involved in the epitope generation, will be discussed. the glycoprotein studied in detail is a truncated form of the wt type glycoprotein, termed poison tail (gpt) . expressed with a vaccinia virus vector, the gpt remains endo h sensitive and never becomes endo d sensitive, indicating that it is restricted to the endoplasmic reticulum. gpt is degraded in the er, and w e believe the degradation products include the immunogenic epitopes recognized by a panel of lad and i-ed t cell clones and hybridomas. lmmunofluorescence studies have confirmed the er localization. flow cytometric evaluations s h o w that the gpt never appears on the cell surface, in contrast to the wt g. the peptides generated are not secreted; using an innocent bystander assay, gpt-infected cells are incapable of sensitizing 'cr-labeled uninfected apc. this contrasts with the rapid ability of supernatants from wt g-vaccinia virus-infected cells to sensitize apc for t cell recognition. investigations of the characteristics of the enzymes contributing to the degradation of the gpt have shown that a reducing environment is essential, as diamide treatment of cells prevents degradation. lysosomotropic drugs (eg. nh,ci and leupeptin) d o not alter the half-life of the protein, but do prevent presentation of the peptides; this is inconsistent with an autophagic component to the proteolysis. ph optima are physiological, as ph environment inhibits the enzyme activity. inhibitors of enzyme classes are consistent with a trypsin-like, and not cystein-, cathepsin b-, or chymotrypsin-like class. supported by nih grant al to csr. (emcv) and mengovirus are related members of the cardiovirus genus of picomaviruses. their rna genomes encode a large polyprotein which is cleaved proteolytically in co-and post-translational reactions to yield all mature viral proteins necessary to establish an infection. although originally thought to be exclusively murine in host range, both viruses actually infect a wide range of mammals. emcv has caused devastating epizootics in captive primates (eg: macaques, chimps and baboons), domestic pigs and exotic zoo collections (elephants, lions and tigers). death, following ingestion of virus-contaminated material, is rapid, and caused by extensive meningoencephalitis and virus-induced damage to the cns. myocarditic lesions are common in older animals. when administered intracerebrally, the ld,, for emcv strain r is about pfu. we are studying the pathogenesis of emcv and mengo with engineered cdna plasmids containing infectious viral sequences. many plasmids contain h'uncated versions of the unusual ' noncoding homopolymeric poly(c) tract that is a hallmark of these cardioviruses. short poly(c) mengoviruses grow very well in tissue culture but are - 'z fold less pathogenic to mice than the wild-type strains. animals receiving sublethal doses of short-tract mengo strains develop high titers of neutralizing antibodies, exhibit potent ctl responses and acquire lifelong protective immunity against challenge with wild-type virus. the genetic stability of the short-tract strains, even upon serial brain passage, mark them as safe, efficacious live vaccines. currently, we believe the poly(c) phenomenon is due to interference by the wild-type virus sequences (long poly(c) tract) with normal cellular cytokine induction mechanisms (ie: ifa and ifp) during the initial stages of animal infection. the targeted cells are probably macrophages, and their singular ability to correctly respond or not respond to poly(c) tract length during the first few hours of infection determines whether an inoculated animal will live (protectively vaccinated) or die. the short-tract viruses probably induce if in the macrophages, and are consequently killed then rapidly cleared from the host in related experiments we've found that attenuated mengo strains can easily carry large heterologous insertions within their genomes, and express these sequences into protein them during replication in animals. the resulting immune response (b cell and ctl) to the chimeras is directed towards the foreign sequences (epitopes) as well as towards the mengo proteins. a chimeric hiv vaccine, a rabies vaccine and an lcmv vaccine have been developed and tested. the lcmv chimera seems especially effective, as a single pfu of this engineered mengo strain, administered orally to a mouse, is sufficient for complete immunogenic protection against intracerebral challenge with wild-type lcmv virus. rsv is the most common cause of serious viral lower respiratory tract disease in infants and children. we have recently renewed our efforts to generate a safe and effective live attenuated rsv vaccine for topical administration that will overcome the deficiencies of previously studied live and non-living rsv vaccines. this vaccine will be a bivalent vaccine consisting of subgroup a and b live attenuated virus components. since the peak incidence of severe disease caused by rsv is in the -month old infant, an rsv vaccine will need to be effective when given to -month old infants. based on the success of live poliovirus vaccines given early in infancy, it is anticipated that the intranasally administered live virus vaccine will infect and induce a protective local and systemic immune response even in infants with passively acquired maternal antibodies. the main approach that we have taken in this effort to develop the live rsv vaccine is to introduce one or more ts mutations by chemical mutagenesis into a cold-passaged virus (cprsv) that had been partially attenuated by the acquisition of host-range mutations selected by passage in cells of a heterologous host species. we have developed a large set of cprsv subgroup a rs mutants (termed cprs mutants) that contain the host-range mutations selected during cold passage and two or more ts mutations introduced by chemical mutagenesis. these mutants have been evaluated in virro for their level of temperature sensitivity and in vivo in rodents, chimpanzees, and humans. a large set of rsv subgroup b cpts mutants has been similarly produced and evaluated. the immunogenicity and protective efficacy of three candidate live attenuated rsv vaccine strains that represent a specaum of attenuation were evaluated for protective efficacy in chimpanzees. prior to infection some of these animals were given rsv immune globulin by the iv route to simulate the condition of the very young infant who possesses passively-acquired maternal rsv antibodies. the three candidate vaccine strains were immunogenic and induced significant resistance to rsv challenge in both groups of chimpanzees. interestingly, the chimpanzees infused with rsv antibodies prior to immunization were primed more effectively for an unusually high serum neutralizing antibody response to infection with challenge virus than chimpanzees which did not receive such antibodies. this high booster response occurred despite marked reshiction of replication of the challenge virus. the evaluation of two candidate vaccines in seronegative human infants will also be described. rs virus is immunologically interesting for at least t w o reasons: ) upper respiratory reinfection occurs despite previous exposure and demonstrable immunological memory: ) humans or rodents previously immunised against virus infection can show enhanced disease during reinfection. others have shown that passive transfer of antiviral antibody either protects against virus infection or has no effect, and there is no evidence of antibody enhancement of disease in vivo. by contrast, t cell immunity appears closely associated with disease augmentation. we have focused on examining the immunological mechanisms of disease enhancement in mice. initial studies showed that transfer of cd + cytotoxic t lymphocytes (ctl) causes rapid virus clearance from the lungs of rs virusinfected mice, but also increased disease severity with alveolar haemorrhage and polymorphonuclear (pmn) cell recruitment to the lung. this disease (reminiscent of shock lung) could sometimes be fatal, whereas normal mice recover well from similar doses of rs virus. next, we compared the effects of cd ' and cd + t cells, using polyclonal t cells separated immunomagnetically from mixed lines grown in vitro with viral antigen. cd ' t cells were more pathogenic than cd + t cells in a dose-for-dose comparison, but that the type of pathology varied depending on the type of cell injected. while testing recombinant vaccinia viruses expressing single rs viral proteins for their ability to protect mice against infection, we observed that animals sensitised t o the major surface glycoprotein g (attachment protein) developed lung eosinophilia after challenge with rs virus intranasally. t cell lines from the spleens of mice sensitised with various recombinant vaccinia viruses were established. those form mice primed with the m ( k) protein were predominantly cd ' ctl, and that produced few cytokines. those from mice primed with fusion protein (f) generated mixed t cell lines with both t h l cd + t cells, and ctl. mice primed to g protein gave rise to predominantly cd ' t cells producing th cytokines. ln vivo transfer of these cell lines into na'ive rsv infected mice reproduces the patterns of disease seen in mice sensitised in vivo with the respective antigens. the mouse model of rs virus disease therefore has excellent potential for illustrating mechanisms of lung immunopathology. the eye is a complex organ whose function is to transmit light images through different cell and tissue layers and liquid media to a neurosensory retina. elements as could occur when invading pathogens arrive and an inflammatory response with its swelling, plasma protein extravasation, leukocyte infiltration and tissue damage results. inflammatory responses when possible and rely on immune defenses which do not involve tissue distortion and damage. restricting tissue damaging responses is not always effective and the process is best developed in response to agents delivered to locations such as the anterior chamber. where an inflammatory response is initiated which may result in ocular impairment. such herpetic stromal keratitis (hsk) is a common cause of blindness in man. animal model studies indicate that hsk is a multi-step process initiated by virus in an avascular structure. hsk fails to occur in the absence of t cells or replicating virus. disappears several days before a visible inflammatory response becomes evident. evidence will be presented that the secondary agonists which drive the inflammatory response may not be viral antigen(s) per s e . multiple cell types are involved in hsk, with the respective role of functional sets of lymphocytes changing according to the clinical phase of the disease. in addition, nonspecific inflammatory cells such as neutrophils and nk cells also influence the severity of lesions. basically the reaction begins with t cells that produce type one cytokines, particularly ifn-y, dominating the scene, but during remission type cytokines, notably il- , appear as mechanistically involved. from the use of knockout mice for various immunological parameters, evidence will be presented that numerous mechanisms of pathogenesis may be at play during hsk. damage to corneal tissues in all systems appear to involve tnfo. a second ocular damaging event in which immunopathology is at least partially involved is herpetic retinal necrosis. evidence that this disease may involve the immunopathological role of cd t cells and protective effects by cd ' t cells will be presented, as will be suggestions by which the pathological events are mediated at the molecular level. thus it is in the eye's functional interest to limit acute viral infections and live vaccines often confer long-term immunity the nature of t and b cell memory is different. b cell memory is manifested not only by the presence of memory b cells but also by continuous antibody production in contrast, the effector phase of the t cell response i s shortlived and long-term t cell memory is due to the presence of 'quiescent' antigen specific memory t cells that are present at higher frequencies and are able to respond faster upon re-exposure to virus due to increased levels of adhesion molecules in this talk i w i l l present our results on. (i) the bone marrow as a major site of long-term antibody production after acute viral infection, (ii) the role of c d ' t cells and b cells (immune complexes) in maintaining cd + t cell memory, (iii) the role offas antigen in regulating t cell responses, and (iv) the efficacy ofvarious antigen delivery systems in inducing long-term t cell memory sendai virus is a natural respiratory viral pathogen of mice. intranasal infection of mice with the virus provokes a virus specific antibody-forming-cell reaction that exhibits a distinct kinetic pattern in the lymph nodes that drain the respiratory tract, in the spleen, and in the bone marrow. the bone marrow afc population is extremely long-sustained, and supports an active humoral response that essentially persists for the lifetime of the infected animal. thus the conventional categories of "primary" and "secondary" response may not apply to the humoral response of mice naturally exposed to respiratory viruses. paradoxically, the population of b cells that reacts most rapidly to sendai virus infection does not itself secrete antibody, but can he demonstrated by the recovery of hyhridomas that secrete "polyspecific" antibodies. the activation of this polyspecific b cell population is, like the humoral response, extremely persistant. viral infection thus sets in train multiple b cell "memory" processes. variation in the rules of development and turnover of different b cell populations constrains the mechanisms that may operate to generate these different forms of memory. establishment and maintenance of t cell memory to respiratory viruses, peter c. doherty, sam hou, christine ewing, david topham, anthony mcmickle, james houston, and ralph tripp, department of immunology, st. jude children's research hospital, memphis, tn . the analysis of the development and memory phases of the cd + "helper" n h ) and cd + cytotoxic t lymphocyte (ctl) responses to the respiratory pathogens, influenza virus and sendai virus (parainfluenza type ) have been characterized by a combination of limiting dilution analysis (lda) for determining th and ctl precursor @) frequency and facs separation of lymphocytes with different activation phenotypes. the interpretation at this stage, largely based on the analysis of the ctl response, is that the development phase of t cell memory and the primary response are synonymous. virus-specific ctlp are produced in considerable excess of the numbers required to provide the effector ctl that terminate the primary infection, with only a fairly small proportion localizing to the target organ (the lung) that supports virus growth. even when many of the proliferating ctlp are killed by administration of a small dose ( mgkg) of the dna-targeted drug cyclophosphamide (cy), there is no indication of immune exhaustion. the cd + th response has, at this stage, not been analyzed through the course of the primary infection. use of the lda approach to determine thp frequencies is inherently more difficult, as the "read-out'' is lymphokine production and there is considerable "bystander" activation in these primary responses to respiratory viruses. memory thp and ctlp are characterized initially by the expression of an "activated" phenotype: cd -high, l-selectin-low, cd d (vla- ) high. after some months, an increasing proportion of the memory t cells revert to the l-selectin-high cd d-low form typical of naive ctlp. the change, which is never absolute, seems to occur first with cd d and the rate varies for different viruses. current experiments are addressing the possibility that intercurrent infection, particularly with the mouse y-herpesvirus which causes persistent infection of lymphoid tissue, may be inducing a switch back to the activated pattern, as a consequence of "bystander" effects, or "low affmity" stimulation via the clonotypic tcr in responding lymphoid tissue. the question of such cross-reactivity and/or exposure to "high lymphokine" environments for the long-term maintenance of memory is also being addressed. to study the factors which regulate the generation and persistence of specific t cell memory we have used model systems utilizing t cell receptor transgenic mice as a source of enriched naive cells which can be either cultured in vitro to generate effector populations or restimulated in adoptive hosts. in either case one can visualize the development of an expanded effector population. we have documented that the proliferation and il- production of the naive t cells depends on their activation by apc expressing high levels of co-stimulatory molecules. we find that b . and icam-i as costimulators strongly synergize and that increased t cell receptor triggering can both increase the magnitude of the response and decrease its dependence on costimulation. when cytokines il- vs il-i /ifny are present at the initiation of the response of either cd or cd cells they dictate that the effectors generated will be polarized either towards il- and il- secretion or il- and ifny secretion, respectively. the fate of the effector population generated and followed in vitro, also is tightly regulated by ag, cytokines and probably by costimulation. cd effector cells not re-exposed to ag, produce no cytokines and they die within - days. effectors restimulated with ag make massive amounts of cytokines, regardless of the presence of cytokines, at low densities of ag and with little dependence on costimulation. when there is little il- produced and no cytokines added, effectors die rapidly by apoptosis. however the combination of il- and tgfp block apoptosis and support expansion of the effector population which is greatly enhanced by periodic ag stimulation. some conditions favor the reversion of effector-like cells to a more resting memory phenotype and these are being further explored. we have also examined the development and maintenance of memory after transfer of effector cells to adoptive hosts. long-lived polarized memory populations are generated from the polarized effectors and these persist for prolonged period in the absence of apparent ag stimulation. this supports the idea that factors other than antigenic stimulation, present in situ can support the expansion and maintenance of memory cells. the rabies glycoprotein (g) is the only external protein of the virion and is therefore responsible for any interaction that rabies makes with the host cell during the first steps of the virus cycle. the g protein is also the target of neutralizing antibodies. there are around trimers of g at the virion surface which constitute the spikes visible by electron microscopy. upon exposure to slightly acidic ph, the glycoprotein undergoes a conformational change which results in ion er and less regular spikes. strikingly and quite differently from influenza hemagglutinin, this conformational change is reversible: if the p d is risen back to . , the s ikes re ain their neutral configuration ( ). probably as a consequence, the viral infectivity is totally preserved after an exposure of hours at p . an cf t, which induces the conformational change, followed by an incubation at neutral ph. since the conformational change is reversible, there is a ph-dependant equilibrium between the native and the low-ph conformation: the higher the ph, the more spikes are in their native configuration. two main antigenic sites and several minor sites have been identified on the native rabies glycoprotein ( ). specific amino acids belonging to each of the two major antigenic sites are important or essential for viral virulence. for instance a lysine in position , which is part of antigenic site , is important, although not essential, for the viral virulence. similarly, the arginine , which belongs to antigenic site , is essential for pathogenicity while dispensable for multiplication in cell culture (reviewed in ). viral strains mutated at arginine have lost the capability to penetrate certain categories of neurons, suggesting that this mutation affected the recognition of specific receptors or subsequent interactions necessaly for the penetration of the virus at nerve terminals. therefore the two main antigenic sites are regions of the glycoprotein which also interact specifically with neurons in the animals. we have found that neutralization requires the fixation of at least one or two igg for every three spikes, irrelevant of the anti enic site recognized by the antibody ( ). most neutralizing antibodies recognize conformational epitopes which are accessible on the native configuration of the protein. some epitopes remain accessible also on the acidic configuration while others are not. in addition, a minority of antibodies recognize epitopes which are only accessible on the acidic conformation. this is not unlikely in view that each spike has a certain probability to undergo a conformational change, even at neutral ph. in consequence the surface of the virus probably fluctuates and g epitopes which are not accessible on the native glycoprotein could be transiently exposed. conformational flexibility at neutral ph and physiological temperatures has also been observed for poliovirus ( ). structural flexibility of external proteins could have important implications in virus-host interactions. katpus, norlhwestern university medical school, chicago, il theiler's murine encephalomyelitis viruses (tmev) are endemic enteric pathogens of wild and colony-reared mice. lntracerebral inoculation of susceptible mouse strains leads to a chronic, progressive inflammatory demyelinating disease of the central nervous system (cns) characterized clinically by an abnormal gait, progressive spastic hind limb paralysis and urinary incontinence, and histologically by parenchymal and perivascular mononuclear cell infiltration and demyelination of cns white matter tracts. demyelination is related to persistent cns viral infection. due to the similarity in clinical and histological presentation, tmev-induced demyelination is considered to be a highly relevant model of multiple sclerosis (ms). our current interests are in determining the phenotype, fine specificity, lymphokine profile and tcr usage of cns-infiltrating cells involved in the effector stages of tmev-induced demyelination. based on a variety of experimental evidence, it is clear that demyelination induced in sjuj mice by infection with the bean strain of tmev is a thl-mediated event: (a) disease induction is suppressed in t cell-deprived mice and by in vivo treatment with anti-i-a and anti-cd antibodies; (b) disease susceptibility correlates temporally with the development of tmev-specific, mhc-class il-restricted dth responses and with a predominance of anti-viral lgg a antibody; (c) activated (le., ll- rc) t cells infiltrating the cns are exclusively of the cd + phenotype, and (d) proinflammatory cytokines (ifnq and tnf-p) are predominantly produced in the cns. we have mapped the predominant thl epitope on the virion to amino acids - of the vp capsid protein. a thl line specific for vp - exacerbates the onset of demyelination in recipient mice infected with a suboptimal dose of tmev. tmev-infected sjuj mice fail to exhibit peripheral dth and t cell proliferative responses to the major myelin proteins, mbp and plp, and pre-tolerization with neuroantigens has no affect on the incidence or severity of tmev-induced demyelinating disease, whereas neuroantigen-specific tolerance prevents the induction of relapsing experimental autoimmune encephalomyelitis (eae). in contrast, tolerance induced with intact tmev virions specifically anergizes virus-specific thl responses and results in a dramatic reduction of the incidence and severity of clinical disease and cns dernyelination in sjuj mice subsequently infected with tmev. these results have important implications for a possible viral trigger in ms as they indicate that chronic demyelination in tmev-infected mice is initiated in the absence of demonstrable neuroantigen-specific autoimmune responses and are consistent with a model wherein early myelin damage is mediated via primarily by mononuclear phagocytes recruited to the cns and activated by pro-inflammatory cytokines produced by tmev-specific thl cells. the concept that prions m novel pathogens which are different fium both viroids and viruses has received increasing support from many avenues of investigation over the past decade. enriching fractions from syrian hamster (sha) brain for scrap= prion infectivity led to the discovery of the prion protein . prion diseases of animals include scrapie and mad cow disease; those of humans present as inherited, sporadic and w o r n neurodegenemive disorders. the inhecited human pion diseases m genetically linked to mutatim in the prp gene that result in non-conswative amino acid substitutions. transgenic v g ) mice expressing both sha and m o w @lo) prp genes were used to demonstrate that the "specie bank?' for -pie prions resides in the primary structure of pip. this concept was strengthened by the results of studies with mice expressing chimeric mdsha transgenes &om which "artificial" prions have been synthesized. similar chimeric mdhuman (hu) rp transgenes were constructed which differ from m o w by amino acids between residues and . au of the tg(mhu m) mice developed neurologic drsease - days after inmulation with brain homogenates from three patients who died of creutzfeldt-jakob disease (cjd). inoculation of tg(mhu m) mice with cjd prims produced mhu mprpsc, inoculation with mo prions produced moprw. ihe patterns of meluzmprpc and mom% accumulation in the brains of tg(mhu m) mice wen differenl about % of tg(huprp) mice expressing huf" and non-tg mice developed neurologic diseane > days after inoculation with cn, prions. the different susce@uies of tg(hw) and tg(mhu m) mice to human prions indiate that additional species specific factors such as chaperone proteins are involved in prion replicaton. diagnosis, prevention and treament of human @on diseases should be faciliated by tg(mhu m) mice. in other sindies, tg mice were compared expressing wt and mutant moprp. overexpression of the wtmoprp-a aansgene - -fold was not deleterious to themiw but it did shorten scrapie incubation times from - d to - d after inoculation with murine m p i e pnons. in contrast, overexpression at the same level of l morp-a transgene mutated at codon (corresponding to codon in hurp) pmdnced spontaneous, fatal neurcdegeneration between and d of age in two lines of tg(mohp-pio l) mice designated and . genetic crosses of tg(moprp-p l) mice with gene targeted mice lacking both rp alleles ( p m -p ) produced anhats with a highly synchronous onset of illness between and days of age. the t g~o p r p -p l o l l ) ~~ mice had numerous prp plaques and widespread spongiform degeneration in contrast the tg and mice that exhibited spongifonn degeneration but only a few prp amyloid plaques. another line of mice designated tg overexpress the mutant transgene - -fold and develop fatal neurodegeneration behveen and d of age. tg mice exhibited the most severe spongiform degeneration and had numerous, large pip amyloid plaques. while mutant moprpccploll) clearly produces neurodegeneration, wtmoprpc profoundly modifies both the age of onset of illness md the mumpathology for a given level of transgene expression. our tidigs and those from other smdies suggest that mutant and wtprp interact, phaps through achaperone-like protein as noted above in sndies of tg(mhu m) mice, to modify the pathogenesis of the dominantly inhe&ed prion diseases. anton, heidi t. link, and jonathan w. yewdell, laboratory of viral diseases, niaid, bethesda, md - . cd ' lymphocytes (tcd +) play an important role in host immunity to viruses and other intracellular parasites. virus-specific tcdi+ recognize mhc class i molecules in association with peptides of to residues derived from viral proteins. this presentation will focus on how and where antigenic peptides are generated by cells. to begin to characterize the nature of proteases involved in the generation of antigenic peptides from cytosolic proteins, we used a panel of recombinant vaccinia viruses expressing different forms of influenza virus nucleoprotein (np). we found that the efficiency of generation of two np peptides is related to the metabolic stability of the source gene product. there has been considerable speculation that such short lived proteins are degraded by proteasomes in a ubiquitin-targeted process. our observations, however, call into question the importance of ubiquitin targeted-proteolysis in generating antigenic peptides from exogenously provided or endogenously synthesized viral proteins. we also examined the extent to which antigenic peptides can be generated in the endoplasmic reticulum (er). we found that antigenic peptides could be produced from short precursors ( residues) hut not from a number of full length proteins (influenza virus hemagglutinin, np, ovalbumin) that are targeted to the er by a nh -terminal signal sequence. peptides were generated much more efficiently from the cooh-terminus of the residue precursor than from the nh -terminus. these findings indicate that the er has a much more limited capacity than the cytosol to generate antigenic peptides, but that er proteases (particularly aminopeptidases) could perform the final proteolytic steps in the generation of class i binding peptides from precursors imported from the cytosol by tap, the mhc encoded peptide transporter. potential advantages of synthetic peptide or engineered recombinant vaccines are that they can be limited to contain only the specific antigenic determinants for desired responses without other determinants that elicit unwanted responses, and that the sequences of the determinants themselves can be modified to enhance potency or breadth of crossreactivity. however, they can have the disadvantage that any single determinant may be presented by only a limited selection of major histocompatibility complex (mhc) molecules of the species. to overcome the problem of mhc polymorphism, we have identified determinants presented by multiple mhc molecules, and have also located multideterminant regions of the hiv- envelope protein that contain overlapping determinants each presented by different class i mhc molecules, so that the whole multideterminant region is presented by multiple mhc molecules of both mouse and human. we have made use of "cluster peptides" spanning these multideterminant regions of the hiv- envelope to provide help for neutralizing antibody (ab) and cd + cytotoxic t lymphocyte (ctl) responses to peptides attached to these helper regions. these synthetic peptide vaccine constructs containing the p peptide from the v loop of hiv- iiib or mn, elicited both neutralizing ab and ctl in multiple strains of mice. the cluster peptides inducing helper t cells were essential for elicitation of ab and ctl to the p segment of both iiib and mn strains of hiv- in mice of several mhc haplotypes. several adjuvants were compared for their ability to elicit both ctl and ab simultaneously, without one response inhibiting the other. a single formulation in incomplete freund's adjuvant (ifa) could elicit all responses, neutralizing ab, ctl, and th helper cells. the ctl specific for the mn strain p peptide crossreacted with strains sc, sf , , and cdc . the peptides in f a also elicit high titers of antibodies in rabbits. boosting was found to enhance ctl responses as well as ab responses. these constructs are being prepared for a human immunotherapy trial. these vaccine constructs are potent and also avoid sites on gp that are known to elicit enhancing antibodies or autoimmune responses that might conmbute to disease pathogenesis. however, we can potentially improve on these by tinkering with the internal structure of the individual epitopes. we have found that replacing a negatively charged glutamic acid residue with an uncharged amino acid in one of the helper determinants makes it to -fold more potent in binding to the class i mhc molecule and in eliciting murine helper t cells that still recognize the natural hiv- sequence. thus, such a modified peptide should be more potent as a vaccine, while retaining the ability to elicit t cells that will respond to hiv proteins that of course do not have the altered sequence. we are currently mapping the critical residues for presentation of one of these peptides by human hla-a , with the intent of developing modified peptides that will be more potent as components of a human vaccine. thus, by leaming how these peptides bind to mhc molecules and tcell receptors, we can design internally modified determinants to construct more potent or more crossreactive second generation vaccines. we are testing these vaccine approaches in a mouse model in which mice can be protected against tumor cells expressing hiv proteins as would an hivinfected cell. dna vaccines, comprised of non-replicating plasmids encoding viral proteins, are capable of generating protective immunity in animal models of several viral diseases. in preclinical models of influenza infection, reduced viral shedding was observed in dna-vaccinated ferrets after challenge with the human clinical virus strain, a/georgia/ . cross-strain protection was conferred by dna encoding the major internal proteins (nucleoprotein, np, and matrix, m ) and the surface protein haemagglutinin (ha) from the antigenically-distinct previous virus strains, a/beijing/ and a/hawaii/ . this protective efficacy was greater than that seen by immunization with the widely-used clinical vaccine composed of killed a/beijing/ virus. thus, compared to a killed virus vaccine, protection seen with the dna vacane against a drifted virus strain was greater. we previously demonstrated that immunization of mice with np dna generated mhc class i-restricted cytotoxic t lymphocytes. mice likewise were protected from death and morbidity following cross-strain challenge'. ha dna vaccines generated neutralizing antibodies in mice, ferrets and primates, and provided protection in m i d and ferret models of influenza. in animal models of other viral diseases, immune responses and protection against viral challenge have been seen after immunization with dna encoding viral proteins. dna encoding hiv gp generated ctl and neutralizing antibodies in monkeys. antigen-specific proliferative responses and, in mice, secretion of high levels of yifn relative to levels of il- , months after immunization were also observed . immunization of rabbits with dna encoding l , the major viral capsid protein of cotton tail rabbit papilloma virus (crpv), resulted in neutralizing antibodies and protected against the development of warts after inoculation with crpv. mice immunized with dna encoding the glycoprotein gd from herpes simplex virus type (hsv- ), developed neutralizing antibodies and were protected from death when subsequently challenged with hsv- . dna vaccines were protective in animal models of various viral diseases. neutralizing antibodies, helper t cells (thl) and cytotoxic t cells were generated. cross-strain protection due to cellular immunity was demonstrated. ' science, - , dna cell biol, the profile of a neurovirulent virus is determined by its mechanism of entry into the cns (neuroinvasion), the type of cns cell in which it replicates (neurotropism) and its ability to cause pathologic effects in the brain (neurovimlence). whereas neuroectodermal cells, especially neurons, are the target cells of most neurovirulent viruses, the main target cell in the brain for siv and other lentiviruses is the macrophage. infection in, expression of viral antigens by and products of siv replication exported from these cells result in inflammation and degenerative changes in the brain and concomitant loss of neurons. siv strains that are mainly t-cell tropic cause transient activation of t-cells and during this period, infected t-cells cross the blood brain barrier and localize in the brain causing persistent hut minimally productive infection and minimal neuro pathologic effects. viral proteins but not virions are produced continuously. by virtue of the tropism of the virus for cd t cells, many infected animals eventually become immunosuppressed and develop aids, but not classical ueurological disease. viruses which are macrophage tropic invade the brain presumably also in t lymphocytes and the viruses infect macrophages in the brain. however, productive virus replication is minimized by antiviral cd t cells which suppress (kill?) all virus producing cells throughout the body, including the cns. productive virus replication in brain macrophages and accompanying inflammatory changes develop only when cd cells fail i.e. after profound immunosuppression sets in. the neurological disease that results from productive virus replication in macrophages in the brain therefore depends on presence of an appropriate macrophage-tropic viral phenotype invading the neuropil and development of immunosuppression in the host. the neurological disease could therefore be defined as one of the aids syndromes. the adenovirus (ad) early transcription region (e ) codes for more than polypeptides, four of which have already been shown to alter the immune response to ad infection. the amount of the class i major histocompatibility complex (mhc) on the plasma membrane can be reduced by the binding of the ad e gpl k protein to the mhc heavy chain, which prevents transport of the complex out of the endoplasmic reticulum. this process interferes with presentation of viral peptides to cytotoxic t lymphocytes. cytolysis by tumor necrosis factor-o (tnf) is inhibited by distinct viral polypeptides, of which (the ad e . k or the complex of the . k and . k proteins) are coded in the e region. the e polypeptides are translated from a family of viral mrnas, that are synthesized from a single viral promoter and processed by alternative splicing. we have studied the functions of the e polypeptides in several murine models. the goals of these experiments were to determine the effects of the ad e polypeptides in acute and persistent viral infections as well as in a transplantation model designed to measure whether these viral immunoregulatory proteins would abrogate allogeneic graft rejection. in a vaccinia virus (v.v.) pneumonia model, in which the isolated ad e . k or ad e gpl k genes were inserted into the v.v. pathogen, the ad anti tnf polypeptide increased viral virulence but the ad anti mhc had no effects. in addition to manipulating the ad e genes in viral constructs, several transgenic mouse lines containing the ad e genes have been constructed for these experiments. the e genomic dna behind the rat insulin promoter (rip) has been used to generate transgenic animals. islets from rip-e transgenic animals (h- b'd) have been transplanted allogeneically to h- d recipients and remained viable, secreting insulin until the end of the experiment at days; in contrast, control nontransgenic islets of the same genotype were rejected by - days. the e genes behind the native e promoter have been inserted into mouse embryos to generate transgenic animals, and the expression of the transgene monitored in multiple organs. the e promoter of the transgene is responsive to stimulation by the ad e a following infection with an e minus ad and can also be upregulated by administration of bacterial lipopolysaccharide. the effects of this transgene on ad pathogenesis are currently being studied. thus, these viral immunoregulatory genes have been shown to alter viral pathogenicity during acute infection and to downregulate the host immune response sufficiently to permit islet cell transplantation. these results on manipulating the ad e genes for the control of the host immune response also have implications for designing adenovirus vectors for gene therapy. "emerging" infections can be defined as infectious diseases that either have newly appeared in the population, or that are rapidly increasing their incidence or expanding their geographic range. recent viral examples include aids, ebola, and hantavirus pulmonary syndrome (fnst identified in a outbreak in the southwestern u.s.). emerging viral infections show a number of common features. most "new" viruses derive from existing viruses that move into new areas or acquire new hosts ("viral traffic"). many are zoonotic (originating from animal sources) (even pandemic influenza appears usually to be a reassortant originating in wildfowl). ecological or environmental changes (either natural, or, often, man-made) may precipitate emergence of new diseases by placing people in contact with a previously unfamiliar zoonotic reservoir or by increasing the density of a mtud host or vector of a pathogen, increasing the chances of human exposure. upon introduction into a human population from a zoonotic reservoir, the newly introduced virus may cause localized outbreaks of disease. some may show rapid variation and evolution upon introduction, and some evidence suggests a role for immune selection in this process. a few viruses (such as hiv) may succeed in establishing themselves and disseminating in the human population, becoming truly "human" infections. human activities can also play an important role in establishment and dissemination. migrations from rural areas to cities, now an accelerating worldwide phenomenon, or other displacements, can introduce remote viruses to a larger population; the virus may then spread along highways and (globally) by air travel. the development of an effective system of surveillance and rapid response is essential, but resources for this are presently inadequate. vaccine development, production, and deployment problems also need to be addressed. immunopathology may be a key feature of many of these infections, a number of which manifest as hemorrhagic fevers. many of the life threatening complications are due to increased vascular permeability. the resemblances to septic shock suggest that cytokines (such as tnf) are likely to be important in the pathogenesis of these infections. the response of cells, such as the macrophage, that induce or synthesize key cytokines, may be an important element, and the ability to infect these cells may be one common denominator. why some viruses elicit this response, while other closely related viruses do not, cannot yet be predicted from molecular data. better understanding of these aspects of the immune response should lead to additional therapeutic strategies. (supported by nih grant roi rr .) genetic approaches have been used to detect and characterize numerous previously unidentified hantaviruses. puumalarrospect hilvsin nombre-like viruses or virus variants are present throughout north and south america, europe and russia. several of the american viruses identified are associated with the newly recognized hantavirus pulmonary syndrome (hps), a severe respiratory illness with high mortality. the genetic relationships of these and previously characterized hantaviruses have been studied by phylogenetic analysis of the nucleotide sequence differences located in pcr bgments amplified from the g encoding region of the virus m segments. the relationships observed are consistent with a long-term association of viruses with their primary rodent reservoirs and suggestive of coevolution of host and virus. a sin nombre virus isolate is now available and its genetic characterization has been completed. various virus antigens have been expressed and are being used to probe the interaction of the virus with the host immune system. hantaviruses cause significant morbidity and mortality throughout the world. more than , cases of hemorrhagic fever with renal syndrome (hfrs) are reported annually in asia, europe and scandinavia. the etiologic agents of hfrs are hantaan, seoul and puumala viruses, with hantaan virus causing the most severe form of the disease. in , a new hantavirus was discovered in the united states (initially termed four comers virus), and was identified as the etiologic agent of hantavirus pulmonary syndrome (hi's). vaccines for hantaviruses are not readily available, although a number of inactivated viral preparations have been made and tested in asia. recurrent problems with inactivated hantaviral vaccines have been lot to lot variability, the need for repeated immunizations, and their inability to elicit long-lasting neutralizing antibody responses in immunized volunteers. because of such limitations on traditional vaccine development for these viruses, as well as the viruses' hazardous nature and slow, low-titer replication in cell culture, we used a recombinant dna approach to develop a vaccine for i-ifrs. our vaccine is a recombinant vaccinia virus expressing the m segment of hantaan virus under control of the vaccinia virus . k promoter and the s segment under control of the k promoter. the m segment, which encodes the g and g envelope proteins, was included because of our findings that: ( ) immunization with vaccinia or baculovirus-expressed g and g induced a neutralizing and protective immune response in hamsters; and, ( ) neutralizing antibodies to g or g could passively protect hamsters from challenge with virulent virus. the s segment, which encodes the nucleocapsid protein (n), was included because of our finding that hamsters immunized with baculovirus-expressed n also were protected from subsequent infection. although the protective immune response to n is probably cell-mediated, the importance of such a response is presently not well defined. assessment of our vaccine in preclinical studies, indicated that immunized hamsters developed neutralizing antibodies and were protected from displaying viral antigen in their lungs after challenge. in a phase i, dose escalation, clinical study, the vaccine induced neutralizing antibodies in individuals immunized subcutaneously with approximately lo pfu of the recombinant virus. in addition to humoral responses, immunized volunteers developed a cell-mediated immune response as indicated in lymphocyte proliferation assays. larger clinical studies, including alternate routes or booster immunizations, are planned. based on these studies, we anticipate that the vaccine will be efficacious for preventing hfrs caused by hantaan and the antigenically closely related seoul virus. we are studying the cross-protective properties of this vaccine with more distantly related hantaviruses such as puumala virus. although we expect this vaccine to be safe as well as effective, we also are investigating the use of more attenuated pox-viruses as vaccine vectors. infection of mice with lymphocytic choriomenigitis virus (lcmv) results in a profound expansion in the number of spleen cd t cells and in the induction of virus-specific ctl activity. thereafter, the cd t cell number declines, and the ctl activity diminishes, though the frequency of lcmvspecific precursor ctl per cd cell, as assessed by limiting dilution assays (lda), is remarkably stable throughout long-term immunity. the decline in t cell and total spleen leukocyte number at the late stages of acute infection is associated with high levels of apoptosis, as detected by the in situ nucleotidyl transferase assay. apoptosis occurred in both the t cell and b cell populations, with the b cells dying in clusters. this apoptosis was also seen in tfansgenic mice ectopically expressing bcl- in the t and b cells and in c bl/ ipr/@r mice, which have a mutation in the fas gene. t cells from the infected animal underwent apoptosis in vitro when stimulated through the tcr with anti-cd , thereby explaining some of the immunosuppression seen during acute viral infections. memory cells persisted for over a year and could be found in blast-size cell populations. challenge of lcmv-immune mice with either pichinde virus, vaccinia virus, or murine cytomegalovirus led to the reactivation of the lcmv-specific ctl response. lda analyses showed unexpectedly that these heterologous viruses crossreacted with subpopulations of lcmv-specific memory t cells. this memory t cell response to virus from an earlier infection was associated with enhanced immunopathology and enhanced clearance of virus during a heterologous virus challenge. over the course of the acute infection, ctl specific for the second virus were preferentially expanded over the crossreactive ctl, and after the acute infection, when the t cell response had subsided, ctl memory to the first infection had decreased. there is therefore a network of memory t cells which contribute to and are modulated by infections with putatively unrelated viruses, and apoptosis plays a homeostatic role in the course of these t cell responses. immune responses to live attenuated retroviral vaccines, r. paul johnson*?, cara wilsont, kelledy mansons, michael wyands, bruce walker?, ronald c. desrosiers* *new england regional primate research center, southborough, ma thfectious disease unit, massachusetts general hospital, boston, ma §tsi/mason, worcester, ma immunization of rhesus macaques with live attenuated retroviruses deleted in nef can induce protective immunity against challenge with pathogenic siv. development of protective immunity in these vaccinated animals occurs only after several months of infection, with maximal protection observed after one year. the specific immune responses responsible for mediating protection have not been defined, and little is known about the cellular immune responses in animals vaccinated with these live attenuated retroviruses. we have analyzed cellular and humoral immune responses in rhesus macaques and chimpanzees infected with live attenuated retroviruses. siv-specific neutralizing antibodies were present in vaccinated animals, but did not clearly correlate with protection against challenge. ctl specific for envelope and gag were identified in vaccinated macaques studied or more months after vaccination. quantitation of siv-specific ctl activity in one of these animals using limiting dilution analysis revealed a relatively high precursor frequency of cytotoxic t lymphocytes, up to moo for gag and / for envelope. cd + lymphocytes obtained from vaccinated macaques were also able to suppress siv replication in autologous cd + cells. suppression mediated by unstimulated cd autologous cells was maximal when cells were in direct contact with siv-infected lymphocytes, but cd + cells activated by an anti-cd -specific monoclonal antibody were able to release. a potent soluble inhibitor of siv replication. in contrast to the relatively vigorous ctl response present in vaccinated macaques, we were not able to detect consistent ctl activity in chimpanzees infected with a hiv- molecular clone (nl ) or attenuated viruses at periods up to one year after infection, despite the use of a variety of stimulation techniques. proliferative responses to hiv p and gp were observed in chimpanzees infected with n u and attenuated variants. although the relative contribution of these immune responses to protective immunity is not known, the relative vigor of the cellular immune responses observed in vaccinated macaques suggest they may play a role in mediating resistance to challenge. obiectives: to analyze the magnitude and specificity of the ctl response to hiv- , and to determine the tcr usage by clonal ctl responses in infected persons, including persons with documented infection of up to years with cd cells > /mm . methods: hiv-l-specific ctl activity was evaluated in pbmc as well as in pbmc stimulated in vitro with hn- infected autologous cd cells, using target cells infected with recombinant vaccinia viruses expressing hn- proteins. ctl epitopes recognized by these individuals were determined using cloned effector cells. quantitative cultures were performed by endpoint dilution, and viral quantitation was determined by qc-pcr tcr analysis was performed by pcr, using both family-specific primers and anchored pcr, followed by sequencing. sequence analysis of ctl epitopes in autologous viruses was determined by pcr amplification and sequencing. clonal frequency was analyzed in pbmc by oligonucleotide probe to the cdr region of the tcr. studies performed in long-term non-progressing persons indicate the presence of a vigorous and broadly directed ctl response. detailed epitope mapping in a person infected for years, who by qc-pcr had <i viral rna molecules per ml and who tested negative by bdna assay, revealed ctl clones directed at six different epitopes, with detectable recognition of three of these epitopes using freshly isolated pbmc as effector cells. the ctl clones were broadly reactive against many hown hiv- variants, and both the p and p epitopes were cross reactive with siv. sequence analysis of autologous virus within the three dominant ctl epitopes revealed the lack of immune escape variants in pbmc, plasma, and virus obtained from coculhue. supernatant. analysis of tcr usage by clones to defined epitopes in persons at different disease stages revealed heterogeneity in tcr usage and heterogeneity in recognition of virus variants withii these epitopes. analysis of clonal frequency using an oligonucleotide probe to the cdr region of the tcr in an individual with a vigorous response to gp suggested that approximately % of circulating t cells were a single hiv-l-specific clone. conclusions: our results indicate that cytotoxic t lymphocytes are part of the immune response in persistent non-progressive hiv- infection, and that prolonged infection can occur without the development of viral immune escape variation within defined ctl epitopes. in addition, vigorous circulating ctl responses can occur in the setting of an e x m e l y low viral load. heterogeneity in the ability of clones from different individuals to recognize sequence variation within ctl epitopes may be important in the pathogenesis of infection. ctl to conserved epitopes or ctl which are broadly reactive with multiple viral mains may be important in conmbuting to maintenance of the asymptomatic state. factor-i (irf- ) gene is essential for the transcriptional activation of inducible nitric oxide synthase (nos) in murine macrophages. it also plays an important role in the activation of interferon and il- and il- receptor genes. to investigate the role of irf- related systems of defense against viral myocarditis, the transgenic mice were innoculated with l o pfu of coxsackie virus (cvb ). homozygous irf- (+) and heterozygous irf- (+/-) mice were randomized into groups to determine heart and spleen viral titres, cardiac pathology and mortality rates at early and late stages of the disease. the mortality was dramatic in the irf- -imice. the hearts of the homozygous animals also had increased mononuclear cell infiltration, necrosis and viral replication. we condude that irf- regulated gene products such as inos and ifn are essential for the early defense against cvb -induced myocarditis by attenuating viral replication and inflammatory responses. the flu season is a yeariy problem, especially in the elderly population. annual vaccination lacks broad protection and antibody titers induced in the elderly are frequently lower than those acheived by younger vaccinees. using the mouse model, we are investigating the differences in immune response between old and young mice to influenza vaccine and at the same time testing the ability of a potent adjuvant to boost the response in the the elderly. our studies show defects in the immune response of the elderly. antibody titers after immunization are lower in the elderly for both seronegative and seropositive mice. cmi studies show that helper t-cell response (proliferation) to in-vitro antigen stimulation is also decreased in the elderly. facs analysis of wbcs show the elderly mice have lower leukocyte and lymphocyte populations, lower cd and b-cell populations and a higher proportion of macrophages and cd cells than young mice. it was also noted that the young mice had very consistent wbc profiles while the elderly profiles had greater variability. serum cytokine studies show lower levels of il , il and il , but higher levels of il and ifng in the elderly as compared to the young mice. the addition of mf to the immunization increased the antibody titers of both naive and seropositive young and old mice. the helper t-cell response of the young mice was unaffected, while the response of the elderly group was increased. cytokine profiles show an increase in il , il and il levels for both young and old, while il and ifng levels went up for young animals but remained constant for the elderly mice. coxsackievirus (cvb ), a member of the picornavirus family, is a common cause of acute or chronic human myocarditis. however, whether cell damage results primarily from direct virusmediated effects or from immune-mediated destruction of the heart tissue during the infection remains unclear. we used a cardiovirulent variant of cvb which is able to infect several strains of mice. the infection results in a disease which strongly implicates immunopathogenetic mechanisms in myofiber necrosis. to characterize the role of the immune system in this disease we used various transgenic knockout (ko) mice with different immune defects. cvb is able to infect normal c bl/ mice and immunocompromised (cd ko and m ko) mice and causes a lethal disease after i.p. injection in a viral dose dependent manner. we found elevated ld os in immunocompromised mice, which also die slightly later than normal mice. this virus replicates in the hearts of all mice used with maximal titers - days pi.. large pathological changes were detectable only in heart tissue of cd ko mice, and were maximal at - weeks after infection. these results suggest the possibility that cd ' t cells may limit tissue damage by an as yet undefined mechanism; the roles of cd + and cd ' t cells in this disease are under investigation. research supported by a grant of the daad, germany. mice lacking p lck are resistant to coxsackievirus b induced heart disease. tamara martino, karen aitken, joseph penninger, jan nikhilanandhan, tak mak, fayez dawood, wen-hu wen, lily wee, martin petric, and peter liu, the toronto and princess margaret hospitals, university of toronto, canada. coxsackievirus (cvb ) causes severe heart disease in adult mice, and is a useful model for the human cardiac conditions of myocarditis and dilated cardiomyopathy. in this study, we have demonstrated that transgenic mice lacking the t-cell signalling molecule p kk are resistant to cvb induced heart disease. the virus replicates in the heart, and is cleared from the myocardium at a rate comparable to "normal" littermates, but there is only minimal mortality, or damage to the cardiac tissue. however, in ick-deficient mice depleted of natural killer (nk) cells prior to viral infection, there is increased cvb replication, and some infiltration by mononuclear cells in the myocardium. to further elucidate the role of the immune system in cvb induced heart disease, cytokine expression in the hearts of cvb infected normal, ick-deficient, and nk-cell depleted mice is under investigation. we conclude that t-cell activation is critical to produce substantive myofiber necrosis after cvb infection, that nk-cells play an fundamental role in protecting the mice from severe virus infection, and that ick transgenic mice serve as an important model for understandina the dathogenic mechanisms involved. we now show that cd + t cells from m-/-mice exhibit antigen specific release of serine esterase and interferon-gamma. further, as quantitated by flow cytometry, the early decrease in cd + t cells seen in normal mice days after lcmv infection, previously presumed to be due to the activity of cd + ctl, still occurs in m-/-mice. however, mice show an earlier rise in percentage and absolute numbers of cd + t cells by days after infection. cd + t cells from am-/mice rise to above baseline levels with kinetics paralleling the early rise in cd + t cells in normal mice after lcmv infection. cd + ciz activity is lower in m-/mice and have a later peak than cd + ctl from normal mice. these differences may explain the lower mortality and longer time course of immunopathologic meningitis in -mice. mice can assume some of the cytotoxic functions of the cd + cell population from normal mice, including immune-mediated pathology. further, cd + t cells from m-/-mice have the capacity to release serine esterase, and show kinetics similar to cd + t cells from normal mice. mice genetically engineered to be deficient in -microglobulin in conclusion, these data suggest that cd + t cells from am-/- australia. molecule is an essential component of the t cell help required for an antibody response. the interaction between cd and its ligand, in the presence of b cell acting cytokines, such as interleukin (il- ), is sufficient to trigger b cells to proliferate and differentiate and to induce immunoglobulin class switching. a recombinant vaccinia virus encoding both factors, cd l and il- , did not, however, stimulate an enhanced antibody response in mice infected with the construct. instead, the antibody levels were lower than those of mice infected with a control virus. the reduced antibody response reflected the diminished growth of cd ol-expressing viruses, to the extent that immunocompromised mice were able to resolve these infections. the kinetics of virus clearance indicate that the anti-viral mechanism of cwol is rapidly activated and has generalized tissue distribution. the cwolmediated clearance of virus is independent of the anti-viral cytokines, tnf and ifn-y. the anti-viral activity of c w l may represent a surprising and potent effector mechanism of t cells activated during a virus infection. mary's medical school, norfolk place, london w lpg, u. k. and *nationallnstirute formedica research, london, nw aa. respiratory syncytial (rs) virus is the most important respiratory pathogen of infants, causing the majority of cases of bronchiolitis, it can be life threatening in very young infants and those with underlying cardiopulmonary disease or immunodeficiencies. t cell immunodeficiency can lead to prolonged infection in children and t cell transfers clear virus in the murine disease model. mice can be readily infected with rs virus and have proved a valuable model of its pulmonary pathology and the t cell response to it. il- has a broad spectrum of activities on proliferation and terminal differentiation of early blast cells and committed progenitors of many lineages. less is known about the effects of il- on t cell development and proliferation and its effect on the host response to infection by common pathogens. the il- gene was disrupted in /sv embryonic stem (es) cells by homologous recombination after transfection with omega-type constructs flanked with a herpes simplex thymidine kinase gene. these cells were used to produce a mouse strain deficient of il- . mice were infected intranasally with a strain rs virus; pathological changes in the lung were monitored by cytology of bronchial lavage, and virus persistence by nested rt-pcr of lavage fluid and lung homogenate. no differences were found in pathology or virus persistence between knockouts and normal mice. il- deficiency is therefore compatible with apparently normal responses to viral infection. recombinant adenoviruses are an atuactive vehicle for gene therapy to lung in the treatment of cystic fibrosis (cf). first generation viruses deleted of ela and elb transduce genes into airway epithelial cells in vivo, however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. in this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive nansfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. our studies indicate that mhc class i resmcted cd + cytotoxic t lymphocytes (ctls) are activated in response to viral antigens leading to destruction of virus infected cells and loss of transgene expression. mhc class ii associated presentation of viral antigens activates cd +t helper (th) cells of the - subset and, to a lesser extent, of the tm subset. ldv establishes a life long viremic infection in mice which is not controlled by anti-ldv immune responses. anti-ldv antibodies are rapidly generated but they neutralize ldv infectivity only poorly. here we show that ldv-specific cytotoxic t cells (ctls)are generated within days pi. c t l s were detected by h- specific lysis of ldv-specific target cells. these target cells were generated by transfection of t -nm cells with a retrovirus vector carrying the gene (ow ) for the ldv nucleocapsid (n) protein. spleen cells from ldv-infected mice were incubated in vitro with ldv-infected macrophages or transfected t -nih cells and cultured for days in the presence of il- . the ctls had largely disappeared in mice by days pi. the following experiments were conducted to further explore the mechanism of immune escape. in situ hybridization was used to localize ldvinfected cells in tissues of infected mice. these were found in persistently infected mice only in the liver. testis, and lymphoidal tissues. in addition large amounts of virion or virion debris accumulated in the germinal centers of the spleen and lymph nodes beginning about days p.i. the accumulation of large amounts of ldv antigen in the germinal centers may be causally related to the suppression of the ctl response as well as to the polyclonal activation of b cells associated with ldv infection. to determine whether immune selection plays a role in ldv immune escape, ldv was reisolated from nude and immunocompetent balb/c mice at various times pi. the ows for the nand the two envelope glycoproteins were r t k r amplified and sequenced. the role of cytokines in initiating the immune response to venezuelan equine encephalitis (vee) was investigated. mice were infected in the left rear foot pad with either cloned virulent vee (v ) or an isogenic single-site attenuated vee mutant (v ) (single amino acid change at e glycoprotein position from glu-lys) and at , , , , , and hours post-infection, the popliteal lymph nodes and spleen were harvested and examined for tnf-a, il- , ifn-y, il- , il- , il- , and il- gene expression using a quantitative rt-pcr. in both strains elevations of tnf-a, il- , ifn-y, il- , and il- were detected in the lymph node, with no changes in either il- or il- ; however, whereas cytokine elevations were detected by hours after injection of v , elevations were delayed until hours following infection with v . cytokine mrna elevations in the spleen were less substantial, but elevated levels of ifn-y, il- , and il- were also observed. these findings suggest that vee infection induces an early highly specific cytokine pattern associated with simultaneous elevations in both ifn-y and il- and that a single amino acid mutation can induce temporal changes in this pattern without alterring either the actual cytokines induced or the degree of their elevation. we are currently performing intervention experiments to investigate the effect of intravenous anti-il- andor anti-ifn-alp antibody administration on this early in vivo cytokine response to examine in particular whether these cytokines may be required for the observed elevations in either ifn-y or il- . to confirm the importance of the immune system in clearing rotavirus infection we infected rag knockout mice with murine rotaviruses. both knockout p u p s and adults developed chronic infections. to determine if a t cell independent antibody could be mediating the clearance of rotavirus infection in nude mice we measured igg, igm and iga in faeces and serum of rotavirus infected mice. a low and transient igm response was found in serum but not faeces of infected nude mice but not in age matched naive nude mice. the protein specificity of these antibodies is being determined. to determine if a primary infection in n u d e mice would induce protection (memory) against a secondary infection, we challenged six week old mice that had been infected as pups with the murine ew strain of rotavirus with the murine cambridge strain of rotavirus. the previously infected mice, as well as naive nude mice, both shed very low but equal levels of rotavirus in the stools. interestingly adult naive heterozygous littermates shed more virus than the nudes. the factors responsible for this relative resistance of adult nude mice to rotavirus infection as well as the mechanism for viral clearence in nude mice will be discussed. symp. , : - ) . the great majority of the poxvirus encoded proteins actively mediating the evasion of host defense are secretory proteins termed "virokines". viruses produce proteins which can either compete with or antagonizz molecules critically involved in generating immune responses. the vaccinia virus complement control protein (vcp) was the first virokine to be reported that has structural similarity to humadmouse complement control proteins, with the greatest similarity to the human complement b binding protein (bc -bp) and the first protein to have a postulated role in the viral evasion of host defense (kotwal and moss, nature , : - ) . bioactive vcp, purified from serumfree medium has been shown to be more potent than the hc bbp ( kotwal, am. biotech. lab., ) and has also been shown to bind to two important complement components, c and c , and block the complement cascade at multiple sites in vitro (kotwal et al., science , - ; mckenzie, kotwal et al., j. inf. dis. , - . the importance of this protein was demonstrated in vivo using recombinant virus lacking an intact dna coding for vcp (isaacs, kotwal and moss, pnas , : - ) and further underscored by the smallpox virus genomic sequence analysis, indicating the presence of a highly conserved homolog of vcp ( massung et al., nature , : - ) . in order to determine the precise effects of vcp at the site of infection, a mouse model has been developed. measurement of the specific swelling response and microscopic examination of the histological changes in balb/c and dbn mice has revealed that vcp is capable of regulating inflammatory response in vivo (jayaraman and kotwal, mol. immunol. : ) . in order to ensure that the possiblity of the unrelated mouse strains sharing identical h- haplotypes but differing at numerous other loci was not overlooked, we performed similar experiments in congenic strains. well characterized c deficient mice injected with poxviruses in comparison to normal mice, produce a significant inflammatory response, accompanied with severe ulceration that persists for several days. the data suggests that the host complement response is a critical factor in egulating poxvirus pathogenesis. previous studies have suggested that immune response was one of the major factors that contributed to the host resistantlsusceptible mechanism to sendai virus infection (brownstein, ) . various immune regulatory molecules were investigated in inbred susceptible /j ( ) mice and resistant c bl/ j (b ) mice which share the same mhc haplotype. when they were given eid, of sendai virus intranasally, mice showed histologically diffuse interstitial pneumonia compared to the local moderate inflammation in mice. virus was eliminated from the infected lung days earlier in b mice than in mice. the cytokine levels and cytokine-producing cells were investigated in the acute pneumonic lung. also the recall cytokine production in the draining mediastinal lymph node (mln) were studied. the maximal numbers of il- , ifn-y, il- , and il- producing cells were comparable for each strains of mice, while more il- producing cells were present in the lung of mice. however, the numbers of these cytokine producing cells peaked in mice days later than in b mice. analysis of soluble cytokines in bronchoalveolar lavage fluid showed that at least two-fold higher levels of ifn-y, il- , and il- were present in mice than in mice. recall cytokine production in the draining mln showed only the presence of il- , ifn-y, il- , and il- , while no il- was detected. generally, higher levels of these cytokines were detected throughout the whole infection process in mice. therefore, the susceptibility of mice to sendai virus is not due to insufficient cytokine production in these two anatomical sites, yet heavier and prolonged virus burden in susceptible mice may drive the more rigorous and protracted induction of immune regulatory molecules. cytokines play a decisive role in determining the type of immune response elicited by an antigen, both in driving th cell differentiation along thl or th pathway and, ultimately, the effector and regulatory activity of these subsets. we have studied the role of cytokines in virus infections by constructing recombinant virus vectors encoding cytokine genes. during replication in mice, these vectors produce the encoded factor which is secreted from the infected cells with profound immunological consequences. cytokines studied todate fall into two categories; either altering viral pathogenesis or selectively stimulating specific immune responses. the expression of il- , ifn-y, tnf-a or il- in recombinant vaccinia virus (rvv) dramatically alters pathogenicity, such that immunodeficient mice resolve the normally lethal infection. on the other hand, expression of il- by rvv suppresses the induction of cytotoxic t cells (ctl) inhibiting the clearance of virus which leads to an increase in viral pathogenicity. the expression of il- by rvv was shown to inhibit the host il- response required for the development of ctl's. no production was also significantly suppressed by il- . rvvs expressing il- or il- , although not affecting pathogenicity, preferentially stimulates iga reactivity to coexpressed antigens. encoding cytokines in recombinant viruses has clearly demonstrated the important role of cytokines as anti-viral effector molecules and in regulating appropriate immune responses. in mice infected with lymphocytic choriomeningitis virus (lcmv), interleukin-i (il- ) doses of >i ngld induce profound immunotoxicities characterized as almost complete inhibition of virus-induced cd + t cell expansion and ctl activation, and up to log increases in viral replication [orange, wolf, and biron, j. immunol. : , . serum tumor necrosis factor (tnf) is also observed under these conditions. the studies reported here further characterize the expression and function of tnf in this context. northern blot and in sifu hybridization analyses demonstrated that il- induced tnf-cx expression and that lcmv infection synergized with il- for this induction. administration of antibodies neutralizing tnf reversed the il- -induced immunotoxicities in lcmv-infected mice and restored anti-viral defenses. the tnf-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and cd + t cells isolated from lcmv-infected mice were more sensitive to tnfmediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. additional physiological changes were observed in il- -treated uninfected mice and were dramatically elevated in il- -treated virus-infected mice, including: ) decreases in body weights; ) elevation of circulating glucocorticoid levels: and ) decreases in thymic mass. these changes were also reversed by anti-tnf. the results delineate a unique tnf-mediated immunotoxicity and have significant implications concerning detrimental consequences of in vivo tnf andlor il- for protective anti-viral responses. lactate dehydrogenase-elevating virus (ldv), a naturally occurring virus, causes a persistent infection in mice and presents an ideal model for the study of immune modulation during acute and persistent virus infections. within a few days following infection with ldv there is a pronounced polyclonal activation of b cells followed by the suppression of primary b cell responses to t-dependent ag. we investigated the effect of acute and persistent ldv infection on the development of a memory b cell response to the model protein antigen, horse cytochrome c (cyt), by employing a modification of the splenic fragment assay. about a % decrease in the frequency of responding agspecific memory b cells was observed in balb/c mice infected with ldv, whether the mice were immunized with cyt at the time of ldv infection or three weeks later. this may be due in part to a defect in t cell help, since in cultures of normal memory b cells and t cells derived from ldv acutelyinfected mice the frequency of responding b cells was also decreased two-fold. in situ hybridization using a cdna probe specific for ldv revealed two patterns of ldv rna within the spleen. twenty-four hr p.i. ldv rna was located within the marginal zone, surrounding each follicle. this pattern is consistent with permissive macrophages. during persistence viral rna could no longer be detected in the marginal zone, but was located within the follicles. the absence of ldv-permissive cells within the follicular region suggests that the source of ldv rna is not due to ongoing viral replication. one possibility is that circulating virus is trapped by a specific cell population within the follicle. the effect of virus trapping within the spleen provides a mechanism by which ldv and other viruses can modulate immune cell function during persistent infections. ifn-y can be produced by activated nk cells. this cytokine enhances immune responses by augmenting macrophage antigen presentation. viral infection induces ifn-dp and nk cell activation. changes in splenic architecture, cell trafficking, and cytokine expression were examined during viral infections of c bu mice. at times coinciding with ifn-dp production and nk cell activation, there was a redistribution of nucleated cells from red pulp to white pulp regions in spleens isolated from mice infected with either lymphocytic choriomeningitis virus (lcmv) or murine cytomegalovirus (mcmv). cell transfer experiments with dioctadecyl- , , ', '-tetramethyl indocarbocyanine perchlorate-or pkh -gl-labeled bone marrow cells isolated from normal mice demonstrated an infection-induced accumulation of non-t/non-b cell populations along recipient splenic marginal zones. flow cytometric analyses demonstrated that approximately % of the transferred bone marrow cells accumulated in spleens after hrs and % of these expressed the nk cell marker, nkl.l+. in vivo antibody treatment procedures, to eliminate cell subsets in donor mice, demonstrated that the cells localizing at the marginal zone were derived from agmi+ and n k l . l + populations. a small subpopulation of marginal zone cells in infected mice were shown to be expressing high levels of ifn- mrna by in sifu hybridization. treatment with anti-agmi or anti-nk .i antibodies eliminated both endogenous nk cells and the ifn-y mrna positive cells. these data demonstrate that newly derived nk cells accumulate along marginal zones. the results also suggest that this trafficking pattern may act to enhance immune responses by facilitating delivery of cytokines to specialized antigen presenting cells. david segal, janet ruby, alistair ramsay and ian ramshaw. depamnent of cell biology, john curtin school of medical research, po box , canberra, act, australia cytokine expression has been shown to correlate with protective or ineffective immune responses in a number of disease models. recently there has been the suggestion that immunity to some retroviruses is associated with the production of ceaain patterns of cytokines. to explore this further we have have used rauscher murine leukemia virus (r-mulv) infection of c bu (resistant) and balb/c (susceptible) mice to elucidate the role of cytokines immunity to retroviruses. initially the in viho proliferation of spleen and lymph node cells from infected mice was examined. in response to stimulation with immohilised anti-cd antibodies the proliferation of spleen hut not lymph node cells from infected mice. was found to he rapidly suppressed. suceptible balb/c mice exhibited a much greater suppression than resistant c bu mice. the cause of this suppression is under investigation however, the immunosuppressive molecules nitric oxide and prostaglandins are not involved. in vitro cytokine production by spleen and lymph node cells from r-mulv infected mice was determined. in response to stimulation with immobilised anti-cd antibodies, spleen cells from infected balb/c mice produced diminishing amounts of ifn- and il- . in contrast spleen cells from infected c bu mice produced ifn-y and l- to levels that were only slightly less than uninfected controls. a- production by spleen cells from infected mice of both strains was at levels higher uninfected controls. anti-cd stimulated lymph node cells from infected mice produced elevated ifn- suggesting that suppressed cytokine production is spleen specific. expression of cytokine genes in vivo is currently being investigated using rt-pcr to detect cytokine mrna in the spleens of infected mice. we have previously shown that primary resting murine b lymphocytes are non-permissive for vesicular stomatitis virus (vsv), however, a productive infection can be induced when infected b cells are activated with anti-immunoglobulin (a-lg) plus il- or lipopolysaccharide (lps). we posit vsv in unactivated primary b cells provides a paradigm of persistently infected lymphocytes and activation dependent recall of an active infection. analysis of the behavior of virus in unstimulated b cells during long term culture and the requirements for subsequent induction of productive infection has been limited by the poor survival of primary cells in culture. we circumvented this limitation by using highly purified small b cells from mice transgenic for the bcl- proto-oncogene, expression markedly extends in vitro survival of unstimulated primary b cells. overexpression of bcl- does not alter b cell infection or induction of a productive infection by activators during acute infection. infection does not effect b cell survival in culture. unstimulated virus infected b cells produce primary viral mrnas but not viral proteins or infectious particles (pfu) during culture. persistently infected b cells stimulated with a-lg plus il- produced a fully productive vsv infection at all times analyzed, up to weeks post infection. in contrast, vsv production in persistently infected b cells activated with lps markedly declined relative to acutely infected activated cells ( - fold by week and , fold by week ). cells were not completely refractory to lps activation as vsv protein was produced. the selective lps deficiency is unique to persistently infected cells as uninfected cultured b cells proliferate and differentiate to produce antibody upon lps activation. these data show that a persistent infection may selectively alter the host cell response to previously productive activators which may as a consequence interfere with immune regulation. rsv-g glycoprotein specific t cells preferentially secrete il- and predispose to pulmonary eosinophillia., anon srikiatkhachorn. and thomas j. braciale, the beirne b. carter center for immunology research and the departments of microbiology, pathology, and pediatrics, university of virginia health sciences center, charlottesville, va we studied the immune responses to two different glycoproteins of respiratory syncytial virus (rsv) in a murine model. balb/c mice were immunized with recombinant vaccinia virus expressing either rsv-fusion glycoprotein ( vac-f), attachment glycoprotein (vac-g) or -galactosidase (as a control). these mice were given rsv intranasally three weeks after priming and then sacrificed or days later. spleens and bronchial lymph nodes were harvested for in vitro culture and lungs were harvested for histologic studies . we found that bulk cultures obtained from both vac-f and vac-g immunized animals secreted both thl and th type cytokines when stimulated with rsv infected spleen cells . however, the levels of - and fn-y were higher in bulk cultures derived from vac-g primed animals while the levels of il- were higher in the bulk culture from vac-f primed animals. the il- and il- production was relatively short lived since spleen cells and bronchial lymph node cells obtaind form mice sacrificed days after intranasal inoculation produced much lower levels of il- and - while the levels of il- and ifn-y production were comparable to bulk cultures obtained from mice at the peak of infection. there was little inflammatory response in the lungs obtained from mice immunized with the control vaccinia. in contrast , lungs from mice immunized with vac-f or vac-g showed significant infiltration of inflammatory cells. there was a striking infiltration of eosinophils in the lungs from mice primed with vac-g. these eosinophils could be detected aroud major bronchi and blood vessels, as well as, in some cases, in lung parenchyma. this study suggests that the immune responses to different viral glycoproteins may be distinct and may play important roles in viral pathogenesis. during infection of normal mice with lymphocytic choriomeningitis virus (lcmv), nk cell responses peak on day and subside as cd + t cell responses are activated at day post-infection. in contrast, m-/-mice, lacking cd + t cells, have dramatically elevated nk cell responses on day postinfection. the m-/-response is evidenced by increased nk cell activity, as well as up to -fold increases in blast and total nki.i+cd -cell numbers. nk cell responses in normal mice are cyclosporin a (csa)-resistant and interleukin (il)- independent, whereas day nk cell responses in m-/-mice are csa-sensitive and il- -dependent. to investigate the role of additional cytokines in regulating cellular responses during acute viral infections, production and function of il- and transforming growth factor (tgf- ) were examined. induction of il- mrna, at late times post-infection of normal mice, was shown by in situ hybridization of t cell-enriched splenic leukocytes and polymerase chain reaction (pcr) amplification of cdna from rna. ellsas of media cor.aitioned with cells isolated on days , , , , , and post-infection demonstrated delayed induction of il- protein as compared to ctl activation. tgf- , evaluated in biological and elisa assays, was induced maximally at days to post-infection. the kinetics of tgf- production by cells from infected m-/mice was similar to that of normal mice. however, cells from m-/-mice produced il- at early but not at late times postinfection. together, these results suggest that either il- is a critical cytokine for shutting off nk cells during normal responses to viral infection, or that the m-l context modulates responsiveness of nk cell subsets to other late cytokines. studies are in progress to distinguish between these two possible mechanisms. the induction of fever in response to infection is an important host defense mechanism that enhances aspects of the immune response and restricts the replication of some microorganism. vaccinia virus, a member of the poxvirus family, is a complex cytoplasmic dna virus that encodes a variety of proteins that interfere with host immune functions, such as complement regulatory factors and soluble receptors for il-lp, tnf and ifny. here we show that expression of the vaccinia virus il- p receptor (vil-lpr) in the w r strain prevents the febrile response and reduces the severity of infection in intranasally inoculated mice. fever was recorded on days - after infection of mice with a vil-lpr deletion mutant, but not in animals infected with wild type wr or a virus revertant. these studies were extended to other virus strains that were used as smallpox vaccines, and expression of the vil-lpr was consistently found to prevent the onset of fever. vaccinia virus induced a severe hypothermia after days in infected mice that was independent on vil-lpr expression and correlated with virus replication in the brain, the organ that controls body temperature. these results represent the first example of a virus mechanism to inhibit the host febrile response and suggest a central role for soluble il-lp in the induction of fever in poxvirus infections. measles virus (mv) infection can depress cell-mediated immune responses for months following clinical disease. mv is known to infect the thymus during human illness and this may contribute to immune suppression. we have used the scid-hu m o w with co-implants of human fetal thymus and liver to determine the effect of virulent and avirulent strains of mv on the thymus. scid-hu mice were. infected by direct inoculation of the graft with pfu of either a wild type strain of mv(chicago- ,chi- ) or an attenu-ated strain (moraten, mor) and sacrificed at intervals over days. peak viral titers, as judged by plaque assay on vero cells, were reached by chi- on d ( . pfu/ third of implant), and moron d ( . pfu/ third of implant). hematoxylideosin stained sections of chi- -infected thymuses showed marked distortion of the cortex and medulla by d with thymocyte poilolosis and decreased cellularity. by d , these. implants were mostly devoid of normal thymocytes. mor-infected thymuses showed relatively preserved architecture and cellularity. suspensions of the cells from implants stained with mabs to cd ,cd and cd were analyzed by flow cytomehy. there were significant decreases in the cd +cd + cell pop-ulation by d with complete loss of all such cells by d with chi- , and only modest reductions with mor. immune fluorescence staining of sections with a mv mab to hemagluttinin(ha) and abs for either human cytokera-tins(ael/ae ) or cd co-localized mv predominantly to epithelial and monocytic cells. additionally, mv antigen was present diffusely by d in both cortex and medulla in chi-i infection whereas mor-infected implants had only patchy distribution by d . only rare cells stained both with mv ha and cd or cd . mv ha was not expressed over background on any cd + cells judged by facs. we conclude that mv replicates in the scid-hu thymic implant primarily in epithelial and monocytic cells, and that the attenuated virus reproduces more slowly and with less cellular disruption. little mv ha could be demonstrated in thymocytes, therefore the data suggest that significant infection of the thymic epithelial stroma disrupts the thymic microenvironment which normally supports and aids in selection of immature t cells. part of the long-term immune suppression seen in mv infection may be due to infection of the thymic epithelial stroma with subsequent loss of thymocytes. it is becoming increasingly evident that many poxviruses contain genes that enable the virus to evade the host's immune system. myxoma virus is a leporipoxvirus and is the causative agent of myxomatosis, a rapidly lethal disease in the european rabbit (oryctolagus cuniculus). one possible mechanism of immune evasion is virus-induced downregulation of cell-surface receptors important for an immune response. cell-surface levels of several receptors on a rabbit t cell lymphoma cell line (rl- ) were monitored by flow cytometry. following infection with myxoma virus, cellsurface levels of cd were found to drop dramatically. other cell surface antigens such as cd , cd , and cd were unaffected during infection with myxoma virus. further more, the downregulation of cd by myxoma virus could be inhibited by treating cells for an extended period of time with pma, suggesting that the downregulation was not simply a masking of the epitope via viral antigens. analysis of cd levels in the presence of cytosine arabinoside indicates that late gene expression is not necessary for the modulation. since the tyrosine specific protein kinase p lck associates with the cytoplasmic domain of cd we have also examined the association of p lck with cd as well as steady state levels of p lck during viral infection. the modulation of surface cd has also been described in hiv infected t cells suggesting that the loss of cell-surface cd may be a common viral immune evasion tactic by lymphotrophic viruses. i n addition, stably-transfected cell l i n e s expressing e i t h e r u s o r us - gene products s i g n i f i c a n t l y reduced l e v e l s of mhc class i heavy chain. studies are i n progress t o f u r t h e r d e f i n e t h e mechanism by which t h e s e v i r a l gene products a l t e r immune recognition. cytotoxic t lymphocytes (ctl) may play a significant role in containing the spread of hiv in infected individuals. although hiv-infection is associated with immune suppression, a vigorous ctl response has been detected in infected adults. hiv can be transmitted from mother to child. one third of vertically infected children has a rapid evolution toward disease, with onset of aids before months. the other two thirds remain asymptomatic for years. the bimodal course of disease evolution in hiv-infected children could be related to differences in the host immune control of viral replication. hiv-specific ctl response from fresh and in vitro activated pbmc of hiv-infected children was measured. the vast majority of infected chidren had detectable hiv-specific ctl, which where cds+cd +. we previously showed that among children with a slow disease progression, fresh ctl were more frequent in the p a(paucisymptomatic) group than in the pl(asymptomatic) and the p b-f groups (symptomatic group). the cohort of children has now been followed during years, and children have been tested at least once. we found that ctl responses were less frequent in the children with a rapid disease progression than in the children with a slow disease progression at the same age. our data suggest that ctl response is an important factor in delaying disease evolution. we, as well as others. have proposed that sag function is critical to the ability of milk-borne m m n to infect mice. to determine whether this is the case, we created transgenic mice (hyb pro/cla) with a frameshift mutation int the sag gene. young hyb pro/cla mice (c weeks of age) showed no deletion of their cognate vp * t cells, unlike transgenic mice carrying a functional sag gene however, a slow, progressive loss was seen in the hyb prolcla mice as they aged, indicating that it was due to expression of wild type sag protein. thus, as the hyb pro/cla mice aged, there was production of virus that appeared to lose the cla mutation. the hyb pro/cla mice produced transgene rna in their lactating mammary gland and shed virus in their milk. their nontransgenic offspring of showed infection with transgene-encoded mmtv because they had the typical slow deletion of vp + t cells characteristic of c h mmtv infection and because we detected transgene-derived m m n rna in their mammary glands. cloning and sequencing of the viral rna produced by the nontransgenic offspring of the hyb pro/cla mice showed that recombination between the mtv- endogenous viral rna and the transgene-encoded rna occurred, such that the frameshift introduced by the cia mutation was repaired. these results show that there is selection of infectious virus that contains a functional sag gene. thus, it appears that the only virus that is capable of being transmitted by the milk borne infection pathway is that which encodes a functional sag protein. hepatitis b virus (hbv) causes acute and chronic liver diseases and is closely associated with hepatocellular carcinoma. in order to understand the cellular immune response against hbv in chronic hbv infection, t cell proliferation, cytotoxicity and cytokine production were studied. we found that although the majority of asymptomatic hbsag carriers and patients of chronic hepatitis b (chb) had no proliferative response to hbsag, some individuals in both groups showed significant t cell proliferation against hbsag. in contrast, the proliferative t cell response to hbcag in asyrnpatomatic hbsag carriers was significantly stronger than that in patients of chb with acute exacerbation. in addition, the frequency of hbcag-reactive t cell precursors measured by limiting dilution assay was much higher in asymptomatic hbsag carriers than in patients of chb. therefore, t cell responses against hbsag and hbcag are regulated differently in chronic hbv infection. furthermore, we demonstrated hbsag-and hbcag-specific cytotoxic t lymphocyte (ctl) activity in asymptomatic hbsag carriers, using autologous hbsag-and hbcag-expressing lymphoblastoid cell lines (lcl) as target cells, respectively. the cloned ctl were able to produce ifn-y, tnf-a or gm-csf after stimulation. these findings demonstrate that t cell response to hbv is not completely suppressed in asymptomatic hbsag carriers. most of them have strong hbcag-specific response and some of them have hbsag-specific response. transcription and tax the human t-lymphotropic virus type i (htlv-i) promoter contains the structural features of a typical rna polymerase i (pol ) template. the promoter contains a tata box bp upstream of the transcription initiation site, binding sites for several pol i transcription factors, and long poly a+ rna is synthesized from the integrated htlv-i proviral dna in vivo. consistent with these characteristics, htlv-i transcription activity was reconstituted in v i m using tbp, tfiia, rtfiib, rtfiie, rtfiif, tfiih and pol . in hela whole cell extracts, however, the htlv-i ltr also contains an overlapping transcription unit (otu). htlv-i otu transcription is initiated at the same nucleotide site as the rna isolated from the htlv-i-infected cell line, mt- , but was not inhibited by the presence of a-amanitin at concentrations which inhibited the adenovirus major late pol i promoter ( pglml). htlv-i transcription was inhibited when higher concentrations of a-amanitin were used ( pglml), in the range of a typical polymerase in (pol ) promoter (va-i). purified tax, transactivates this promoter -to -fold in v i m . interestingly, basal and tax,-transactivated transcriptional activity of the htlv-i ltr could be reconstituted with the . m phosphocellulose fraction. these observations suggest that the htlv-i ltr contains overlapping tax,responsive promoters, a typical pol i promoter and a unique pol i promoter which requires a distinct set of transcription factors. tax, further in vifro transactivates a polymerase i template containing the base pair repeats cloned upstream of the ovalbumin promoter and g-free cassette. tax,-transactivated transcription was concentration dependent and inhibited by low concentrations of a-amanitin. flaviviruses are arthropod-borne viruses whose route of infection is via the skin. they are mostly neurotropic and responsible for significant human morbidity and mortality. the classic cell-mediated immune response to a viral infection may be influenced by the ability of these viruses to modify expression of cell-surface molecules involved in the presentation of antigen to, and activation of, t cells. the skin langerhans cell is the prototypic nonlymphoid dendritic cell and as such is uniquely placed to participate in a response against epidermally-acquired viral infections. the migratory properties of these cells contribute to their role as initiators of t cell-mediated immune responses within the draining lymph node. we have previously shown infection of epidermal cells in vifro by the flavivirus west nile (wnv) results in an increase in mhc class i and i expression on the majority of epidermal cells and langerhans cells respectively. in this study a technique for infecting the epidermis with wnv in vivo was developed. tme-dependent increases in the surface expression of a number of antigens which are involved either directly or in a co-stimulatory capacity in initiating a cell-mediated immune response, were detected on both the majority of epidermal cells and the langerhans cell population using flow cytometry. these increases were detectable as early as hours after infection. a significant decrease in the percentage of langerhans cells remaining in the epidermis was observed within hours of infection. the phenotypic changes observed in vivo are analogous to those described following in vifro culture of langerhans cells. these results, together with the reduction in langerhans cell numbers, may represent the in situ maturation and concomitant migration of these. cells as a consequence of virus-induced cytokines within the skin microenvironment. which cause a wide variety of illnesses with high morbidity and mortality in humans throughout the world. their high genomic stability argues for a survival strategy related more to interaction with the vertebrate host immune response, than a dependence on viral genetic mutation. our previous work has shown that west nile virus (wnv) infection of many cell types directly induces functional increases in class i and mhc expression. we report here that wnv infection of human embryonic fibroblasts (hef) results in the increased expression of cd by two distinct mechanisms. an early, direct cytokine-independent mechanism operates within h of virus infection, while an indirect mechanism, regulated by type interferon (ifn), operates within h of virus infection. cd expression increased by - fold within h of wnv infection on hef, and by - -fold within h. wnv-inactivated, conditioned supematants removed from infected hef cultures after h incubation did not alter cd expression on unqimulated hef. whereas conditioned supernatants from h-infected cultures increased cd expression by about . - -fold after incubation for h, but not after h, similar to cd induction by ulml of ifn-p. increased cd expression on hef by wnv was also cell-cycle dependent. cd increased only in quiescent, contact-inhibited infected hef in go phase. in contrast, induction of cd by types and ifn was not cell-cycle dependent. other viruses, including double-stranded dna viruses, vaccinia, and adenovirus and , and the single, positive-stranded rna alphavirus, semiliki forest virus, did not induce cd expression on hef after h. another alphavirus, ross river, was able to induce cd but only by the indirect mechanism of type ifn-dependent release. poly i.c, also, increased cd expression to the same extent as ifn-p after h, making it unlikely that the early increase was due to a nonspecific viral effect. the closely related flavivirus, kunjin, induced increased cd expression in a manner similar to wnv. the ability of flavivhses to induce increased cd expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infectiodreplication. recognition of viral peptides presented on the cell surface in association with class i mhc molecules leads to lysis by cytotoxic t cells (ctl) and forms an important part of the immune response to hiv infection. hiv virus has a high mutation rate and variation in the region of the viral epitope may allow evasion of this immune response. variation could theoretically affect processing of the antigen, binding of the epitope to the hla molecule or recognition of the presented epitope on the cell surface. we have studied proviral sequence variation in gag and ctl responses in a number of hla b patients infected with hiv. amino acid substitutions, such as a lysine to arginine change at position of the pi gag nonamer cckkkyklk, lead to loss of recognition of the peptide by ctl from the patient whose provirus contained this sequence. these variant peptides bind to hla with comparable affinity to the index peptide suggesting that this loss of recognition is likely to be caused by changes in the interaction between the hla-peptide complex and the t cell receptor. other changes, such as lysine to arginine or glutamine at position , not only cause loss of recognition, but also lead to inhibition of lysis of targets bearing the index peptide. thus it appears that in addition to loss of recognition by cytotoxic t cells, naturally occurring epitope variants may act as "antagonists", as has been demonstrated in mhc class ii systems. antagonism may be an important mechanism allowing immune escape by the hiv virus. genes. subsequent complex formation between peptide, class i and p microglobulin in the er results in stable cell surface expression of the trimeric mhc- molecule. in previous studies we showed that in hpv- positive cervical carcinomas there was a loss of mhc- protein expression, which correlated at the single cell level with loss of tap protein. in this study we investigated whether loss of tap and mhc- is mediated by an hpv- encoded protein. human keratinocytes were transfected withvarious hpv- constructs including pat , the full length genome, pat esx the full length genome with a premature stop codon in e , puc.et , the e and e oncogenes only, and pkve , expressing e from mouse moloney ltr the different constructs were transfected into primary keratinocytes, cloned cells grown in medium supplemented with and without y-interferon ( y -a r ) for hours. cells were harvested and total rna and protein harvested for northern and western blots respectively. western blots showed very low steady state levels of tap- and mhc- heavy chains in the cells with pat as well as those containing es alone, which was marginally increased by y-lfn. in contrast, primary keratinocytes, pat esx and puc.et lines showed comparable tap- and mhc- protein levels, which increased a & y-ifn treatment. northem blots showed no differences in the amounts of tap- and mhc- mrna between the different cell lines. the data indicate that expression ofhf'v- e leads to post-transcriptional loss of mhc- , presumably by interfering with tap. to map and characterize functional differences between e a of ad and adl , we previously constructed a series of hybrid ad / e a genes and used them with ad e b to transform primary hooded lister rat kidney cells. at least two regions within the first exon of ad e a were identified which influenced tumorigenicity. this study further examines the role of these regions in tumorigenicity by analyzing their affect on cell surface mhc class i expression and sensitivity to class i-restricted cd + as well as to non-class irestricted nks. the bcrfl open reading frame of epstein-barr virus exhibits remarkable sequence homology with the coding sequences of interleukin- from a variety of organisms. many of the numerous immunological properties ascribed to interleukin- are shared by the product of bcrfl and this has led to it being termed viral interleukin- . in order to investigate the activity of viral interleukin-i (vil- ) and its interactions with the human interleukin- receptor we have expressed the protein in a bacterial and the eukaryotic cos- expression systems. the bacterially expressed vil-i was partially purified and used to set up two assays to measure i l l o activity: i)the increase in igm secretion from an ebv transformed b cell line -mt .l and ii)the downregulation of class ii hla expression on the human monocytic cell line thp- . a series of deletion mutants (both n-and c-terminal as well as an internal deletion to remove a putative heparin binding domain) were constructed to identify possible domains within the vil- protein that interact with the hil- receptor and confer its biological activity. a number of these mutants have been expressed in the cos- expression system and their structure and biological activity are currently being assessed. the identification of the domains within vil- that interact with the receptor or accessory proteins may aid in the understanding of the possible role of vil-i within the ebv life cycle and in the pathogenesis of the numerous diseases associated with the virus. generation. to further test the role of ctl in ad pathogenesis, viruses lacking the cll epitopes were tested when mutants that lack the immunodominate ctl epitope in eia where used, a second immun-ssive epitope in elb becorns the predominate target of clu. these findings arc important since human ad is currently being tested as a vector for gene therapy of cystic fibrosis. our data suggest that when consuucting ad vectors to be. used for gene therapy, one must retain either the . k or . k genes to decrease pathology and that meting the genes that encode the antigens that a n recognized by clu does not prevent the generation of ad specific clu. the interferons (ifns) a n ? a family of cytokines whose functions include the protection of cells against viral infection. type i ifns include the ifna subtypes and ifnp that compete for binding to the same cell surface receptor, while type ii ifn (ifny) binds to a different receptor. the orthopoxviruses, of which vaccinia virus (vv) is the prototypic member, have developed a number of anti-ifn strategies. the vv e l protein competitively binds dsrna and prevents the activation of ifninduced and dsrna-activated protein kinase (pkr), while the vv k l protein shows sequence similarity to the eukaryotic initiation factor a (eif a) that is phosphorylated and inactivated by pkr. the k l protein competitively binds the kinase and blocks host eif a phosphorylation and hence ifn-induced inhibition of host protein synthesis. onhopoxviruses also suppress cytokine action by expressing soluble cytokine receptors that bind and sequester the ligand; to date soluble receptors for interleukin- , tumour necrosis factor and ifny have been described. supernatants from vv-infected cells were found to contain a soluble inhibitor of type i ifn that was conserved in most of the orthopoxviruses tested. the inhibitor was produced early in infection and did not inhibit ifny. the ifna/p inhibitor was mapped and the gene expressed from recombinant baculovirus. the inhibitor blocked the binding of i-ifna to u cells and binding of i-ifna to supernatants from baculovirus and vv-infected cells demonstrated that the inhibitor functioned as a soluble receptor for fnc fp. direct binding of -ifna to vv wr supernatants revealed that the soluble ifna/p receptor had a high affinity for type i ifn. deletion of the gene from the vv genome and ligand blotting of the soluble receptor demonstrated that ifn binding was encoded by a single protein. competitive binding curves using ifna from other species revealed that the poxvirus soluble ifndp receptor bound human and bovine ifn with high affinity but murine ifn with relatively low affmity. interestingly, the soluble ifncrip receptor is highly conserved in variola virus. given the importance of ifn in antiviral defense it is likely that the soluble ifndp receptor plays an important role in the virulence of the orthopoxviruses. endogenous processing of a viral glycoprotein for presentation t o cd + t cells has defined a previously under-investigated pathway in antigen processing and presentation. it may be important not only for pathogens, but also for self-proteins, and thus may be involved in self-tolerance. we have been characterizing the processing o f the er-restricted gpt glycoprotein of vesicular stomatitis virus (vsv) biochemically and enzymatically, by cellular localization using confocal immunofluorescence, cellular fractionation, and by t cell recognition assays. by flow cytometry, gpt is undetected on the plasma membrane; in contrast, the wild type protein (g) is readily found following infection of a cells with a vaccinia virus vector, leading t o endogenous synthesis. the gpt can be found exclusively in the er compartment using co-localization with markers for er (signal peptide binding protein, calnexin), and not in the golgi compartment (a-mannosidase , wheat germ agglutinin), endosome, lysosome, or surface plasma membrane. this is consistent with the characteristics o f the localization of the proteases which appear to be responsible for its degradation. work is in progress to localize the site of peptide binding to mhc heterodimers. supported by nih grant a t o csr. presentation of an out-of-frame class i restricted epitope. t.n.j.bullock and l.c.eisenlohr, department of immunology, thomas jefferson university, philadelphia, pa . antigen presentation by class i mhc molecules is thought to require the degradation of fully formed proteins in the cytosol. this degradative process supplies oligopeptide epitopes for transport into the endoplasmic reticulum (er) where they can interact with and stabilize class i molecules. stable class i molecules, associated with p -microglobulin, can then proceed to the cell surface where they present the epitopes to t cell receptors. the generally accepted model for protein translation, the scanning hypothesis proposed by ko&, is thought to describe the traditional method of translation for the majority of proteins. we wished to test the hypothesis that any internal methionine that is in good translation initiation context can be a source of short peptides, which may then be processed into class i epitopes. nucleoprotein gene (np), the target of the ctl response of several inbred mouse strains. np contains three class i restricted epitopes at amino acids - (h -kk), - (h -kd) and - (h -db). the frameshift was introduced amino acids upstream of the h -kd epitope. the mutated genes were then recombined with vaccinia virus and tested for presentation using ctl restricted to each of the epito s described above. we found that, whilst presentation of the h -i@ epitope was unaffected by the frame shift, the epitope proximal to the frameshift (h -kd) was no longer presented to appro riately restricted ctl. however, presentation of the distal h -dg epitope was retained. therefore we have shown, using a viral protein and a viral expression system, that out-of-frame epitopes can be processed and presented to ctl. work is ongoing to c o n f m that internal methionines are capable of providing a platform for the initiation of translation for in-frame and out-of-frame epitopes. we have created a frameshift mutation in the influenza pr the fine specificity of t cell recognition of peptide analogues of the influenza nucleoprotein epitope np - srywairtr was studied using hla b -restricted influenza-specific cytotoxic t cell (ctl) clones, of defined t cell receptor (tcr) usage, derived from unrelated individuals following natural infection. synthetic analogue peptides were synthesized containing single amino acid substitutions, and tested both for binding to hla b' in vitro, and for presentation to ctl clones by hla positive targets. even conservative amino acid substitutions of the peptide residues p , , and profoundly influenced ctl recognition, without affecting binding to hla ' . these amino acid side chains are thus probably directly contacted by the tcr. ctl clones which used the tcr v a l gene segment (but not those using tcr va ) were also sensitive to p substitutions, suggesting that the tcr alpha chain of these clones lies over the n terminus of bound peptide, and that the "footprint" of certain tcrs can span all exposed residues of a peptide bound to mhc class . these results, taken together with previous structural and functional data, suggest that, for nonarner peptides bound to hla , p i , p and p are "flag" residues with tcr accessible side chains. the e / k protein of human adenovirus type (ad ) is a resident transmembrane glycoprotein of the endoplasmic reticulum. its capacity to associate with class i histocompatibility (mhc) antigens abrogates cell surface expression and the antigen presentation function of mhc antigens. at present, it is unclear exactly which structure of the e / k protein mediates binding to mhc molecules. apart from a stretch of approximately conserved amino acids in front of the transmembrane segment, e / k molecules from different adenovirus subgroups (b and c) share little homology. remarkably, the majority of cysteines is conserved. in this report, we examined the importance of cysteine residues (cys) for structure and function of the ad e / k protein. we show that e / k contains intramolecular disulfide bonds. by using sitedirected rnutagenesis, individual cysteines were substituted by serines and alanines, and mutant proteins were stably expressed in cells. based on the differential binding of monoclonal antibody tw . and cyanogen bromide cleavage experiments, a structural model of e / k is proposed, in which cys and cys as well as cys and cys are linked by disulfide bonds. both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human mhc antigens. this was demonstrated by three criteria: loss of e / k coprecipitation, lack of transport inhibition and normal cell surface expression of mhc molecules in cells expressing mutant e / k molecules. mutation of the three other cysteines at position , and had no effect. this indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the e / k molecule, namely, to bind and to inhibit transport of mhc antigens. previous studies have suggested that several abundant cmv proteins are major immunogenic targets in seropositive adults. we are interested in defining the major viral protein targets of a cd ' ctl response, in order to derive a vaccine strategy for individuals who are unable to mount immune responses which are lymphokinedependent because of immunosuppression. hla-typed and cmv-pgsitive normal volunteers who have hla-a alleles that represent - % of the u.s. population are being tested to determine which of abundant cmv proteins they recognize by a cd ' ctl response: p , p , p , ie, and gb. t cell lines will be derived in order to unambiguously determine the hla restriction of the cd ' ctl response to each of these proteins. proteins which are recognized by the most hla diverse population will be further characterized in terms of mapping of class epitopes through the use of t cell clones derived from the polyclonal cell lines by limiting dilution. the defined epitopes will form the basis of a vaccine strategy to augment the memory responses of seropositive volunteers against cmv. these epitopes will be used to boost the ctl precursor frequency of bone marrow transplant donors as a means to transfer cellular immunity to immunosuppressed hematologic transplant recipients. an alternative strategy is to immunize seropositive individuals with recombinant viral proteins as a means to boost immunologic memory. we are pursuing that strategy in a transgenic murine model of hla-a . developed by dr. l. sherman (scripps institute, la jolla). we are vaccinating the transgenic mice with two well defined cmv proteins, p and gb together with either of two lipid-based adjuvants, commercially available d tapm (bcehringer-mannheim) or mf gth (chiron, emeryville, ca). our preliminary studies with hsv- gb demonstrate that both adjuvants are effective at eliciting murine class i restricted responses against the protein. current studies are evaluating the recognition properties of the adjuvant-cmv protein complexes by hwa as a restriction element in the transgenic model. the ctl response to sendai virus in c by mice is directed almost exclusively to a single h- kb-restricted epitope derived from the virus nucleoprotein, npj - (sev- ). analysis of independent t cell hybridomas generated from c by mice following primary sendai virus infection has shown that a very diverse repertoire of tcr is selected in response to this epitope. crystallographic analysis of sev- bound to kb has shown that the side chaiis of peptide residues phpi, gl , a d s , and alaps protrude towards the solvent and are potentially available for recognition by the tcr notably, residues gi and a d protrude prominently from the peptide binding site due to their l o c a l i o n on a bulge in the center of sev- . to determine the importance of each of these residues for t cell recognition, we analyzed hybridoma responses to sev- analogs substituted at each of these four positions. preliminary data showed there generally appeared to be dominant recognition of glyp and asnm. however, individual hybridomas exhibited distinct patterns of fine specificity for residues phep and alaps. thus, individual hybridomas were dependent on one, both, or neither of these residues for recognition of sev- . these data are consistent with a critical role for the gi and a d in governing tcr-sev- eb recognition and suggest a structural basis for the diversity of the tcr repertoire selected by this @tope. previous results from this laboratoty demonstrated that the dominant influenza a epitope recognized by hla . restricted ctl from hla-a . uansgenic mice was the m peptide epitope that is immunodominant in human ctl responses. however, analysis of a large number of ctl lines revealed a subset of influenza a/pr/ / -specific murine ctl that recognized an hla-a . restricted epitope distinct from m . using recombinant vaccinia viruses encoding werent influenza gene segments, the epitope recognized by these ctl was shown to be derived from the a/pr/ nsl protein. because these ctl did not recognize targets infected with the a/alaska/ / saain of influenza, candidate peptide epitopes were synthesized based on sequences that included an hla-a . specific binding motif and that differed between a/pw and nalaska all of these ctl recognized a nonamer and a decamer peptide which contained a common amino acid sequence and two distinct sets of bmding mtif residues. however, the n name.r peptide was able to sensitize ctl for half maximal lysis at - fold lower doses than either the octamer or decamer. the homologous peptide derived from nalaska nsl contained conservative amino acid changes at positions and and was not recognized at any tested concentration, although it bound with higher &ity to hla-a . than the peptide from a/pw . the a/pr/ nsl nonamer epitope was also recognized by human influenza a specific ctl derived from two individuals. these results substantiate the general utility of hla class i aansgenic mice for the identification of human cn epitopes for other pathogens. furthemore, the recombinant dhfr was functional in the induction of gb epitope-specific ctl response upon immunization of c bv mice. these results indicate that an viral epitope expressed in a cellular protein can be. efficiently processed, presented and recognized by epitope-specific ctl, and suggest that the cellular proteins can be used to express ctl epitopes for induction of cd + immune responses. virus-specific cytotoxic t lymphocytes (ctl.) were generated a day later at this site. to determine which apc was capable of stimulating virusspecific ctl precursors in the mln, b, t and dendritic cells from the mln of influenza-infkcted mice were separated and examined for the presence of virus. the predominant cell type which contained infectious virus was the dendritic cell. b and t cells from the mln contained little, ifany, virus. the apc capacity ofthese populations was tested by their ability to stimulate vir~~-~pecific t cell hybridomas. only dendritic cells from the mln of influenza-infected mice were able to stimulate virusspecific t cell hybridomas, althwgh all apc populations from both naive and influenza-infected mice were effective stimulators after in y h pulsing with the appropriate intluenza peptide. potential apc populations were also separated from the lung. v i s was detected in bronchioalveolar macrophages and dendritic cells but not b or t cells. both macrophages and dendritic cells isolated from intlum-infected lungs could stimulate virus-specific t cell hybridomas. the ability of the mln and lung apc populations to stimulate naive cd ' t cells and generate virus-specific ctl is currently being examined. virus infected cells present only a very limited number of peptides intracellularly processed from a viral protein to ctl even when many peptides hearing the mhc class i-restricted binding motif are present in the protein. infection of h- b mice w i t h lymphqtic choriomeningitis virus (lcmv) induces a cd + ctl response directed against three wellcharacterized epitopes presented by h- db molecules: " - (fqpq-ngqfi), gp - (kavynfatcgi) and gp - (sgven-pggycl). the h- db motif is characterized by a sequence of to a.a. with two anchor residues: asn at position and hydrophobic (met, ile, leu) at the c-terminus. the lcmv np and gp proteins contain thirly-one other peptides exhibiting the db motif. however, no ctl response against one (or more) of these peptides has been characterized. peptide binding to mhc is a critical step in antigen presentation. the aim of this study was therefore to analyze the binding properties of the potential db lcmv peptides. the lcmv peptides and known db-selective peptides were synthesized and their mhc binding affinities measured in two db-specific binding assays. most of the lcmv peptides ( / ) did not bind to db. the other (including the epitopes) and all the known db peptides showed good affinity. comparison of the sequences (good vs. non binders) allowed the identification of auxilliary anchors required for high binding affinity or of negative elements hampering mhc binding. in addition to the main anchors, the positive and negative factors at secondary residues play a crucial role in governing peptidemhc interactions. knowledge of such factors might he of importance for the prediction of mhcrestricted ctl epitopes. etienne joly, andrea gonzalez, carol clarkson, jonathan c. howard and geoffrey w. butcher. laboratory of immunogenetics, department of immunology, the babraham institute, cambs cb at, uk. tap transporters from rats can be divided into two allelic groups, depending on their capacity to provide the rt .aa molecule with an appropriate level of suitable peptidesl. recent results suggest that this might correlate with the rt .aa molecule requiring arginine-ended peptides (powis et al., manuscript submitted), which the tapb allele of the transporter is unable to translocate across the er membrane efficiently ~ . rt .a alleles are naturally linked with the tapa or the tapb allelic group . we have set out to characterise various alleles for the rt .a molecule, and find that, for the majority of tapaassociated rt .a molecules, acidic residues line the c/e pocket, dictating arginine as c-terminal anchor residue for the bound peptides. on the other hand, in tapb-associated rt .a molecules, one acidic residue at the most is found in the c/e pocket, which certainly results in a different anchor residue for the bound peptides. the selective pressure of viral infections must have driven this coevolution which affects dramatically the array of peptides presented to cytotoxic t lymphocytes. cytotoxic t lymphocyte responses in hiv infection can be impaired due to variation in the epitope regions of viral proteins such as gag. we show here an analysis of variant epitope peptides in three gag epitopes presented by hla b . seventeen variant peptides were examined for their binding to hla b ; all but one bind at concentrations comparable to known epitopes. all except two could be seen by ctl clones grown from hla b positive hiv- infected patients and were therefore immunogenic. however, in one haemophiliac patient studied in detail, there was a failure to respond to some of the peptides that represented virus present as provirus in his peripheral blood. in one case his ctl had previously responded to the peptide. thus there was a selective failure of the ctlresponse to variant epitopes. this impaired reaction to new variants and failure to maintain responses to some epitopes late in hiv infection could contribute to the loss of immune control of the infection. pira, anna ferraris, daniele saverino, peifang sun and annalisa kunkl; dept. immunology, san martino hosp. univ. of genoa, genoa, italy. th epitopes present on viral proteins can be recognized by specific th cells if appropriately expressed by antigen presenting cells (apc) as a result of uptake and processing. since viral epitopes are not simply present in the context of viral proteins, but also in the context of whole viral particles, it is important to determine the role of the molecular and/or structural context on antigen uptake-processing-presentation. therefore we have generated panels of cd + human t cell lines and clones specific for different hiv antigens (gp , p , p ), in order to test their ability to respond to the same epitopes present within synthetic peptides, recombinant proteins or inactivated virions (provided by g. lewis, dept. microbiology, univ. maryland, baltimore). we could identify t cell lines and clones that were able to discriminate the molecular and structural context of the epitops. certain t cells, in fact, responded to peptides and proteins, but not to viral particles, whereas other t cells were also able to proliferate when challanged in vitro with autologous apc and viral particles. the data suggest that in the human th cell repertoire specific for viral antigens t cells exist that can discriminate the molecularstructural context of th epitopes. it will be interesting to ascertain whether t cells specific for epitopes that can only be recognized when provided in the context of a soluble molecule, but not of a viral particle, have any relevance in viva protection, or are a simple by-product of the cellular immune response. eric g. pamer, merceditas s. villanueva, section of infectious diseases, yale university school of medicine, new haven, ct listeria monocytogenes is a gram positive bacterium that infects macrophages and secretes proteins into host cell cytosol. the murein hydrolase p is secreted by l. monocytogenes and is required for complete bacterial septation. in the infected macrophage secreted p is processed by the host cell into the nonamer peptide p - and is presented to cytotoxic t lymphocytes by the h- kd mhc class i molecule. we have used strains of l. monocytogenes that secrete different amounts of p to show that the rate of p - production is proportional to the amount of antigen secreted into the host cell cytosol. p is degraded in the host cell cytosol with a half life of minutes. the appearance of p - is coupled to the degradation of newly synthesized p . we have determined the rate of intracellular p secretion and by accounting for the rate of p degradation we estimate that approximately p molecules are degraded to produce one p - epitope. this ratio is maintained over a range of intracellular antigen concentrations. our findings provide an estimate of the efficiency of antigen processing and demonstrate the remarkable capacity of the mhc class i antigen processing pathway to accommodate new epitopes. we have isolated and characterized three cytotoxic t lymphocyte (ctl) clones from the peripheral blood of two acute seroconversion patients and one patient in the first trimester of pregnancy. these clones were cd + and class i hlarestricted by the b molecule. all three clones recognized lllb and rf but not mn strains of hiv- . using vaccinia vectors expressing truncated versions of the hiv- envelope, the clones were found to recognize an epitope within amino acids - , but not including - of gp . further mapping of the epitope with synthetic -mer peptides overlapping by , or -mers overlapping by , was unsuccessful. the sequence of the region of gp recognized by these clones was compared to the predicted hla- peptide binding motif and a possible matching region was found. using shorter peptides corresponding to this potential epitope recognition site, the minimum epitope recognized by the clones was determined to be the aa sequence rpnnntrksi spanning amino acids we have further pursued a strategy to define a minimal cytotoxic epitope for a vaccine against cmv infection using t cell clones derived from individuals who have the mhc gene (kind gifts of drs. riddell and greenberg, fred hutchinson cancer research center and dr. robert siliciano, johns hopkins university medical center). we tested by chromium release assay (cra) the recognition of a series of allelic variants of ebv-lcl. by restricted and cmv or hiv-specific t cell clones. several conclusions quickly became apparent. the previously described ' peptide epitope from pp was not able to prime the autologous ebv-lcl for killing by the pp -specific ctl, whereas a recombinant vaccinia virus expressing whole pp could cause the same cell line to be recognized and killed in the same experiment. in addition, an hiv gp -specific cd ' ctl which has a defined minimal cytotoxic epitope will only recognize and kill a subset of ebv-lcl. the two t cell clones will not recognize each other's autologous ebv-lcl. the resolution of this interesting phenomena comes from sequence analysis of the hla class i b genes from both ebv-lcl. ebv-lcl which contain the b' allele are recognized and killed by the pp -specific t cell clone, and cell lines carrying ' alleles are recognized by the hiv gp -t cell clone. we conclude that the reported cmv pp b" restricted epitope is not correct, since the ctl in question will only recognize ' alleles in combination with the correct pp epitope. fragments with or without a signal sequence sensitize rma-s/kd to a similar limited extent. this data i s consistent with an inefficient movement of peptides from the cytoplasm into the er by a tap independent mechanism and does not reveal a processing competent compartment within the secretory pathway. peptide transport by the transporter associated with antigen processing (tap) was studied using a microsome system as previously reported by heemels et. al.. in this system, a radiolabeled synthetic peptide which can be n-link glycosylated is used as the indicator peptide for the transport studies. the transport efficiency of synthetic peptides corresponding to antigenic peptides restricted to the murine kd molecule was measured by inhibition of labeled peptide transported into the microsomes. the transport efficiency of three kd epitopes in the type a influenza virus " - , ha - and ha - was found to be similar. an amino acid peptide corresponding to ha - which contains the - epitope was transported at a similar efficiency as the amino acid minimum epitope. however, when the peptide sequence is further extended by one amino acid to residue , this peptide is poorly transported. these results suggest that the flanking region of an epitope can dramatically influence the transport of the epitope. when the transport kinetics of tap was studied using the microsome system, the vmax for transporting the indicator peptide (a variant of np epitope that has the sequence tynrtrali) was found at . fmolelminute (+/- . ). the km for this peptide was found to be . nm(+/- . ). bypassing a block in antigen processing for class i-restricted cytotoxic t cell recognition. amy j. yellen-shaw and laurence c. eisenlohr. thomas jeferson universitv. hiladelphia, pa., . previous work from our laboratory showed that processing of an influenza nucleoprotein (np) epitope (amino acids - ) expressed endogenously from a recombinant vaccinia virus "minigene" is severely impaired when a flanking sequence (the dipeptide threonine-glycine) is appended to the cterminus of the construct ( - /r-). the inhibition of processing is overcome by placing the unprocessed peptide in the context of the fulllength np molecule, demonstrating that regions of a protein outside the epitope itself critically affect the ability of the proteolytic machinery to fragment the protein appropriately. to determine the requirements for bypassing the block in antigen processing, we have constructed an array of "minigene"-expressing vaccinia recombinants in which the unprocessed epitope is extended by varying lengths toward either the c-terminus or the n-terminus of the np molecule. our results show that while an extension of the c-terminus by only one amino acid restores processability, a much longer extension of the n-terminus ( < n < amino acids) will also allow the substrate to be processed. it is therefore clear that a full-length, properly folded molecule is not required for liberation of the blocked epitope, and that probably more than one mechanism can contribute to enhancement of substrate proteolysis. we hypothesize that the c-terminal extension allows recruitment of an endopeptidase versus exopeptidase ("trimming") activity which is capable of cleaving the difficult bond. we considered the possibility that the n-terminal extension rescues processing by recruitment of the ubiquitin-dependent degradation system. to address this possibility we replaced all available ubiquitination sites (lysine residues) in one of the rescued constructs ( - /r-) to see if the construct would still be processed and presented. the six available lysine residues were changed to arginine using pcr-based mutagenesis. the resulting construct (termed r) was recombined into vaccinia virus and tested for presentation to np-specific ctl. the r construct was presented at a level equivalent to that seen with the wild-type - /rconstruct. this result provides clear evidence that entry into the ubiquitindependent degradation pathway is not responsible for rescue of presentation in this system and more importantly, that ubiquitination is not required for processing of all large substrates. chia-chi ku, li-jung chien ,and chwan-chuen king, institute of epidemiology, national taiwan university, taipei, taiwan, r.o.c. dengue virus (den) can cause dengue fever (df) and dengue hemorrhagic fever (dhf) i dengue shock syndrome (dss) and den- was the most common serotype found in dhf outbreaks globally. current hypotheses suggested that dhf may be associated either with antibodydependent enhancement (ade) or with viral virulence. den can replicate predominantly in monocytedmacrophages (mim), but whether peripheral blood lymhocytes (pbls) are the target cells of den still remain controversial. in order to compare whether various clinically derived den- will interact with mim and lymphocytes in different manners, we used two isolates --plo strain (obtained from a df patient during taiwan outbreaks) and strain (isolated from a dhf patient in thailand by cdc, usa) to infect primary mim and lymphocytes as well as several types of cell lines. primary lymphocyte culture was nonadherent cells obtained after hr adherence of pbmcs, whereas the primary mim culture was collected by depletion of lymphocytes using anti-cd icdi mab and complement prior to adherence procedure and the purity of mim culture was checked by cd surface marker staining. supernatants (sn) of virus were harvested at various time points post infection after with several or without treatments. our prelimanary data showed that dhf-associated den- strain had higher viral yield in certain age of mim and a promonocytic cell line (hl-cz) than taiwan df-associated den strain. in addition, this dhf-den strain was more likely to infect the promonocytic (hl-cz) than well differentiated monocytic (ctv- ) and lymphocytic (h ) cell lines and also had higher peak yields than den-i virus in hl-cz cells. interestingly, dhf-den strain replicated much more efficiently in primary lymphocytes no matter these cells were activated with pha or not, whereas taiwan df-den strain virus was hardly detectable in sn of both activated and non-activated lymphocyte cultures. therefore we conclude that ( ) different strains of dengue virus could orchestrate quite differently with immune cells, ( ) different stage of mim differentiation might be an important permissive determinants for dengue virus infection and replication, and ( ) den virus strain virulence -a more important factor than lymphocyte activation status -seemed to determine whether this strain would infect human pbls. further studies should be focused on searching for detaied mechanisms of virus and immune cell interactions. ( ) when viral yields were enhanced early than day post infection, it provided tremendous opportunity to attack the immune system and finally may lead to severe disease. hiv- using recombinant immunoglobulin molecules, marie-claire gauduin, graham p. allaway, paul j. maddon, carlos f. barbas, dennis r. burton, and richard a. university school of medicine, new york. ny . primary isolates of hiv- have been shown to be less sensitive to neutralization by immune sera, monoclonal antibodies and cd -based molecules than t cell line-adapted strains of hiv-i. we studied two immunoglobulin molecules for ability to neutralize primary isolates of hiv-i. lgg is an immunoglobulin molecule created from a combinatorial phage expression library and reacts with the cd binding site (cd -bs) on gp . cd -lgg is a recombinant molecule in which the variable domains of both heavy and light chains of lgg were replaced with the first and second immunoglobulin-like domains of human cd . both molecules have been previously shown to effectively neutralize hiv-i in vitro. ex vivo neutralizations were performed as follows: lgg and cd -lgg were added at pg/ml to wells containing serial dilutions of plasma from hiv-i-infected patients and phastimulated peripheral blood mononuclear cells from seronegative donors. p production was measured over days of culture and an end-point titer of hiv- in the presence and absence of added antibody was determined. both igg and cd -lgg were found to reduce the original hiv titer from seven plasma samples with high virus titer (> tcid /ml) by up to -fold. this is in comparison to soluble cd which only reduced viral infectivity by -fold at the same concentration. in vitro binding and neutralization assays on isolates recovered from plasma confirm the potency and breadth of neutralization by these two molecules. these studies suggest that recombinant antibodies directed at the cd -bs of hiv- gp are able to effectively neutralize primary isolates of hiv- and may be useful in dissecting the mechanisms of resistance to neutralization by other antibodies. dillner and p. heino, microbiology & tumor biology center, karolinska institute, stockholm, sweden hpv the major cause of anogenital precancers in man. the search for neutralizing epitopes that could form the basis for a preventive vaccine has shown that the surface-exposed imunodominant epitopes of the capsid are strongly conformationdependent, which has precluded detailed epitope analysis. similarly, immunization with whole, denatured capsid proteins has only identified linear immunodominant epitopes positioned on the inside of the capsid. reasoning that linear surface-exposed epitopes should exist, but might be cryptic, a set of overlapping synthetic peptides corresponding to the entire hpv capsid proteins was used to generate hyperimmune sera. several antisera against different peptides were reactive with intact hpv capsids at titers up to : . . hiv- serum antibodies and mucosal iga. basil golding, john inman, paul beining, jody manischewitz, robert blackburn and hana golding. div. of hematology and viral products, cber, fda, and lab. of immunology, niaid, bethesda md . previously, we showed that hiv- proteins conjugated to . abortus (ba) could generate anti-hiv- neutralizing antibodies in mice even after depletion of cd * t cells. in this study a -mer peptide from the v loop of hiv- (mn) was synthesized ) and coupled to ba and klh. balb/c mice were immunized twice i.p. with these conjugates at two week intervals. v -klh induced mainly igg , whereas v -ba induced all igg isotypes but lgg a predominated. fecal extracts from mice immunized with v -ba were shown by elsa to contain iga antibodies. sera from these mice bound gp , expressed on the surface of infected cells. sera from mice immunized with v -ba inhibited syncytia formed between cd ' t cells and chronically infected [hiv-i (mn)] h cells. inhibition of syncytia, formed by other hiv- lab. strains correlated with the degree of their homology with the v region of hiv-i (mn). to mimic the efffect of hiv- , mice were depleted of cd ' cells using anti-l t at the time of primary or secondary immunization. following primary immunization, cd + t cell depletion abrogated v -klh antibody responses, whereas responses to v -ba were retained and sera from these mice were able to inhibit gp- mediated syncytia. in secondary responses, cd ' t cell-depletion prevented boosting to v -klh, but v -ba increased anti and syncytia-inhibiting antibodies. these results suggest that: . . abortus, can provide carrier function for a peptide and induce both serum and mucosal antibody responses, and . that infection with hiv- with subsequent impairment of cd ' t cell function would not abrogate anti-hiv- antibody responses if . abortus is used as a carrier to stimulate memory responses. nucleotide sequence analysis of the vh genes revealed the usage of one particular vh germline element (vh - p) in all clones. this finding allowed the determination of somatically mutated positions in the vh regions. two vsv-ind neutralizing antibodies expressed vh and vl genes in complete germline configuration whereas the rest of the clones showed somatic mutations which obviously were antigen dependently selected for. however, binding affinities of mutated and unmutated antibodies were comparably high. in order to determine the influence of somatic point mutations on one single antibody we generated a monovalent single chain antibody (fv-ck) of a mutated clone and reversed it stepwise to germline configuration by means of site directed mutagenesis. surprisingly, already the germline configuration of fv-ck could neutralize vsv-ind, even though the binding affinty was lower than that of the mutated fv-ck. every single somatic point mutation tested improved the binding avidity although some mutations reduced affinity. thus, during the course of vsv-ind infection some antibodies are subjected to avidity maturation although this is not required for the generation of high affme, efficiently virus neutralizing antibodies, lisa hyland'", sam hou'.~, and peter c. doherty'. 'department of immunology, st. jude children's research hospital, memphis, tn i, departments of immunology and microbiology, and 'pathology, university of otago,dunedin, new zealand. the b and t cell responses in c bl/ j(b ) mice treated with the mab mel- to l-selectin have been analysed following i.n. infection with sendai virus. mel- treatment caused a - % decrease in the lymphocyte recruitment to the mediastinal (h ln) and cervical (cln) lymph nodes following infection with sendai virus. the cellularity of the spleen was unchanged. the clonal expansion of cd + ctl precursors in the mln was slightly delayed, but potent ctl effectors were present in the virusinfected lung by day after infection and the overall magnitude of the response was not compromised. the prevalence of iga antibody forming cells (afcs) was greatly increased in both the mln and the cln of the mice given the mel- antibody. the igm response was prolonged and the igg response, particularly iggl, was delayed compared to controls. the altered pattern of the antibody response may reflect the limited availability in mel- -treated mice of th cells secreting lymphokines which are involved in ig class switching, by blocking the entry of cd + th precursor cells into lymph nodes. facs sorting for l-selectin+, +, and l-selectin-, b + cell populations from the mln and the cln of normal b mice days post sendai virus infection, showed that the afcs were from the l-selectin-, b + cell population, a population which comprised - % ofthe total cell population. we have distinguished targets of broadly neutralizing antibodies present in hiv- infected individuals by imunoselection in vitro and by the use of chimeric virus. one target of neutralizing antibodies, defined by an escape mutant with an ala to thr substitution at position in gp , is resistant to human monoclonal antibodies that map to a site closely congruent with that for cd binding. substitution of gly, ser, and val fail to confer resistance. a second, defined by an ala to val substitution at position , upstream from the v loop, does not involve the same site and does not involve v . substitution of thr or ile also confers resistance. replacement of the v loop of hiv-l(mn) into a clone of hiv-l(iiib) allows the detection of two other broadly neutralizing targets. one recognizes the v peptide of mn but is affected by regions outside v . the other appears to be conformational and outside v , but its functional recognition is influenced by the v loop. all of these sites seem to depend on the overall conformation of the envelope protein rather than a single discrete linear epitope. antibodies against amino acids - of the hiv transmembrane (tm) glycoprotein have been shown to enhance hiv infection in vitro in the presence of complement. there has been no study demonstrating that enhancing antibodies to this region of hiv, despite increasing levels of infectious virus to fold in vitro, adversely affect disease pathogenesis. in two separate studies reported herein, it is shown that animals which have high levels of antibody against this region of siv, amino acids - of the envelope, fair poorly compared to animals with lower antibody levels against this region when subsequently challenged with siv. when actively immunized with a synthetic peptide from this region of siv, animals died earlier and failed to clear antigen at two weeks after infection compared to animals that received a control peptide (p< . ). when animals were passively immunized with antibodies from a longterm survivor of siv infection, those animals that received higher levels of antibody against the tm peptide died within six months compared to longer intervals for those animals that had lower levels of antibody to this region. when taken together, these data suggest that antibody to the tm region of siv and hiv in general, and to this highly conserved peptide in particular, are detrimental to the host. therefore, immunization strategies that minimize the immune response against tm or treatment protocols that decrease antibody levels against tm may lead to prolonged survival following exposure to lentiviruses. we have developed a mouse model to examine the immune response to hpv proteins when these proteins are presented to the immune system via the epithelial route. in this model animals are grafted with keratinocytes expressing hpv e a n d e genes using a transplantation procedure which permits epithelial reformation. animals so grafted when challenged intradermally with e either as protein or via a recombinant vaccinia virus exhibit a delayed type hypersensitivity response which is e -specific and cd + t cell mediated. animals grafted with a sub optimal priming inoculum of cells develop immune non-responsiveness and have an abrogated dth response when challenged subsequently with a priming cell graft. in the present study w e have examined the antibody status in these animals. the e protein of hpv was expressed in e. coli as a maltose binding fusion protein using the plasmid vector pmalc. after cleavage and affinity purification this protein was used in a n elisa assay to measure antibody levels in groups of mice ( ) those not challenged with e ( ) mice not grafted but challenged with e protein in the ear ( ) mice primed by grafting with hpv e expressing cells and challenged with e protein ( ) mice primed by grafting with x hpv e cells on day , grafted again with lo hpv e cells on day and challenged with e protein in the ear. mice optimally grafted and challenged (group ) exhibited high titres of igg antibodies, particularly elevated levels of iggza. mice sub-optimally grafted (group ) exhibited igg antibody levels comparable to the control group ( ). the possible mechanisms of this immune attenuation are discussed. the hepatitis c virus is a frequent cause of chronic liver disease. a proposed mechanism responsible for virus persistence is evasion of the host immune response through a high mutation rate of crucial regions of the viral genome. the portion of hcv genome coding for the amino-terminal part of the putative envelope protein (gp ) undergoes frequent mutation during the course of infection. we have cloned and sequenced the hypervariable region (hvri) of the virus isolated from an hcv asymptomatic patient at three time points during months follow up. sequence analysis has allowed the identification of variants of this region and multiple antigenic peptides (map), corresponding to three hvrl variants, sequentially foundin the blood stream of the patient, have been synthesized. maps have been used as antigens for detection of specific antibodies in elisa. our results show that anti-hvri antibodies and their cognate viral sequence coexist in the blood stream but a viral sequence becomes undetectable when the specific antibodies reach maximum levels of reactivity. thus humoral immunity against the hvrl may play a role for virus clearence. the presence of anti-hvr antibodies was also investigated in hepatitis c viremic individuals and non-viremic patients. a high frequency of positive reaction ( %) against at least one of the three hvrl variants analysed in this study was detected in the viremic patients. finally, competition experiments show that antibodies crossreacting with more than one hvrl variant are produced by hcv infected individuals. this results suggest that complex cross-reactivity exist between hcv isolates for antibodies against the hvrl region as described for antibodies against the gp v loop of hiv. we propose as mechanism for viral escape in hcv chronic infections the one described as the "original antigenic sin", observed firstly in influenza, in togavirus, paramixovirus, enterovirus, and recently in hiv infection. using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge witb the murine rotavirus strain edim (ward et al., ) . to determine i f a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of reassortants between the fully protective edim strain and a partially protective heterologous rotavirus strain (rrv-g). reassortants that contained genes for edim proteins responsible for protection were anticipated to provide complete protection; however, no edim proteins were found to be both necessary and sd cient for full protection. instead, protection was found to be highly correlated with viral shedding (p = , ) and with serum rotavirus iga titers stimulated by the different reassortants (p < , ). this indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of edim proteins. this conclusion was supported by the finding that the titers of serum rotavirus i& but not igg, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against edim. if these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization. h a l l medical center, individuals with adequate serum samples were identified as either rapidly progressing (rp) or slowly progressing (sp) by clinical and surrogate marker criteria. anti-v profiles were determined using synthetic proteins derived from the amino acid sequences of the v region of laboratory strains of hiv- in standard capture elisa format. serum obtained from each patient at multiple different time points was screened against these peptides. the majority of individuals in both groups demonstrated broad recognition, with reactivity to peptides corresponding to the v regions of mn, sf , ny and han/sc. less than % of individual in each group recognized the v peptide derived from iiib, @=ns, between groups). as the rp progressed to aids there was significant nonspecific narrowing of response, while the sp remained broadly reactivity (p< . ). in v i m neutralizing activity of the homologous laboratory isolates was determined with cytotoxicity, cytopathic effect and p ag inhibition assays. although most patient serum was capable of inhibiting p ag production in homologous lab strains while aids-free, there was no relationship with the ability to inhibit homologous virus effects on target cells and anti-v profiles. model, we show that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appear in the bone m w and constitute the major sou~ce of longterm antibody production. following infection of adult mice, wspecific antibody secreting cells (asc) peaked in the spleen at days postinfection, but were at this time undetectable in the bone marmw. the infection was essentially cleared by days and the asc numbers in the spleen rapidly declined while an increasing population of lcmv-specific asc appeared in the bone marrow. when compared to the peak response at days post-infection, timepoints from days to more than one year later demonstrated greater than a l@fold reduction in splenic asc. in contrast, jltvlv-specific plasma cells in the bone marrow remained at high numbers and correlated with the high levels of antiviral serum antibody. the prewnce of antiviral plasma cells in the bone marrow was not due to a persistent infection at this site, since virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and pcr assays. the igg subclass profile of antibody m e t i n g cells derived from bone manuw and spleen correlated with the igg subclass distribution of lcmv-specific antibody in the serum. upon rechallenge with w , the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. bone marrow asc populations and lcmv-specific antibody levels in the serum did not change during the early phase of the reinfection but both increased about -fold by days post-challenge. after both primary and secondary viral infection, lcmv-specific plasma cells were maintained in the bone marrow showing that the bone marrow is a major site of long-term antibody production after acute viral infection. "memory" t cells, and associated with responsiveness to soluble and recall antigens. cd + lymphocytes staining bright, dim, or negative (equivalent to an isotype control) for cd were evaluated in uninfected controls (group l), hiv- positive patients with % cd + t cells (group ), and hiv-i-infected patients with ~ % cd + t cells (group ). most of these subjects also had -color staining for cd \cd ro\cd ra. the appearance of positive cd and cd ro on hivinfected and uninfected cells correlated well (r=. p<.ool). the percentage of cells staining cd +\cd +.(bright plus dim) was . ( %cl . - . ) in group , . ( . - . ) in group , and . ( . - . ) in group . the respective values for these groups that were cd +\cd gbwm was . ( . - . ), . ( . - . ), and . ( . - . ). values for cd +\cd ro+ were . ( . - . ), . ( . - . ), and . ( . - . ), respectively. in single factor discriminate function tests, the %cd +\cd + cells best predicted subject group ( % correct), proving to be a better discriminator than %cd +\cd b'h' ( % ~orrect),cd +\cd ~"" ( l%), cd +\cd ro+ ( %) and cd +\cd ro+\cd ra-( %). overall, no advantage was seen to splitting the cd +\cd + cells into bright and dim positive subsets in the subjects studied for the purpose of stratifying early vs. late hiv infection. likewise, splitting the cd +\cd ro+ compartment into cd ra+ subsets did not improve the ability to distinguish between uninfected and early or late hiv- infected patients. the relationship between the virus-specific cytotoxic response in hiv infected patients and disease progression support the concept that a vaccine candidate should also induce a virus-specific ctl activity. immunization of uninfected adult volunteers by a hiv-gpl recombinant canarypox virus was carried out in a phase i trial.two injections of a recombinant canarypox expressing the hiv-l/mn gp were performed at month and and two boosts of recombinant gpl mn/lai at month and in alum or incomplete freund adjuvant( fa). hiv-envelope specific cytotoxic activities were detected from ctl lines derived from pbmc stimulated by specific stimulation with autologous hiv infected blasts. ctl lines were obtained from out of donors : seven out of eighteen ( %) were found to present envelope specific cytotoxic activity at months , , or post immunization ; this activity was characterized as a cd +,cd +, mhc class-i restricted cytotoxic activity, and for at least two volunteers, this activity was still present two years after the first canari-pox/env injection. because avian poxviruses are incapable of complete replication and undergo abortive replication in mammalian cells , this is a n example of the persistence of long term memory cd + cytotoxic t lymphocytes in the absence of the priming antigen, indicating that t-cell memory might be independent of continued antigenic exposure. the university of alabama at birmingham, al . mhc class i restricted cd ' ctl activity plays an important role in the control of influenza virus infection as indicated in studies in mice and humans. cytokines such as il- and ifn-y regulate the generation of virus-specific ctl responses. we recently demonstrated a good correlation between the induction of influenza virus-specific ctl activity and the production of ifn-y by the cd ' t cells at the single cell level using an if?-specific elispot assay, secreted ifn-y by an elisa, and ifn-y specific mrna expression by rt-pcr. several recent studies have characterized cd + and cd ' t cells by their expression on the surface of distinct d r isoforms. cd ra is expressed on naive or virgin t cells, while cd ro is expressed on memory t cells. in the present study, pbmc of healthy young adult subjects were stimulated with influenza a virus and then enriched for cd + t cells. the cd ' cells were stained for cd ro' (pe) and cd ra' (fitc) cells and sorted. ctl activity against virus-infected autologous target cells was determined in a hour 'lcr release assay while ifn-y production and expression was assessed by elispot and quantitative rt-pcr, respectively. cds+/cd ro+ (memory) cells exhibited significant mhc class i ctl while cds+/cd ra+ cells exhibited no lytic activity. no activity was exhibited by freshly isolated or unstimulated cd +/cd ro+ t cells. similarly, cd +/cd rot t cells contained significantly higher numbers of ifn-y spot forming cells and higher quantity of ifn-y-specific mrna than cd +/cd rac cells. these data support our previous findings that ifn-y may serve as a useful surrogate marker for influenza virus-specific ctl activity in humans. in studying the kinetics of the cd + t cell response in lcmv infection we have observed a profound activation and proliferation of cd + t cells with a - fold increase in total number peaking at day - post infection. in c bw mice, most of the viral antigen is cleared by day seven, and after day the total cd + number per spleen drops about -fold. however, the relative specificity of the viral peptidespecific precursor ctl frequencies @ctwf) per cd + cell remains remarkably stable between day - of the acute infection and for many months thereafter. thus, the decline in the cd ' t cell number is not a function of the tcr specificities but is rather an across-the-board event. in contrast, we found that subsequent to the decline of the ctl response to a second heterologous virus infection such that the mouse was in a "resting, immune'' state, there often was a reduction in pctl/f to the first virus. for example, infections with w or mcmv substantially reduced the pctuf to lcmv or pv in all memory compartments, including spleen, lymph nodes, peritoneal exudate cells. reinfection with the original virus substantially elevated its pctuf and restored the pctuf that had been reduced by a heterologous viral infection. analyses of the progression of ctl responses during a heterologous virus challenge of a virus-immune mouse indicated a high frequency of crossreactive ctl appearing early during infection, but as the infection progressed there was a higher proportion of ctl specific only for the second virus. thus, we believe that when the across-the-board apoptosis of t cells occurs late in the infection, ctl specific for the first virus are diluted by those responding to the second virus. this may cause the reduction in memory to the first virus and may be one of the mechanisms contributing to the waning of secondary immune responses to certain viruses over time if there is no re-exposure to the original infectious agent. t-cells which arise after virus infection will aid our understanding of tcell memory and be useful in the design of vaccines which augment the memory response. to estimate the sendai virus specific precursor frequency in memory mice, cd + cells from c bl female mice which had been infected with sendai virus intranasally (i.n.) more than two months earlier were subjected to limiting dilution analysis. responder cell populations were enriched for cd + cells either by magnetic bead depletion of non-cd + cells, or by facs after staining with anti-cd monoclonal antibody these enriched (> % cd +) responders were cultured with sendai virus-infected, irradiated, t-cell depleted splenic antigen presenting cells (apc). supernatants from these cultures were tested for activity on the cytokine-dependent ctll cell line. duplicate cultures of responders on uninfected apc were used to set the level of rejection (mean cpm + x std. dev.). using this type of analysis we were able to demonstrate a frequency of memory thp at cd + cells, compared to a frequency greater than / ooooo in naive controls. the memory cd + cells were further characterized as cd rb-low ( / ) , cd -high ( / ), lselectin-low ( / ), and cd d-high (vla- -high) (v ). this is close agreement with other phenotyping studies on cd + memory cell specific for soluble antigens. t cells, ralph a. tripp, sam hou, anthony mcmickle, james houston and peter c. doherty, department of immunology, st. jude children's research hospital, memphis, tn . the immune response of influenza a and sendai-virusspecific, memory cd ' cytotoxic t lymphocyte precursors (ctlp) have been analyzed in c bu mice infected intranasally with unrelated or cross-reactive respiratory viruses. the numbers of influenza a-specific memory t cells increased in the regional lymph nodes (ln), spleen and bronchoalveolar lavage through the course of an irrelevant infection (influenza b). memory t cells showed evidence of enhanced steady-state activation. profiles of ctlp recruitment were analyzed in association with t cell proliferation and activation to determine whether signaling via the t cell receptor is necessary to induce "bystander" stimulation of the memory t cell pool. the extent of t cell proliferation was addressed by treating mice with low doses of cyclophosphamide (cy). "resting" sendai virus-specific memory t cells were unaffected by cy treatment, however upon challenge with influenza and treated or days later, the emergence of influenzaspecific ctlp was severely diminished. cell cycle analysis showed that cy eliminated the majority of cd ' t cells from the ln and spleen resulting in dna fragmentation of - % ofthis lymphocyte subset. a decrease (though smaller) in the numbers of sendai virus-specific ctlp indicated that some of the cycling cells killed by cy were memory t cells, presumably activated in a "bystander" manner. the decrease in ctlp numbers for both influenza and sendai virus-specific ctlp was still apparent days after cy treatment, long after the viral elimination. thus, immune responses to unrelated antigens may be a mechanism involved in maintaining the pool of memory t cells. experimentally vsv can result in an acute cns infection of mice. data from our in vitro experiments indicate that no has inhibitory effect on productive vsv infection. vsv infection at neuroblastoma nb a cells was significantly inhibited by loopm of a no donor s-nitro-n-acetylpencillamine (snap), while oopm of the control compound n-acetylpencillamine (nap) had no effect. when vsv infected nb a cells were treated with pm of a constitutive no synthase (cnos) activator n-methyl-d-aspartate (nmda), a significant inhibition of vsv production was observed. inhibition by pm of nmda was reversed by pm of nos inhibitor n-methyl-l-arginine (l-nma). work is in progress to determine the effects of inducible nos (inos) in a glioma cell line c on vsv infection. levels of no and expressions of both cnos in neurons and inos in glial cells in the cns following vsv will be further investlgated. supported by nih grant a to carol s. reiss. pediatrics, university of iowa, iowa city, ia. mouse hepatitis virus, strain jhm (mhv-jhm), is a neurotmpic coronavirus which causes acute encephalitis and acute and chronic demyelinating encephalomyelitis in susceptible rodents. . % of suckling c bu (kbdb) mice inoculated intranasally with mhv-jhm at days and nursed by dams immunized against the virus develop a chronic demyelinating encephalomyelitis characterized clinically b hindlimb paralysis, at - weeks postinoculation. the chronic demyelinating encephalomyelitis nor the clinical symptoms. recently, it was shown that lymphocytes isolated from the central nervous system (cns) of c bu mice both acutely and persistently infected with mhv-jhm display a cytotoxic t lymphocyte (ctl) response to the s protein of mhv-jhm. this response was further characterized by identifying the ctl epitopes that are recognized by a bulk population of ctls from the cns of mhv-jhm infected c bv mice. three epitopes were identified using synthetic peptides and truncated forms of the s protein in primary ciz assays. the epitopes recognized were amino acids - (cslwngphl, db), - (rcqifani, kb), and - (nfcgngnhi, db). thus, the results indicate that cytotoxic t lymphocytes responsive to the s protein of mhv-jhm in c bu mice recognize both kb and db-restricted cil epitopes. ctl lines and clones specific to these peptides and the entire s protein are being developed to test their biological significance in vivo with respect to the acute encephalitis and chronic demyelinating disease caused by mhv-jhm. a marked change in susceptibility to some neurotropic viruses during the first few postnatal weeks has long been recognised in rodents. infection of neonatal or suckling mice with the neurotropic alphavirus, semliii forest virus results in lethal encephalitis. infection of weaned animals is not lethal. earlier investigations focusing on changes in specific immunity have shown this not to be the explanation. infection of - week old mice with severe combined immunodeficiency does not result in acute rapidly fatal encephalitis. we have studied mortality, neuroanatomical distribution and spread of infection in mice of different ages and the effect of gold compounds on rendering infection of - week old mice lethal. neuroanatomical distribution of infection correlates with synaptogenesis. as this is completed in different systems within the first two weeks postnatal, systems no longer transmit virus and infection switches from disseminated to focal and restricted. complete productive replication and transmission of infection require smooth membrane synthesis which is present in neurones undergoing synaptogenesis, absent in mature neurones but inducible by administration of gold compounds. infection of neurones undergoing synaptogenesis is productive and virus is transmitted along neuralpathways, infection spreads rapidly around the brain, destroys cells and animlas die of a fulminant encephalitis. in mice infected after days of age replication in mature neurones is restricted, nonproductive, cannot be transmitted, does not spread, is non-destructive and non-lethal. as a consequence, in the absence of immune responses virus can persist in isolated cns cells for life and can even be detected by reverse transcriptase pcr in immunocompetent mice months after infection. in the presence of an immune response, cd + t-cells recognise and destroy infected glial cells leading to dem yelination. a~k e r m a n n ,~ virology swine,' virology cattle,' and avian diseases research units, national animal disease center, usoa, agricultural research service, ames, ia a recombinant pseudorabies virus (prv) (lltbap) was constructed which contains a . kb deletion spanning the standard recombination junction of the unique long and internal repeat sequences replaced by e lacz expression cassette. this deletion interrupted the large latency transcript gene (llt) and truncated one copy of the diploid immediate early iel gene. replication and viral gene expression of lltbaz in madin-darby bovine kidney cells was similar to that of the parental virus and a virus rescued for the deleted sequences (lltbres). when inoculated intranasally in -week-old or -day-old pigs, lltba replicated efficiently at the site of inoculation yet caused markedly reduced fatality when compared to the parent or lltbres viruses. in particular, the lltba -infected pigs did not exhibit neurological symptoms characteristic of prv infection. to further examine the pathogenesis of lltba , -day-old pigs were infected intranasally with lltpa or lltbres and necropsied at various times postinfection. virus isolation from the nasal turbinate, tonsils, and trigeminal ganglia was comparable between the two viruses. although both viruses spread to the brain and induced an inflammatory response in cns tissues, virus isolation from brain tissues was reduced about -fold for lltpa . abundant prv antigen was detected in the cerebrum and cerebellum of lltpresinfected pigs, but only a few antigen positive neurons were observed in the cerebrum of lltba -infected pigs. while replication of lltbres in the brain progressed until death at days post-infection, replication of lltpa in the brain ceased by days post-infection and the pigs exhibited only mild clinical signs. since lltba is capable of spread to the cns, reduced neurovirulence of lltbaz is likely the result of its decreased ability to replicate in cns tissues. the cns is a target for hiv infection, and in individuals with aids this can lead to a devastatin dementia. only certain viral variants appear capable invading the cns and infecting microglia and brain macrophages. in order to determine whether the virus entering the brain may be particularly pathogenic to the cns, we isolated microglia from the brains of siv-infected rhesus monkeys. transfer of these cells into naive animals indicated that productive siv infection could indeed be transferred. furthermore, cns infection occurred within a relatively short time span, and was associated with viral gene expression in the brain and pathology characteristic of hiv encephalitis. serial transfer of microglia into additional animals also resulted in successful transfer of infection, neuroinvasion, and neuropathology. behavioral analysis in a trained group of animals is ongoing. this result demonstrates that neuropathogenic virions partition into the cns during natural siv infection, likely driven by mutational events that occur during the course of infection. molecular characterization of the microglia-associated virus has revealed that a distinct pattern of sequence changes in the envelope gene occurs concomitantly with this in vivo selection. our approach will allow the dissection of functional neuropathogenic elements present in these viruses. in non-specific host defense mechanisms. ifn-y-induced nitric oxide (no) in murine macrophages was previously shown to inhibit the replication of poxviruses and herpes simplex virus type (hsv- ) . we now demonstrate that murine macrophages activated as a consequence of vaccinia virus (vv) infection in viva express inducible nitric oxide synthase (ios). the vvelicited macrophages were resistant to infection with w and efficiently blocked the replication of w and hsv- in infected bystander cells of epithelial and fibroblast origin. this inhibition was arginine dependent, correlated with no production in cultures and was reversible by the nos inhibitor nqjmonomethyl-l-arginine. the mechanism of no mediated inhibition of virus replication was studied by treating vv-infected cells with the noproducing compound, s-niuoso-n-afetyl-penicillamine. antibodies specific for temporally expressed viral proteins, a vv-specific dna probe and transmission electron microscopy were employed to show that no inhibited late gene protein synthesis, viral dna replication and virus particle formation, but not expression of the early proteins analyzed. further, we have also identified putative enzymatic targets of inactivation by no that results in inhibition vv replication. although antiviral ctl are important for virus elimination. they can only halt further virus spread, and cannot reduce the number of infectious particles already present. the beneficial effect of ctl-mediated lysis is apparent only if infected cells are lysed before assembly of progeny virus. if infectious virus was released from infected cells in solid tissues before the generation of neutralizing antibody or in sites where antibody did not readily penetrate, then recruitment of mononuclear phagocytes, which phagocytose and destroy infectious material and/or become non-productively infected, would definitely help control virus dissemination. in this context, inos induction in macrophages may be an important antiviral strategy. in addition, the inhibition of virus replication in infected contiguous cells by inos-expressing macrophages at infectious foci would prevent release of mature viral particles after lysis by nk cells and ctl. since viral early proteins are expressed in such infected cells, their recognition and subsequent lysis by ctls will not be hindered. cns persistence, tropism and genetic j. pedro s i s , anthony a. nash and john k. fazakerley, department of pathology, university of cambridge, cb iqp, uk theiler's murine enchephalomyelitis virus, a natural occuring enteric pathogen of mice, is a picomavirus belonging to the curdovirus genus. following intracerebral inoculation of - week old cba or balb/c mice, the bean strain causes a chronic persistent cns demyelinating infection in a proportion of the cba that survive acute infection. balb/c mice are resistant to chronic demyeliating disease. we have studied the tropism, persistence and genetic variability of bean, in cba and balb/c mice in the chronic phase of this disease. by in situ hybridisation and reverse transcription (rt) pcr and southern blot analysis, no viral rna could be detected in the cns of any balb/c mice later than day post-infection. in contrast, in a large group of cba mice studied up until days post-infwtion, viral rna could be detected by both techniques in % of mice until as late as days post-infection. by employing a combination of, in situ hybridisation for viral genome followed by immunocytochemistry for cell phenotypic markers, bean rna was observed predominantly in oligodendrocytes and occasionally in astrocytes during persistent infection, in both brain and spinal cord. in the persistently infected mice, the striking total destruction of the pyramidal layer of the hippocampus, substantia nigra and anterior thalamic nuclei indicated that these were the mice that had had greatest dissemination of virus and highest virus titers during the preceeding acute phase of infection. direct pcr t h d cycle sequencing of uncloned rt-pcr products, revealed that during persistent infection, loops i and ii of the vpi capsid protein gene did not undergo any genetic variability. furthermore, no changes were detected in this region in sequenced pcr products amplified from the cns of mice with severe combined immunodeficiency in which no selective immunological pressure would have been operative. infection, thomas e. lane, michael j. buchmeier, dorota jakubowski, debbie d. watry, and howard s. fox, department of neuropharmacology, the scripps research institute, la jolla, ca our laboratory is interested in the effects of siv infection in the central nervous system of rhesus macaques. to enrich for neuroinvasive and neurovirulent viruses, microglia were isolated from infected monkeys and used t o infect new, uninfected monkeys. such microglia-mediated infection resulted in the production of neuropathological changes, including giant cells, macrophage infiltrates and microglial nodules in recipient animals within months. microglial cells isolated from siv-infected monkeys produced virus in vitro as measured by reverse transcription (rt) and p production. treatment of microglia with recombinant human interferon alpha (rhulfn-a) resulted in a sharp decrease in viral activity (both rt and p production) suggesting that rhulfn-a is able t o modulate viral activity in infected microglia. we have analyzed slvenv sequences by pcr amplification directly from microglia dna preparations from monkeys. nucleotide sequence analysis results in an enrichment of unique sequences in the v region of the siv env gene. the majority (> %) of nucleotide changes encoded amino acid changes, indicating that these envelope sequences evolved as a result of selection. moreover, sequential passage of sivassociated microglia resulted in an increase in potential n-linked glycosylation sites within the v region of the env gene when compared with the parental virus. these data suggest that sequential passage of microgliaassociated siv may select for neuroinvasive, neurovirulent variants. the adoptive transfer of ctl specific for an ld-restricted epitope within the nucleocapsid protein of the jhmv strain of mouse hepatitis virus both protect from acute infection and reduce virus replication in the mhc class positive cells within the cns. the source of these ctl and the route of their delivery is critical in the outcome of this protection. for example, fold less spleen cells activated in vitro with the pn peptide are required for protection via the direct i.c. route than the i.v. route. in addition, ctl clones are unable to protect via the i.v. route and are very efficient via the i.c. route. these data suggested the possibility that the cd + t cells within the polyclonal activated spleen cell population derived from in vitro culture on the pn peptide were facilitating access to the cns. to examine this question, polyclonal pn-specific t cells were either depleted of cd + t cells prior to transfer to infected recipients or untreated cells were transferred to recipients depleted of cd + t cells with monoclonal antibody gk . . both of these treatments eliminated the ability of the ctl to reduce virus replication within the cns, suggesting that cd + t cells in the peripheral compartment are required for the entry of ctl into the parenchyma of the cns during acute cns encephalomyelitis. division of retrovirology, walter reed army institute of research and henry m. jackson foundation, rockville, md ; department of retrovirology, armed forces research institute of medical sciences, bangkok, thailand background the hn- epidemic in thailand is largely due to two highly divergent subtypes of virus, b and e. dual infection with distinct hn- subtypes, which has not been reported previously, would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. merhoak: pcr typing and serologic typing were used to screen a panel of non-random convenience specimens from hiv- infected subjects in thailand. specimens that showed dual subtype reactivity in these assays were subjected to differential pmbe hybridization and nucleotide sequence analysis of multiple molecular clones to c o n f m the presence of dual infection. results. two individuals were shown to simultaneously harbor hiv- of env subtypes b and e (table) . additionally, both subtypes were identified in co-cultured pbmc from one individual. conclusions. these data provide the fmt evidence of dual hiv-i infection in humans and reinforce the need for polyvalent vaccines. infection by herpes simplex virus wsv) induces in man and in mice cytolytic t lymphocytes (ctl) which recognize the immediateearly protein icp . because of its early expression during the hsv replication cycle, lcp represents a prime target for specific t cell responses susceptible of controlling virus replication. we have expressed in e. coli a remmbinant construct coding for a fusion protein consisting of a fragment of influenza virus non-structural protein (nsi) and the lcp sequence of hsv- . the nsi-icp protein was purified by preparative eleclmplmresis and formulated in oil-in-water emulsions with monophosphoryl lipid a (mpl) and qszl adjuvants. balwc mice were immunized by two intrafootpad injections of formulations containing pg of nsi-icp . responder cells obtained from draining lymphnodes were re-stimulated in vitro with p cells lransfected with icp and then lesled for cytolytic activity on icp -p and control p . the induction of icp specific ctl by different formulations was observed and will be discussed. the induction of heterologous cytotoxic t lymphocytes (ctl) using cassettes of multiple conserved t cell epitopes derived from different proteins and/or virus strains is envisioned as a promising vaccine approach. to study the effects of antigen processing on peptide presentation from chimeric epitope precursors we are using a model system comprising two distinct viral epitopes which are immunodominant in the h- d haplotype: a dd restricted epitope from the gp protein of hiv- and an ld restricted epitope from the murine hepatitis virus nucleocapsid protein (mhv n). the influence of proximity and flanking sequences of epitopes on antigen presentation was analyzed using vaccinia virus (vv) recombinants in which the epitopes were expressed as chimeras containing the individual epitopes in reverse order or separated by different spacer residues. whereas individually expressed epitopes were efficiently recognized by protein-specific ctl, recognition of peptides derived from tandem constructs varied significantly with closer epitope proximity and sequential order. following immunization with the recombinant viruses, the chimeras were all able to induce antiviral ctl specific for the native proteins. however, cn, frequency analysis indicated that the number of responder cells to the same epitope dramatically depends on its context within the chimera and correlates with antigen recognition in vitro. the profound effect of flanking regions on ctl induction suggests that the context of an epitope will require careful evaluation in the design of recombinant multivalent minigene vaccines to induce an optimal t cell mediated immune response. and robert e. johnston', departments of 'microbiology & immunology and %iochemistxy, univ. of north carolina, chapel hill, nc a hll-length cdna clone of venezuelan equine encephalitis virus w e ) has been altered to contain two strongly attenuating mutations and a second subgenomic rna promoter immediately downstream of the structural gene region. expression ofthe influenza ha protein from this second promoter in baby hamster kidney (bhk) cells was approximately ?? of the level in influenza virus-infected cells, as measured by immunoprecipitation. fourweek-old cd- mice were inoculated subcutaneously with x ' p h of the ha vector, vector alone or diluent. expression of ha mrna was detected in the draining lymph node of ha vector-inoculated mice by in situ hybridization, consistent with the organ tropism of vee. mice were challenged three weeks after imnmization by intranasal administration of lo ed, of influenza h s . au corn mice suffered severe disease and % died. only one of ha vector-inoculated mice died, and another exhibited signs of disease for one day and recovered. the geometric mean elisa titer of anti-ha serum igg in the ha-vector inoculated mice was , while only three control mice had measurable serum reactivity, and that was at the lowest dilution tested, . in a parallel experiment, no influenza infectivity was detected in the lungs of ha vector immunized mice at days postchallenge. in contrast, / pbs-inoculated mice and / inoculated with vector alone were positive for influenza infectivity and had geometric mean titers of . and . x lo pwgm, respectively. this vector also has been used to express the h n w c a protein in a form recognized by patient sera and a specific antibody on western blots. these experiments demonstrate the feasibility of using vectors based on attenuated vee cdna clones for protective immunization against heterologous human and animal pathogens. dose/response curves have been used to compare different routes of immunization with plasmid dna encoding the h hemagglutinin glycoprotein of influenza virus. routes of inoculation included intramuscular, intradermal and gene gun delivery of dna. from to . ug of dna was inoculated by intramuscular and intradermal routes. from . ug to . ug of dna was inoculated by gene gun. each route was evaluated for single and boosted immunizations. antibody titers were followed over a week period, following which animals were evaluated for protection against a lethal challenge. each of the routes raised both antibody and protective responses. gene gun-delivery of dna required to , times less dna to raise responses than the intramuscular and intradermal inoculations. boosts did not have much of an effect on antibody titer or protection except at low dose inoculations ( ng and lower for the gene gun). for each of the routes, antibody responses showed good persistence over the weeks of the experiment. inoculation of mice with plasmid vectors carrying a microbial gene under the control of an appropriate promoter results in a full spectrum of immune responses to the vectorencoded antigen. using a murine rabies model a plasmid termed psgsrab.gp expressing the full-length rabies virus glycoprotein regulated by an sv promoter was shown to induce upon inmuscular inoculation a rabies virus specific t helper cell response. of the thl type, cytolytic t cells and virus neutralizing antibodies resulting in protection against a subsequent challenge with live rabies virus given either peripherally or directly into the cenaal nervous system. a response comparable in magnitude was also induce upon inoculation of a vector expressing a secreted form of the rabies virus glycoprotein. the immune response to the dna vaccine could be modulated by co-injection of the rabies virus glycoprotein-expressing vector with plasmids expressing mouse cytokines. inoculation of mice with the psg rab.gp vector and a vector expressing granulocyte/macrophage colony stimulating factor (gm-csf) enhanced both the t helper and the b cell response to rabies virus thus improving vaccine efficacy. co-inoculation with vectors expressing interferon-g failed to improve the response. co-inoculation of the antigen-expressing vector with a plasmid encoding mouse i l caused a reduction of both the t helper cell response and the b cell response to rabies vlns. hpvl e hpv associated cervical cancer cells express hpv e protein and antibody to hpv e can be detected in the blood of cancer patients, yet the twnours are. not rejected. a mouse transgenic for the e protein of hpv , and expressing e protein in the skin, has recently been described ( ) and these mice develop spontaneous humoral immunity to e protein similar to patients with cervical cancer( ). to determine whether immunisation could induce immunity to e sufficient to allow tumour rejection, we firstly demonstrated that immunisation of h-zb mice with hpv e protein with quil a as adjuvant could induce cytotoxic t cells able to kill hpv e expressing tumour cells in nrro. we then used similar immunisation with e iquil a to induce e specific immunity in fvb (h- ) mice. h- qskin @s expressing e were not rejected by e immunised h-zq mice, though immunisation induced antibody to e , and similar grafts were rejected, as expected, across an doantigen mismatch in h-zb mice. we conclude either that hpvl e lack a tc epitope in the context of h-zq, or that expression ofe in the skin from the e transgenic mice is insufficient for recognition by primed effector cells, and further experiments will address this distinction. cervical carcinoma is strongly associated with infection by human papillomavirus (hpv) types or , and continued expression of the e and e gene products. this provides an opportunity for an immunotherapeutic approach to the treatment of cervical carcinoma by activation of immune reponses directed against these virally encoded tumour specific antigens. we have constructed a recornbinant vaccinia virus expressing e and e from hpv and with the aim of inducing e and e specific hla class i restricted cytotoxic t lymphocytes (ctl). the sequences have been inserted into the wyeth vaccine strain of vaccinia virus at a single locus in the form of two separate fused e /e reading frames, each under the control of an early v a d n i a promoter, and each modified to inactivate the rb binding site. the virus has been characterised with respect to its ability to synthesise the expected hpv proteins, its genetic stability, and growth and virulence in a mouse model prior to use in human clinical trials. analysis of hpv € specific ctl from c bu mice immunised with this recombinant virus show the response to be equivalent to that generated by a control vaccinia recombinant expressing non-modified hpvi e alone, with similar recognition of the defined immunodominant h- db restricted epitope, e residues - . ability of mice to resist influenza challenge, arthur friedman, douglas martinez, john j. donnelly and margaret a. liu, department of virus and cell biology research, merck research laboratories, west point, pa mice infected with the laboratory strains of a/pr/ / (hln ) or the mouse adapted a/hk/ (h n ) show complete protection against challenge with a different strain of influenza a. humans, however, undergo multiple influenza infections as previous infections appear to provide weak or short-lived protection against the continual antigenic change of strains. we have previously shown that immunization of naive mice with dna encoding the conserved internal antigen nucleoprotein (np) provides protection against both h and h strains of a/influenza. although such mice became infected they were resistant to weight loss and death this differed substantially from a/pr and a/hk recovered mice which were resistant to subsequent infection. to produce a more representative model of human infection, we infected the lungs of mice with currently circulating strains of human influenza. mice that had been given lung infections with a/beijing/ were susceptible to subsequent infection with the a/hk/ strain although they were resistant to weight loss and death. other strains such as a/beijing/ or a/georgia/ provided only marginal protection against weight loss and death against a/hk challenge. mice that were immunized with np dna had greater resistance to weight loss and death after a/hk/ challenge than mice previously infected with a/bei/ and a/ga/ , and were similar to mice that had been previously infected with a/bei/ . thus, infection with different virus strains provide various levels of cross strain protection and the level of protection provided by immunization with dna can exceed that induced by live influenza infection. the development of sendai virus-specific cytotoxic t lymphocyte (ctl) effectors and precursors (p) has been compared for mice that are homozygous (-/-) for a disruption of the h- -ab class ii major histocompatibility complex (mhc) glycoprotein, and for normal (+i+) controls. the generation of cd + ctlp was not diminished in the (-/-) mice, although they failed to make virusspecific igg class antibodies. while the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (bal) of the infected lung was not modified and potent ctl effectors were present in bal populations recovered from both groups at day after infection. there was little effect on virus clearance. as found previously with cd -depleted h- b mice, the absence of a concurrent class il-mhc-restricted response does not compromise the development of sendai virus -specific cd + t cell-mediated immunity. the importance of cytoxic t lymphocytes in defense against acute and chronic viral infections is gaining increasing recognition. our approach to investigating the structure-function relationship between immunogens and their in vivo ability to elicit cytotoxic t lymphocyte responses has been to formulate simple, well-defined structures that vary in their ability to introduce associated antigens directly into the cytoplasm of antigen presenting cells. we have introduced methods for the preparation of unique, lipid-matrix based immunogens, which are highly effective in mice and monkeys for stimulating strong cd + cytotoxic t cell responses, (ctl). antigens used have been proteins or peptides derived from influenza, parainfluenza, and hiv viruses, and whole formalin-fixed siv. ctl can be induced by parenteral as well as oral administration. comparing the physical and chemical nature of our formulations with those from other laboratories which have reported the use of subunit preparations to induce cd + ctl, leads us to propose that a minimal immunogenic formulation capable of eliciting cd +, mhc class i restricted cytotoxic t lymphocytes includes: i) a peptide that represents a mhc class i epitope; ii) a component that enhances the aftinity of the immunogen for mhc class i positive antigen presenting cells ; iii) properties that can compromise the integrity of a lipid bilayer, facilitating delivery of the antigen directly into the cytoplasm for class i presentation. cd + responses to peptides, glycoproteins, and even whole fixed viruses, makes them attractive candidates for diseases where clearance of infected cells is important in protection and recovery. cani ne rabies is uncontrolled. rabies also is epizootifally active in several species in most areas of the wor!d. thm, vaccination of animals, both wild and domestic, as well as postexposure treatment of humans remains a global concern. unfortunately, in those countries in which,people most need postexposure prophylaxis, the best vaccines are expenswe and in limited supply, whereas available vaccines are of questionable immunogenic efficiency, are otten contaminated and may produce neurological complications. the goal of this study was to determine whether a rabies vaccine for global use is complete one round of replication have the potential to be used as vaccines. we have previously reported the abiliity of a ghdeleted herpes simplex virus type (hsv- ) to protect mice and guinea-pigs from subsequent challenge with wild-type hsv. this virus, which we have called disc (disabled infectious single cycle) virus, can infect normal cells but the absence of gh in the progeny virus prevents further rounds of infection. as disc hsv clearly has potential as a vaccine, it is important to determine the durability of the immune response elicited by this virus. we have investigated the ability of disc hsv- to protect mice from a wild-type virus challenge six months post vaccination using the ear model of hsv infection. two immunisations on day and day resulted in a considerable reduction in virus titres in the challenged ears, and an almost complete absence of virus in the dorsal root ganglia. hsv-specific antibody titres as determined by neutralisation and ellsa were maintained for the six months period. it was possible to demonstrate an hsvspecific cytotoxic t-cell response in the disc hsv- vaccinated mice following challenge; this ctl activity was similar to that observed in mice vaccinated with wild-type virus and challenged after the same time period. animals vaccinated with inactivated virus or control mock-vaccinated mice showed a low level of ctl activity typical of a primary ctl response following challenge. these results indicate that an effective cell-mediated and humoral anti-hsv immune response can be maintained for at least six months following vaccination with disc hsv- . viruses which lack an essential gene and thus can only the lmmunogenicity of two ctl @topes. influenza npl - and plasmodium berghei cs protein - were studled in balblc mice. paptides were formulated as a) a iipopepudepeplkle conlugated to irlpalmltoyl-sgly~~l cysteine (pam cyj) and dissolved in a % dmsolglycerol solution. b) micmparticles prepared with poly @.l ladide-coglywlide) using a solvent evaporation technique. the micropaltides were administered as a suspension in phosphate buffered saline or c) an emulsion prepared wilh egg lecithin and % soya oil in water. wpg of peptide or controls (the welght equivalent of blanks) were administered to groups of mice intra-peritoneally or subcutaneously at . and days. days following the last immunization splenocytes were cukured in vlro in the presence of appropriate pepwe or wntml m h rat con a supematant as a source of omwth fadors. ctl adivity was measured in a standard hour chromium release assay and results expressed as % specific lysis. ctl could be elicited in vivo with all three formulations. at an ewedoctarget ratio of w:l the plasmodium berghei peptide encapsulated in micmpartides gave % iysls on peptide pulsed target calls. levels of lysis were similar for the peptide in emulslons. the iipopeplide p ccs - gave a level of lysis of % at an e:t ralioof w . these results demonstrate that peplides edminldered in a variety of formulations can induce a systemic ctl response in vivo. peplide vaccines using such formulations wuld be used to stimulate ctl responses as part of a prophyladic vaccines or as immunotherapeulics. attenuated the attenuated sabin strains of poliovirus have been used for many years to elicit protective immunity to poliovirus. oral vaccination with replicating polioviruses generates both mucosal and systemic immunity. therefore, use of recombinant polioviruses expressing heterologous antigens as vaccine delivery vectors should provide a system for generating protective immunity to those antigens. cdna copies of the poliovirus genome has been used to construct vectors containing a multiple cloning site for insertion of heterologous genes. a pilot enhanced-potency inactivated poliovirus vaccine (ejpv) with assumably improved immunogenicity containing win-treated type poliovirus (shah sauketf) together with the regular type and type canponena was subjected to s*mdard safety and potency tesls in the labmatory and laken through wase i and i clinical aials. in balb/c mice, the lrypin-mcdit%d e-if' v cfryipv) was found to induce antibodies targeted ouaide the uypsin-sensitive bc-loop of capsid protein vp . as previously shown for hypsin-mated type poliovirus @vm alone. trypsin used to modify the type component at the bulk phase was removed by the vaccine manufacturer (rivm) in the regular purification process. absence of uypsin in the final product was further confumed by immunizing mice and rabbits with -fold concentrated type component of tryipv. assays for lrypin antibodies using eia and westem blot techniques were newve. in the clinical phase aial six adult volunteers with existing immunity to poliovirus were given increasing doses of tryipv. already one tenth of the regular dose induced a booster effect in neuarlizing antibodies to both intact and mypsin-treated type poliovirus. no unexpected sideeffects were recorded phase i trials comprised adult volunteers with at least years since the last dose of poliovirus vaccine and children who were due to receive the third dose of the regular immunization schedule at about years. in both groups. individuals received tryipv and were injected with the regular enhanced potency ipv (e-ipv). serum specimens drawn before injection and one month after were tested for neunalizing antibodies using standard microneuwlization assays (all mutypes) and the racina test (intact and uypsin-mated type poliovirus). in all volunteers tryipv was at least as immunogenic t w the regular e-ipv according to all assays. no statistically significant differences in side effects were reported. a murine/influenza virus model has been used to evaluate the longevity of antibody and protective responses raised by gene gun delivery of a hemagglutinin-expressing dna. mice were immunized and boosted at one month with . ug of an h expressing plasmid dna (pcmvri ). antibody responses and protection against a lethal challenge were followed over the next year. antibody responses had good longevity exhibiting comparable titers at one year post boost as at days post boost protection against the lethal challenge was complete at days, month and four months post boost, but only partial at one year. a transgenic mouse model for identifing htlv- t-cell epitopes: generation of hla-b* -restricted ctl directed against synthetic peptides and naturally processed viral antigens, christian schiinbach*, ai kariyone*, kiyoshi nokiharaa+, karl-heinz wiesmulle and masafumi takiguchil, departments of tumor biology* and immunology#, institute of medical science, university of tokyo, tokyo "tokyo university of agriculture and technology, tokyo +biotechnology instruments department, shimadzu corp., kyoto, japan $natural and medical science institute at the university of tubingen, reutlingen, germany the majority of human t-cell leukemia virus type-i (htlv-l), hla class i-resmcted t-cell epitopes have been identified by cloning htlv- patient-derived t cells. here we describe for the frst time a rapid method (reverse immunogenetics) for identifing t-cell epitopes, together with a transgenic mouse model as a guide for testing the cellular immune response to a mixture of the lipohexapeptide immunoadjuvant pamgcys-ser-(lys) and synthetic htlv- peptides which seem suitable for vaccine design. htlv- amino acid sequences were searched for eight to mer patterns carrying the anchor residues of the hla-b* peptide motif at positions two and eight to fourteen. candidate peptides were synthesized according to the matched sequence patterns. their hla-b* affinity was quantitatively analyzed in an indirect immunofluorescence peptide binding assay using rma-s-b* cells. the fourth group (controls) were inoculated with h n (in) thereby providing heterotypic ctl immunity in the context of a natural infection without the confounding effects of humoral immunity against surface antigens. all four types of inoculations have been shown to protect normal (class i expressing) mice from a lethal challenge with influenza, presumably mediated by class i restricted cytotoxic t cells. the two groups inoculated via the intranasal route may gain additional protection by activating the mucosal immune system (iga). none of these types of inoculations has been evaluated in the context of class i restricted cytotoxic t cells, the only ctls found in class i deficient mice. for all four types of inoculations, mhc class i deficient mice lost significantly more weight than the class i expressing control groups (seven mice per group) indicating the importance of class i restricted t-cells in protection. within the class i expressing groups, there was no significant difference between the four types of inoculations; within the class i deficient groups the vac-np im immunized mice lost significantly more weight than the h n group;the other two groups, vac-np in and genetically immunized groups had intermediary results. these data lend support for a protective role for mucosal immunity. results on both class i and class i ctl activity for the four types of inoculations will also be presented. we tested the pbmcs of patients participating in two vaccine therapy trials for their ability to recognize overlapping peptides of the gp la sequence. seventeen patients participating in a phase i gp protocol and patients participating in a phase i gp protocol had their pbmcs isolated by ficoll separation of heparinized venous blood. the fresh pbmcs were plated, in triplicate, into well plates containing peptides overlapping the la sequence of gp , pulsed on day with tritiated thymidine and harvested and counted on day . results: the percentage of patient's pbmcs from each trial with an lsi to each peptide are depicted below. conclusions: the pbmcs of hiv-infected volunteers who have been multiply immunized with either gp or gp proliferate to multiple peptides within the gp molecule. reactivity from the end of c through early c (lai #i - ) is particularly prominent and contains previously undescribed th epitopes (asterisks). conspicuously missing is reactivity to the v loop peptide (la # ). although the percent reactivity to the entire gp molecule is similar between the immunization groups, there is differential recognition of some of the individual peptides, particularly peptides in early c (lai # - ). the intracytoplasmic lifecycle of listeriu mmcytogenes (lm) enables it to be a convenient vaccine vehicle for the introduction of foreign proteins into the mhc class i pathway of antigen presentation. taking advantage of these properties, we have inserted the nucleoprotein (np) gene from lymphocytic choriorneningitis virus (lcmv) into the lm chromosome by site specific homologous recombination. infection of mice with recombinant lm expressing lcmv-np elicited a virus-specific ctl response. we were able to recover lcmv-np specific ctl precursers from recombinant lm vaccinated mice as shown by vigorous secondary ctl responses after in vitro stimulation. in contrast to mice immunized with wild type lm, mice vaccinated with np-recombinant lm were protected against challenge with immunosuppressive lcmv variants. protection was demonstrated by reduced viral titers or complete clearance of lcmv from serum and various organs including, spleen, liver, lung, kidney, and brain. the kinetics of the lcmv challenge indicate that mice vaccinated with recombinant lm were able to arrest viral growth early in the infection due to a strong ctl response and did not exhibit the immunopathology associated with infection of naive mice. since lm not only delivers antigens into the mhc class i pathway but also induces il- production, it has the potential to function simultaneously as a vehicle for expressing foreign antigens and as an adjuvant promoting cell mediated immunity. , latent membrane protein [lmp] and a) in chromium release assays. we were fortunate in identifying one child from whom cryopresetved pbmc samples were available before. and during ebv seroconversion. ebv-specific ctl activity was demonstrated concurrent with initial detection of virus in the peripheral blood by ebv-dna pcr. in the absence of detectable serum antibody. ctl lines from all nine children recognized one or more ebv latent gene prcduct(s). all children demonstrated ctl responses against one or more ebna proteins ( a, b, c). and ebna c was recognized most frequently. no ctl responses were detected against the ebv latent proteins ebna , , lp or lmp . the ebv-specific ctl lines expressed cd /cd and mab blocking experiments demonstrated that the majority of target cell lysis was inhibited by antibody against mhc class-i but not antibody against mhc class- . these results represent one of the first reports characterizing ebv-specific ctl responses in young children. the striking similarity between ebv-specific ctl responses described here in young children and those reported for adults suggests that the ebna family of proteins and lmp a should be considered for inclusion in candidate ebv vaccines. evaluation of cellular immune responses to adenovirus vectors in the cotton rat. soonpin yei,' gary kikuchi,' ke tang' and bruce c. trapnell.' departments of virology' and immunology, genetic therapy, inc., gaithersburg, maryland replication deficient recombinant adenovirus (av) vectors are efficient gene delivery vehicles currently being developed for a variety of in vivo gene therapy strategies such a s for the fatal pulmonary component of cystic fibrosis. the cotton rat (sigmodon hispidus) is one of the most widely accepted animal models for studying these av vectors because wild type human adenovirus replicates in cotton rats and because of the histopathologic similarities of infected respiratory epithelial tissues from humans and cotton rats. despite this, methods for studying immunologic responses in the cotton rat have not been developed. importantly, recent studies in the cotton rat (gene tber. : - ; ) in our laboratory suggest that a dose-dependent specific immune response to av vectors can limit expression of the transgene. in this context, w e have established methods to evaluate cytotoxic lymphocyte (ctl) responses to av vectors in the cotton rat. to accomplish this, a ctl target cell line was established consisting of primary cotton rat lung fibroblasts (crlf). splenocytes from cotton rats exposed previously to an av vector were harvested, cultured in virro with irradiated, addb nfected crlf. cultured (effector) splenocytes were then incubated with s'cr-labelled crlf (target) cells a t effectoctarget (e:t) ratios of , and . in parallel, splenocytes from naive cotton rats served as negative controls. results demonstrated vector-specific ctl lysis of target cells significantly greater than controls: . f . % vs . * . %. . * . % v s . * . %. and . * . % vs . * . % (meanrts.e.m., n= ; p<o.ol, all comparisons) a t e:t ratios of , and , respectively. thus, a specific ctl response does develop in cotton rats following in vivo av vector administration. this observation will be useful in guiding vector modifications in the rational development of improved vectors for clinical use. the identification of ctl epitopes is essential for subunit vaccine design against aids. a major portion of the anti-viral ctl responses after infection with htv in humans or siv in rhesus macaques is made up by anti-gag specificities. here, effector cell populations were established from hiv seropositive individuals by specific stimulation of pbl with pfa fixed autologous b-lcl infected with tw gag. expanded cultures were then tested for ctl activity against autologous b-lcl pulsed with overlapping -mer peptides derived from p . two individuals ( and ) had ctl against p . (a(mino) a(cids) - , krwiilglnknrmysftsi), two others ( and ) recognized p . (aa - , frdwdrfyktlraeqasqd). similarly we found two siv infected rhesus macaques reacting against the analogous aa sequences of siv; it. p . ( ) and p . (ivy). this led us to further fine map the human and rhesus ctl reactivities using sets of aa overlapping -mers and to test for hiv- isiv crossreactivity. both two p . epitopes (possibly restricted by hla-a ) and the p . epitope were non-overlapping, and all three epitopes were non-crossreactive. finemapping of the p . epitopes is in progress. the sn p . epitope was also non-crossreactive, despite only one aa difference (position , s+t) between the siv and hiv sequence. others have also mapped cl'l epitopes to the same regions of p in humans (johnson ji , , , buseyne jv , , and to p . in cynomolgus macaques (gotch jmprim . , ) . in conclusion, multiple non-cross reactive ctl epitopes are clustered within corresponding regions of hiv and siv gag. ctl hotspots may be important for inclusion into vaccines. forming virus) with cd- and cd- -cells, b-cells and adherent cells, while an average of % of virus was associated with lps-stimulated -cells and adherent cells. in the second experiment the combined function of antigen presenting cells, -helper cells and -cells (humoral immune response) to a third party antigen (srbc) during the acute phase of enterovirus induced disease was increased at day , and decreased at days , and post-cvb , infection relative to unlnlected animals. conclusions: these data indicate that cvb , associates with and replicates in rnurine splenic immune cells, and that infection of susceptible mice with cvb , modifies the immune response to a third party antigen during the acute stages of disease. an understanding of such cvb ,-i mmune cell interactions is criitical for gaining insight into the pathogenesis and persistence of enteroviral infection both within and outside of the immune system. understanding the pathogenic mechanisms of virus infections depends upon identification of viral gene products involved in host-virus interactions in vivo. many viral gene products which affect pathogenesis in vivo are nonessential for virus replication in vitro. therefore, we constructed insertion or deletion mutants in the hind i j and i regions of murine cytomegalovirus (mcmv) to assess the importance of these genes in in vivo replication of virus as well as interaction with immune regulatory and effector cells. three mutants replicated in nih t fibroblasts as efficiently as wild-type virus, thus confirming the nonessential nature of the genes. deletion of . kb in hind i j and of . kb in hind i j and i resulted in mutants with reduced replication in target organs in vivo. an insertion in hind i j had no significant effect on replication in mouse salivary gland, but curtailed growth of the virus in footpad fibroblasts and subsequent priming of mcmvspecific cpl precursors in the draining lymph node. in addition, the . kb deletion mutant was unique in its failure to replicate in a permissive, differentiated macrophage cell line. current studies aim to further define the role of this gene region in hcmv pathogenesis. that in vv - progression o f v v life cycle is blocked at the the uncoating ii or replication stage. furthermore, host protein synthesis in v v - cells is not shut-off after v v infection suggesting uncoatingll/replication is important for determining cell fate. since this mutant clone vv - was isolated b y retroviral insertional mutagenesis. a cellular gene that was integrated by single retrovirus was isolate and codes for a kb transcript in northern blot analysis. a kb cdna was isolated and codes f o r a novel polypeptide o f amino acids that show no homology with known proteins. experiments are in progress to understand t h e role of this cellular gene in vv infection. in addition, a two-hybrid system will be employed to identify a n y viral gene product interacting with this cellular target u s i n g o u r laboratory's s t a n d a r d s t r a i n o f a/japan/ / in in uitro assays of viral infectivity of the mouse b-lymphoma a - . . we have found that although infection sensitizes the a cells for recognition by both class i and class i restricted ctl.'s and leads t o high surface expression levels o f hemaglutinin (ha), the infected a cells grow through t h e infection, ha expression declines, and few of the infected cells die. in contrast, using a second s t r a b of a/japan/ / from a different passage history, we find that n o t only are the infected a cells sensitized for lysis by both class i and class ii restricted ctl's, exhibiting even higher levels o f ha surface expression, but in addition, most cells in the infected population die with characteristics of apoptosis. the two virus strabs appear to be highly related because in various assays we have found t h e m antigenically indistinguishable in both their t-and b-cell epitopes. using these two virus strains in a n in uiuo dose response assay, we have found that o u r laboratory's standard strain will kill all mice infected with a - the therapeutic and prophylactic antiviral efficacy of interleukin- (il- ) was studied using murine models of herpes simplex virus (hsv) and murine cytomegalovirus (mcmv) infection. therapeutic intraperitoneal administration of il- commenced hours after mice were infected w i t h hsv, and was continued daily for a total of days. il- therapy improved the survival rates of mice w i t h systemic hsv infection, compared to that of placebo treated infected mice. since neutrophilia and thrombocytopenia were common in dengue patients,we are investigating whether bm may be accompanied with abnormal hematopoiesis. using ficoll-hypaque separation, mononuclear cells prepared from the total bm cells. . x we~ells/well of both adherent and nonadherent cell types were infected with two different den- virus strains : ( ) thiland strain isolated from the dengue hemorrhagic fever(dhf patient and ( ) taiwan strain isolated from the dengue fever(df) patient at moi=l and collected the suprnatants at various time points for assay. the preliminary data showed that : (a) adherent cells were infected with den and dhf virus strain replicated much more efficiently ( . x pfu/ml on day p.i. than df strain; (b) nonadherent cells were times less efficient to produce viral yields than adherent cells (dhf and df strains peaked at . ~ ' pfu/ml and . ~ pfu/ml on day p.i., respectively); (c) addition of gm-csf to nonadherent cells increased virus infectivity and productivity. in conclusion, den viruses were able to infect bm cells and the more virulent virus strain isolated from dhf patients had better replication capability in human bone marrow cells. future studies on cytokines and immunologic evaluation are in progress. number of gene products whose function is to modify host responses. recent evidence suggests that a subclass of poxvirus-encoded host response modifiers interferes with host defense responses, and thus can be termed immune or inflammatory response modifiers (irms). several poxvirusencoded irms possess homology to mammalian proteins which are associated with host defenses, while other poxvirus irms have little sequence homology to immune-effector molecules or their receptors. the ultrecht strain of rabbitpox (rpv) virus induces a large reddish pock on the chorioallantoic membrane (cam) of the chicken embryo. rpv mutants lacking the spi- /crmn k gene trigger an inflammatory response by - hr after inoculation, similarly to that found for cowpox virus (brighton red strain) mutants. rpv mutant viruses with a deletion of the ps/hr gene, a gene which has recently been associated with virus host range and virion maturation, elicited an inflammatory response on the chicken embryo cam at - hr after virus inoculation. thus, another poxvirus-encoded gene product has been identified that has irm functional activity which would not have been predicted by sequence analyses. poxviruses have recently been found to possess a herbert w. virgin iv, center for immunology and division of infectious diseases, washington university school of medicine, st. louis, mo murine cytomegalovirus (mcmv) follows three stages of infection in the immunowmpetent host. following the initial acute infection, a persistent infection remains in which lytic virus resides in the acinar cells of the salivary glands. finally a third stage of infection exists which is characterized by a lack of detectable infectious virus. it is not known whether mcmv remains persistent at some low level during this thiid stage or establishes molecular latency. to test this, spleens, kidneys and salivary glands from mice - months post infection were evaluated by two sensitive assays. sensitivity was tested using virus co-cultured on murine embryo fibroblasts (mefs) for days. by this method, pfu mcmv was reproducibly detected in the presence of sonicated spleen, kidney and salivary glands diluted :lo. scid mice were used as a second detection assay for mcmv. using scid mice and different doses of virus, the lds, of mcmv in scid mice was - pfu. mcmv infections were detected when - pfu were added to sonicated spleens, kidneys, and salivary glands prior to injection into scid mice. using both co-culture with mefs and injection of tissue into scid mice, we have tested a total of spleens, kidneys, and salivary glands and have detected no persistent virus. while no preformed virus existed in these tissues, reactivation from explanted spleen, kidney and peritoneal exudate cells occurs in culture after - days. in addition, lytic mcmv was detected in scid mice days after injection with ( )' kidney cells from latently infected mice. following identical transfers of latent spleen cells, mcmv was not detected in scid mice although explants from the same organs reactivated in vitro. using facs sorting, panning, magnetic bead separation, and reactivation assays, individual cell types have been analyzed for the presence of mcmv genome and the ability to reactivate virus. heat shock proteins (hsp) are expressed in all organisms in response to a variety of stresses, including virus infection. however, since viruses are not known to encode hsp genes, this represents expression of cellular hsps. we have found a dramatic induction of the kda hsp in the ovaries of w-infected mice, and using primary ovarian fibroblasts, demonstrated hsp mrna within virus-infected cells. the physiological role of hsps expressed during virus infection is unknown, although hsps act as molecular chaperones to facilitate protein folding and assembly in unstressed cells, and a number of bacterial hsps are essential for bacteriophage replication and morphogenesis. in order to determine whether hsp expression was beneficial to vv replication, we constructed a recombinant vv which encoded murine hsp , but found that this virus grew to similar titres as control viruses, both in vitro and in vivo. in particular, kinetic studies of virus growth in an hsp -negative cell line showed no difference from control viruses, and the presence of hsp did not influence the virulence of infection in immunocompromised nude mice. these results are interesting given that hsp proteins act as molecular chaperones and that hsp can be found in association with vv proteins. we suggest that this association may simply reflect the inherent affinity of hsp for non-native proteins, and the close physiological proximity of hsp and nascent viral proteins within the virus infected cell. our results indicate that the functional significance of these interactions are minimal with respect to the outcome of virus infection. furthermore, although the expression of hsw in vv-infected mice coincides with the peak anti-viral cellular immune response, hsp does not appear to be immunologically significant, as we cannot demonstrate any immunological responses to hsp during the course of vv infection. the effect of in vivo neutralisation of ifn-y on cytokine production during influenza infection was compared in cs /bl and balb/c mice. similar cytokine profiles were obtained on in v i m restimulation of mln or spleen cells from virus-infected mice of each strain. at days and postinfection, mln or spleen cells from both strains of mice produced il- , il- , il- and ifn-y, but little or no il- or il- . however, following in vivo treatment with anti-ifn-y antibody, production of l- and il-s was induced in mln and spleen cells of balb/c mice whereas those from cs /bl mice showed little change in cytokine profile. an increase in il- and l- production was also observed on in virro restimulation of splenocytes from balb/c mice. however, significant amounts of l- and ifn-y were still secreted in these cultures. in v i m neutralisation of ifn-y in the restimulated cultures had little effect on the production of other cytokines, regardless of whether this cytokine had also been neutralised in vivo .despite the change in cytokine profile, anti-ifn-y treatment had no effect on the kinetics of viral clearance in balb/c mice. a similar situation was seen for c /bl mice. congenic strains of mice are currently being used to determine whether the effect on cytokine profiles is associated with the mhc haplotype. wunner, and steven l. spitalnik, department of pathology and laboratory medicine, university of pennsylvania and the wistar institute, philadelphia, pa rabies virus glycoprotein (rgp) is the only glycoprotein on the viral surface, is the target of viral neutralizing antibodies, and is the viral protein that interacts with the host cell receptor. rgp of the era strain is a amino acid type i membrane glycoprotein with potential n-glycosylation sites at asn , asn , and asn . due to rgps central role in the immunology and biology of rabies, it is important to determine its three-dimensional structure. towards this end we produced a recombinant form of rgp that may be more amenable to analysis by x-ray crystallography. to avoid the use of detergents, we constructed a soluble amino acid form of rgp lacking a transmembrane domain (rgpt ). rgpt was secreted by transfected cells and was appropriately glycosylated, assembled, antigenic, and immunogenic. since n-glycosylation was required for rgpt secretion, we minimized the number of n-glycans by site-directed mutagenesis. interestingly, rgpt was secreted only when asn l was n-glycosylated. in contrast, full-length rgp was expressed at the cell surface a any of the three potential sites was n-glycosylated. soluble inhibitors of n-glycosylation were used to simplify the structures of the n-glycans on rgpt . although inhibiting alpha-glucosidases with castanospermine blocked secretion, inhibiting any further processing with deoxymannojirimycin had no effect on secretion. finally, to simplify the purification process, a tail of his residues was added to the cooh-terminus of rgpt . this modification did not affect secretion, antigenicity, of assembly of the recombinant glycoprotein, but did allow purification using ni+ -agarose chromatography. in summary, we constructed a soluble form of rgp with a minimal number of homogeneous n-glycans and an "epitope" tag. this form of rgp is structurally similar to the extracellular domain of the full-length protein and may be amenable to analysis by x-ray crystallography. endogenous retroviral genes (ev) are well characterized in chickens. our laboratory has established chicken lines with single and multiple ev loci and, compared to ev negative birds, these lines exhibit a delayed and suppressed titer of neutralizing antibody in responses to avian leukosis virus (alv) challenge. to address the effects of ev genes on the cell mediated immune response we have developed an in-vitro assay to monitor the in-vivo induction of mhc restricted cytotoxic t cells (ctl) induced by alv. using this assay, we find the ctl response in birds expressing the ev- locus is reduced to % compared to ev negative birds having nearly identical (eighth backcross) genetic backgrounds. other ev loci ( , , and ) also reduce the ctl response to alv in mhc matched chickens with different genetic backgrounds. ' national public health institute. helsinki. finland universities of tampere, qulu and 'helsinki, finland coxsackie b virus infections have been associated with clinical iddm in seveml previous studies but their initiating role in the slowly progressing beta-cell damage was for the fmt time directly implied in our recent prospective. nationwide dime study. siblings of the index iddm cases who progressed to clinical iddm experienced enterovim infections more frequently than nonprogressing siblings during prospective follow-up period. enterovim infections were initially demonstrated by detecting significant increases in serum class-specific cross-reactive enteroviral antibodies by using radioimmunoassay (ria) with the heavy-chain capture principle. causal association benveen these infections and the pathogenesis of iddm was suggested by temporal association of sercconvenions of viral antibodies with increases in islet cell antibody levels which are considered to be markers ok ficell damage. the possibility of the existmce of specific diabetogenic s & s of enternviruses was then considered. to identify the serotypzs responsible for the infections, successive sera from subjects exhibiting seroconvenion of autoantibodies coinciding with an increase in enteroviral antibodies were anlyzed by plaque n e u n a t i o n assay using a set of different enterovhes. the results indicate that several enternvirus serotypes may be associated with the progressive prediabetic p~ocess. not only different coxsackie b but also coxsackievirus a -infections were found to coincide with seroconvenions of islet-cell and insulin auwantibodies. it is known that an immunogenic region of gad . one of the major isletcell antigens, shows a high degree of homology with the c protein of cbv- . studies on potential immunological cross-reactions between enteroviruses and two significant autoantigens, hsp and gad , are in progress. this is carried out by using immunohistochemistry in infected cell lines and in frown sections of human pancreas with antibodies induced by synthetic peptides and natural and expressed viral proteins, the role of p alterations in human malignant pleural mesotheliomas (mpm) is unclear. immunostaining (ipox) of snap frozen mpm specimens revealed p expression in of ( %). p detection by ipox is usually associated with mutated p . sscp for exons - revealed p alterations in of specimens, and loh at exon was seen in only of evaluable specimens. thus, most of these specimens appeared to contain wild-type (wt) p . we recently reported that sv induces mesotheliomas in rodents, and that sv -like sequences are present and expressed in mpm. of note, sv large t antigen (tag) binds and inactivates wt p i n ! , abolishing its tumor suppressor function. we theorized that the immunohistochemical persistence of p may result from its physicdl binding with tag. tag was detected by ipox in ( %) of these mpm specimens. expression of tag was significantly associated with coexpression of wt p (p = . , fisher's exact text), and preliminary experiments suggest co-precipitation of p and tag. finally, we demonstrate that waf- is not detectable in mpm expressing wt p suggesting that p is inactive. we speculate that tag expression in mpm binds to and inactivates p contributing to the malignant phenotype. human papilloma virus (hpv) infection is involved in % of cervical carcinomas and possibly % of cervical intraepithelial neoplasia (cin). at present it is unclear whether patients infected with the transforming hpv types can mount efficient t cell responses. to address this issue we examined the ctl response of peripheral blood lymphocytes from patients attending a cervical neoplasia outpatient clinic. all were diagnosed hpv positive with various stages of c m and nine patients were selected for hla-a expression by facs analysis. pbmc were stimulated with caski, a cervical carcinoma cell line demonstrated to be hf' v type ' and hla-a '. culture media consisted of rpmi- % human serum supplemented in three cases with u/ml il- , in another three cases with u/ml il- and u/ml il- , and in the final three cases with u/ml il- and u/ml il- . the ctl were screened for reactivity to caski and in addition the cervical carcinoma cell line c a (hpv, hla-a ') transfected with either the hpv type e or e genes. one patient's cells maintained with uiml il- and ulml il- showed hpv e protein specific cytotoxicity in a hla-a restricted fashion. these ctl lysed the caski stimulator cell line but not the nk sensitive target k- . the ctl efficiently lysed a c a-e clone but in contrast a vector only transfectant was not lysed. the lysis of c a-e was blocked with a-cd and a-cd antibodies at % and % respectively. facs analysis determined that this ctl population was . % cd 'cds' and . % cd 'cd '. limited e recognition was also observed but has not been hlly characterized to our knowledge this report represents the first demonstration of hpv e responsive ctl isolated from the peripheral blood of hf' v infected patients. we have previously shown that proviral variants found in the peripheral blood mononuclear cells (pbmc) of hiv- infected individuals can interfere with recognition by autologous cytotoxic t-lymphocytes (ctl). abolition of recognition could be caused by interference with t h e processing of viral peptides, by a failure of the variant peptides to bind to mhc class-i molecules or by a failure of the mhc class-vpeptide complex to efficiently activate the tcr (t-cell receptor). these mechanisms could allow the virus to escape the immune surveillance by ctl. most studies so far have addressed the question of viral variation in t-cell epitopes by analysing proviral dna, extracted from the pbmc of hiv- infected individuals. however, the analysis of virion-associated rna offers several advantages. sequences found in viral rna are likely to be functional, a t least u p to the point of packaging into virus particles. furthermore, rna sequences represent virus which is currently actively produced. here, we present the sequence variation i n two hla-b restricted epitopes: p - gag and amino acids - of the pol gene. both proviral dna from pbmc and viral rna sequences from plasma are discussed. the ability of viral variants to hind to mhc class-i molecules was assessed and the recognition of variant peptides by ctl was tested. variants were also tested for their ability to antagonize recognition of the index sequences. we show that viral variants with the ability to interfere with recognition by ctl are not only found in proviral dna but also in virion associated rna. objective: clinical course of hiv- infection may be influenced by an effective host immune response against hiv- and/or by viral properties. to investigate the cellular immune response to hn- we analyzed ctl activity against different hiv- proteins in long-term asymptomatic hiv- infection as well as during rapid progression to aids. methods: longterm asymptomatic individuals with cd counts > celllpl after more than years of infection were selected from the amsterdam cohort study on aids versus subjects who progressed to aids < years. ctl activity was measured on "cr labelled hla matched or autologous b-lcl, infected with rvv expressing hiv- ag. both bulk and limiting dilution ctl assays were performed longitudinally with pbmc after ag-specific stimulation. sequences of ctl epitopes were determined in homologous virus isolates resulrs: different kinetics of anti-gag ctl responses were observed in rapid progressors. in any case ctl responses disappeared during progression to aids. in long-term asymptomatic subjects persistent ctl responses were observed together with low viral load. conclusions: sustained, broad anti-hiv cellular immunity may correlate with maintenance of the asymptomatic state in long-term survival by controlling viral replication. enteroviruses are a large group of positive stranded rna viruses known to be responsible for a number of distinct disease entities. recombination is thought to be capable of generating new enterovirus strains that cause significant morbidity. for example, enterovirus which was responsible for a pandemic of haemorrhagic conjunctivitis and poliomyelitis is thought to have originated by recombination between a coxsackie b like virus and another unidentified enterovirus. we are studying a group of echovirus isolates from an outbreak of disease in southem india. sequence analysis within the ' untranslated region reveals that these isolates fall into two groups that differ by - % (equivalent diversity to that seen between between published sequences of poliovirus and coxsackie a virus). these two groups of viruses also differ in their cell tropism. isolates defined as group by their 'utr sequence grow equally well on ht cells (a human colon carcinoma cell line) and vero cells. isolates of group , with one exception, grow only on ht cells. analysis of the structural proteins of these isolates revealed differences in migration that correlated with their cellular tropism. thus, significant genotypic and biological diversity exists amongst these virus isolates. one virus isolate had the ' untranslated region sequence of a group virus but the protein profile and cellular tropism of a group virus. the best explanation of these findings is that this anornolous isolate is a natural recombinant between the parented strains. both the ease with which viable recombinants are generated and the diversity present within this one enterovirus serotype increase the potential for the production of novel pathogenic enterovirus strains. dominant susceptibility to polyoma tumors in inbred wild mice, sharon r. nahill, yupo ma, john carroll and thomas l. benjamin, department of pathology, harvard medical school, boston, ma polyoma virus (f' y) is a mouse dna tumor virus which, under appropriate conditions, causes tumors in a wide variety of cell types. generation of tumors is a function of both the viral and host genomes. lukacher et al. have recently described a dominant gene, pyv', carried by the c -i mouse strain, which confers susceptibility to py-induced tumors mapping and immunological analyses indicate that py is the mouse mammary tumor virus superantigen (mtv sad gene, which deletes t cells required for py tumor immunosurveillance in h- ' mice. to determine the generality of endogenous superantigens as determinants of susceptibility and to reveal potentially novel mechanisms of susceptibility, we have looked for dominant susceptibility @ s ) gene(s) id newly established and genetically diverse inbred wild mouse strains, czech i and pedatteck (peru). both strains are susceptible to py as % of infected animals develop a full profile of tumors. crosses between cs br, whose resistance is contributed by the major histocompatibility (mhc) locus, and susceptible peru or czech , yield f progeny which are fully susceptible, indicating a dominant inheritance pattern of susceptibility the incidence of tumor-bearing backcross animals [((peru x cs br) x c br) and ((czech i x cs br) x c br)i suggests that ds is due to at least one, but not more than two genes. amplification of genomic dna from the czech i and peru mice by pcr using primers specific for mtv sag indicates that both strains are negative for proviral mtv sag. furthermore, the mechanism ofds in these mice may be independent of all mtv sag as pcr using primers specific for the highly conserved region of mtv sag is unable to amplify mtv dna from peru or czech i genomic dna. these results indicate that, like the c hibi, the pedatteck and czech i contain gene(s) which overide the resistance to py-induced tumors contributed by the mhc of the c br parent and which may cause tumors via a novel, mtv sag-independent mechanism. we have initiated efforts to map the ds in peru and czech i mice using pcr and primer pairs flanking simple sequence length polymorphisms. fis- is a low leukemogenic, but relatively strong immunosuppressive variant of friend murine leukemia virus (f-mulv). this variant was originally isolated from t-helper cells of flc-infected adult nmrl mice. compared to f-mulv, fis- suppresses primary antibody response more efficiently in infected mice. some of the fts- infected adult nmri mice developed a disease resembling the acquired immunodeficiency syndrome induced by hiv. restriction mapping and nucleotide sequence analysis of fis- show a high degee of homology between this variant and the prototype f-mulv clone . in this study we have attempted to localize the genomic determinant of fis- which is responsible for induction of a strong suppression of primary antibody response. six chimeric viruses of fis- and f-mulv were constructed. the primary antibody response of the mice infected with these chimeric viruses were investigated. the results of these experiments will be presented. anti-fmdv antibodies, as measured in an elisa capture assay, were cross reactive. b) cellular: proliferative (cd ) t cell responses of peripheral blood mononuclear cells (pbmc) were low or undetectable during primary responses to vaccine or virus, and frequently low during secondary responses. for good t cell proliferation in vitro, multiple immunisation is required. this may reflect preferential stimulation of the th cd t cell subset. interestingly, when cd responses were observed, cd tcell responses were also detectable. . recognition of individual viral proteins a) expression cloning: structural and non-structural protein pseudogenes were cloned from cdna by pcr. expressed in pgex- xuc. and purified by sds-page. b) humoral: structural and non-structural proteins were recognised by infected animals. a good anamneetic antinon-structural response was only observed when the boosting serotype differed from the serotype stimulating the primary response. c) cellular: both structural and non-structural proteins were recognised and some were cross reactive. interestingly, vp was strain specific, and the polymerase ( d) was the most immunogenic and cross reactive. d) a construct comprising d and the immunodominant vp epitopes was prepared and tested. in common with other herpesviruses, the envelope glycoproteins of equine herpesvirus (ehv- ; equine abortion virus) are major determinants of the infectious process and pathogenicity, and are inducers of humoral and cell-mediated immune responses. as such, they are candidates for components of subunit vaccines against ehv- . to generate useful amounts of individual ehv- glycoproteins, we have constructed recombinant badoviruses capable of expressing glycoproteins c, d, h (gc, gd, gh ) in insect cells, and have evaluated the recombinant products as innnunogens in a murine model of ehv- infection. au three glycoproteins induced serum (elisa) antihodies to ehv- , and ehv- gc and gd also induced neutralizing antibody responses. following intranad challenge with infectious ehv- , protective immunity, as demonstrated by acelerated clearance of virus fiom respiratory tissues to below detectable levels, was evident in mice immunized with either recombinant gc or gd. in contrast, gh-immmkd mice did not develop detectable neutralizing antibody, and did not clear challenge virus more rapidly than controls. delayed type hypersensitivity and lymphoproliferation responses to ehv- antigen were observed for each of the ehv- glycoproteins, and in experiments with gdimmunized mice, a role for cell mediated immunity in protection was confirmed by adoptive transfer and t-cell depletion experiments. the data provide support for the potential of glycoproteins c and d as a subunit vaccine against ehv- . molecular pathogenesis of ural infeetiom - enterovirus-immune cell interactions: implications in enterovirus-induced diseases we have also evaluated the effect of virus infection on the humoral immune response to cvb , infection in adolescent c h/hesnj mice. antigen presenting cell, -helper cell and -cell function were evaluated utllizlng a sheep red blood cell (srbc) plaque assay. mice were injected intrapentoneally (ip) with lo plaque forming units of cvb , at day and with lo srbc's at days , , and post-cvbb, infection. splenocytes were harvested days post-srbc injection, mixed with target srbc's and guinea pig complement and incubated. plaques were then quantitated. results: cvb , was associated with . % to . % of cd- positive t-cells and w a % to % of adherent splenocytes. after mitogen (lps and con a) stimulation, b-cells and adherent cells were demonstrated to be permissive for viral replication. a % and % under non-stimulated conditions. an average of % of virus is cell-associated (plaque north america. bruce anderson, teny yates, norah torrez-martinez, wanmin song, brian hjelle. university of new mexico, albuquerque, n.m.we recently identified a new species of hantavirus (hmv) associated with the harvest mouse reithrodontomys megalotis (hjelle b et al, j. viroj. , in press ). an arizona woodrat (neotoma mexicana) was found to he infected with hmv, presumably through "spillover". hmv is most closely related to the four comers hantavirus (fcv) of deer mice (genus peromyscus). the nucleocapsid gene and protein of hmv differ from those of fcv by % and % of residues, and the nt s genome is shorter by nt. we surveyed reithrodontomys animals captured in the u.s. and mexico for hantavirus antibodies; ( . %) were positive. s segment cdnas were amplified and sequenced from seropositive animals captured in california ( ), arizona ( ), new mexico (l), and mexico ( ). a monophyletic clade of hmv-like agents was identified at all sites, although an r. megalotis infected with an fcv-like virus was also identified in the state of zacatecas, mexico. nucleotide sequence distances among members of the hmv clade were up to . %. but amino acid distances were less than %. hmv is enzootic in harvest mice throughout much of north america, and can also infect wood rats. htlv i-associated myelopathy/tropical spastic paraparesis (hamnsp) is a slowly pro ressive neurological disease characterized by perivascuaar mononuclear infiltrates in the cns. htlv i-specific cd + ctl are found in pbl and csf of infected patients with htlv i-associated neurological disease but not in htlv i seropositive individuals without neurological involvement. previous studies have shown that in hla-a + patients, htlv i-specific cd + ctl restricted by hla-a recognize a peptide derived from the htlv i tax protein (tax - llfgypvw). in the present study, we have analyzed the potential of these tax-specific ctl to recognize addtional peptides. our results demonstrate that a subpopulation of high affinity cd ' tax - specific ctl clones cross-react on a self peptide derived from the se uence of myelin-associated glycoprotein (mag - vl&sdfri) presented by hla-a . these obsenatlons suggest that the demyelination process in hamltsp may be,due, in part, to virus-specific ctl recognition of a self myelin component that is independent of htlv i infection. development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of lymphocytic choriomeningitis (lcm) virus. the c hebfej and blo.br/sgsnj mouse strains have been of special interest because they display autoimmune hemolytic anaemia with varying degrees of apparent immunological involvement. in this study, we examined the role of cd + t helper cells in this autoimmune response by treating mice with the cw-specific gk . monoclonal antibody. we also determined if polyclonal activation of b lymphocytes, induced either by lcm virus or by lactate dehydrogenase-elevating virus, another well known b cell activator, correlated with the development of anaemia in these mice. our results strengthened the central role of the immune system in the anaemia in c h mice by showing that depletion of cd + cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. as reported by others, we found that the anaemia was more mild in b o.br mice than in c h mice. however, we could not confirm the difference in the degree of b lymphocyte polyclonal activation between these mice. furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma igg levels. one of the two class i mhc (h-pkd)-restricted immunogenic sites identified on the influenza strain aijapanl (h n ) hemagglutinin (ha) encompasses two distinct partially overlapping epitopes, mapping to residues - and . . when we investigated the magnitude of the ctl responses of balwc mice to the two overlapping epitopes, we found that while the nhrterminal nonamer epitope is immunodominant, eliciting vigorous ctl responses in njapanl -immunized balb/c mice, the ctl responses to the cooh-terminal decamer epitope are weak and variable. the c-terminal epitope subdominance seems to be due to factors other than inefficient processing of the epitope in vivo because ctls generated by priming mice with recombinant sindbis viruses expressing only one of the ha - subsites displayed patterns of responsiveness similar to that of influenza virus primed ctls. limiting dilution ctl assays showed that the ctl precursor frequency (pctl) of the nterminal epitope is at least ten fold higher than the pctl of the cterminal epitope, implying that the low and variable pattern of cterminal specific responsiveness was due to the limited t cell precursors in the c-terminal specific ctl repertoire. this was further confirmed by the limited heterogeneity in the cross reactivity patterns displayed by the c-terminal specific ctl for an ig vh fragment and the ha - epitope of influenza strain a i m in short term bulk cultures, and the facs analysis of tcr vg chain usage. taking these together with our previous observation that some jha - specific ctls can also crossrecognize an ig vh fragment. these studies had provided a strong evidence that ig gene products may influence t lymphocyte function and repertoire development. we have previously described the identification of homologous regions in the c-terminus of hiv- gp and in the n-terminus of hla class i beta chains. forty percent of patients infected with hiv-i virus were shown to have antibodies which bind to the homologous sequences, as well as to native hla class i molecules. affinity purified crossreactive antibodies (crab) were shown to have direct blocking effects on normal t cell responses to recall antigens, and could mediate adcc of hla class ii+ cell lines.in order to determine the contribution of such antibodies to disease progression, we obtained longitudinal plasma samples from patients in the macs study. in a first study, it was found that the presence of high titers crabs correlated with a more rapid disease progression (p = . by fisher two tail analysis)in a second, year-longitudinal study of progre.ssors and stable patients we found: ( ) the production of crab was seen in - % of rapid progresson, while the true stables produce only infrequent low-titers crab. ( ) in rapid pmgressors, production of crab preceded by - years the marked drop in cd counts. ( ) crab production did not correlate with the degree of hyperglobulinemia in these patients. ( ) the presence of crab during the asymptomatic stage correlated with early loss of t-helper responses to recall antigens.we are currently establishing whether periodic measurements of crab in patients sera could be valuable in predicting a drop in cd counts and disease progression. the lymphokine ifn-y is i pleiotropic insnunomodulator and possesses intrinsic antiviral activity. we studied its significance in the development of antiviral immune responses using ifn- receptor deficient (ifn-yr-'.) mice. after inoculation with live attenuated pseudorabies virus (prv) the mutant mice showed no infectivity titers in various tissues and transient viral ag expression only in the spleen similar as in wild-type mice. however, the absence of the ifn-yr resulted in increased proliferative splenocyte responses. the prv-immune animals showed a normal ifn- and - production, without detectable - , and with decreased - secretion in response to viral ag or con a. immunohistochemically, an increased ratio of ifny/i - producing spleen cells was found. after immunization with either live attenuated or inactivated prv, ifn-yr"' mice produced significantly less antiviral antibody (ab), and more succumbed to challenge infection than the intact control animals. the reduction in ah titers in the mutant mice correlated with lower protection by their sera in transfer experiments. thes? findings are in line with the strong enhancing effect of exogenous ifn-y on rabies virusand prv-specific igg responses. our data demonstrate that a physiological ifn-y system is surprisingly not critical for the generation of antiviral th-i-type and the suppression of th- -type cytokine responses. the lymphokine, however, is an important mediator in the generation of protective antiviral ab. key: cord- -ceahghs authors: epel, elissa s. title: the geroscience agenda: what does stress have to do with it? date: - - journal: ageing res rev doi: . /j.arr. . sha: doc_id: cord_uid: ceahghs geroscience offers a counterpoint to the challenged pursuit of curing diseases of aging by focusing on slowing the biological aging process for extended healthspan earlier in life. remarkable progress has led this field toward animal trials and the next challenge lies with translation to humans. there is an emerging number of small human trials that can take advantage of new models integrating behavioral and social factors. understanding dynamic aging mechanisms, given the powerful social determinants of aging (crimmins, ) and human variability and environmental contexts (moffitt, ), will be critical. behavioral and social factors are intrinsic to aging. toxic stressors broadly defined can lead to stress-acceleration of aging, either directly impacting aging processes or by shaping poor behavioral health, and underlie the socioeconomic disparities of aging. in contrast, hormetic stressors, acute intermittent stressors of moderate intensity, can produce stress resilience, the ability for quick recovery and possibly rejuvenation of cells and tissues. although health research usually examines static biomarkers, aging is reflected in ability to recover from challenges pointing to new interventions and targets for examining mechanisms. a fuller model incorporating stress resilience provides innovative biobehavioral interventions, both for bolstering response to challenges, such as covid- , and for improving healthspan. circumstances and substances. hormesis traditionally described a cells or organisms bi-phasic response to an external chemical or stressor. there is indeed overlap between stress processes and aging processes, and two become intertwined with the concept of hormesis. toxic stress includes traumatic or ongoing adversity for months on end, and the psychological responses--chronic high perceived stress, burnout, or depression. many large scale studies demonstrate that traumatic or chronic psychosocial adversity, including low socioeconomic status, predict higher allostatic load, whereas high levels of psychosocial resources are associated with lower allostatic load, with small but reliable effects (danese & mcewen, ; wiley et al., ) , described further under "reserve capacity" (section ). homeostasis to allostatic load. stress research started with examination of the acute stress responses to acute stressors in rodents. cannon's stress studies led to the popular concept of homeostasis (cannon, ) but a simple linear model of homeostasis does not explain the range of human stress responses, and there have been many elaborations of this concept. selye described the continuum from acute stress to chronic stress (selye, ) . acute stress can be hormetic when there is quick recovery back to homeostasis. given the complexity of physiological regulation, and that our body mounts a response in mere anticipation of threat, sterling and colleagues have described allostasis as a more encompassing description of the body's regulation-the constant fluctuations to meet expected demands (schulkin & sterling, ) which in biogerontology has been called "homeodynamics." chronicity of stressor exposure reveals a "fragility in homeostasis" (ramsay & woods, ) when physiological signs of 'exhaustion' appear, such as, in rodents, damage in organs. mcewen and colleagues have labeled this cost of adaptation--the dysregulation and damage across systems--as allostatic load (mcewen, ) . the concept of allostatic load, whether it is at a systemic or cellular level, gives us an intermediate phenotype of aging, an early step toward development of diagnosable disease. this is a critical concept to geroscience, and in fact many of the actual measures of allostatic j o u r n a l p r e -p r o o f load used in from the psychology and public health literature are actually indices of aging (entringer & epel, ) . geroscience leaders have started to identify the biomarkers important in geroscience trials, as those that can predict aging outcomes and mortality, and are responsive to interventions, and this short list includes glucose control and inflammation (justice et al., ) . thus, there is potentially great overlap between geroscience biomarkers and the stress-related allostatic load markers described in section (cellular, multi-system, and measures of recovery), and these fields can inform each other. . an integrative model of stress and aging. stress acceleration (toxic stress) and stress rejuvenescence (hormetic stress). given the important role of social stress in aging, we need a deeper understanding of types of stress exposures. an overarching model explains the range of stress exposures, from toxic stress to acute hormetic stress, and our body's diverging responses to these exposures. our stress responses are not typically thought of as basic mechanisms of aging but indeed they are actively shaping rate of aging. as shown in figure , the dose and intensity of the stressor determines in part whether the organism responds with positive physiological changes or impairments in aging processes (modified from franceschi et al., ) . the exact timing of stressor exposure is an important consideration of a hormetic or pre-conditioning effect, as some stressors lead to sensitization across stressors, rather than habituation (i.e., belda et al., ) and this may differ by species, stage of development, and stressor paradigm. therefore, a general model cannot determine the exact parameters of intensity and dose for hormetic stressors. it is important area of future research to identify the boundary conditions and inflection points for the range of potentially hormetic stressors . moderate stressor exposure can lead to both housecleaning in the cells, making them appear younger or rejuvenated, as well as growth of new neural pathways. over time, the accumulation of hormetic stress can promote slowing of aging processes. j o u r n a l p r e -p r o o f . hormesis is a form of stress resilience. this paper brings together the hormesis literature with the broader stress resilience literature. the cellular biology of hormetic responses is well mapped, and has general common responses as well as stressor specific responses. the acute stress response has a common pathway of creating calcium influx, oxidative stress, and energetic stress. this increases transcription factors such as nrf- , foxos, creb, and nf-kb, leading to many hormetic effectors, such as chaperone proteins (eg, heat shock proteins which help fold proteins efficiently and prevent protein aggregation), er stress, endogenous antioxidants (sod, glutathione), growth factors, and mitochondrial proteins (mattson, a) . after moderate doses, the cells become resistant to many other types of stressors (heat, uv, oxidative stress, metals), and to death (murakami et al., ) . hormesis is a universally observed phenomena across types of cells and types of stressors, including psychological stress. in model organisms, short manageable stressors lead to improvements in aging, although this depends on types of stressor and species (lagisz et al., ; rattan, ) . for example, low dose gamma radiation over time can extend average lifespan up to % in mouse studies (calabrese & baldwin, ) . in humans, there is evidence of hormetic stress, such as the effects of exercise, although this is not typically labeled as hormesis. hormesis naturally applies to humans-not just to cells but to physiological and psychological regulation. a typical example is vaccinationwhich leads to enhanced immune responses later. here we expand the definition of hormetic stress to include the positive stressors that humans engage in-such as short term bodily stressors like exercise and temperature stress, but also novel challenging experiences that expand coping resources, knowledge, generativity, and feelings of accomplishment, described further below (section . ). since hormetic stress has traditionally been applied to cellular physiology, we use the larger concept of 'stress resilience' as the widest umbrella term for describing when humans recover quickly, in any system, from various exposures. as shown in the appendix, there are many j o u r n a l p r e -p r o o f overlapping terms that relate to the concept of stress resilience. just as the term "stress" is a multi-level construct that needs to examination in a sophisticated interdisciplinary manner, stress resilience is also a multi-level concept that encompasses the full range of human exposures, responses, and inter-related systems. the term stress resilience thus subsumes psychological resilience, physiological resilience/enhanced allostasis, and social resilience. this model of stress resilience can thus be applied to most processes--at the cellular level, physiological level, and psychosocial level. psychosocial stress resilience here refers to the dynamic recovery in psychological, behavioral and social processes and related physiological processes. high stress resilience is reflected by quick physiological and affective recovery from psychological stressors. the neurochemistry of psychological resilience has also been described, based on rodent models (cathomas et al., ) . whether a stressor leads to a hormetic or toxic response is not solely determined by the chronicity of the stressor. it is also determined in part by the psychological appraisals, which is shaped by the context, culture, personal history and personality of the individual. when one feels demands exceed resources, in any situation, this creates a physiological and emotional stress response (folkman et al., ) . if it is perceived as a threat they cannot cope with, this repeated stress response over time will last longer and be more wearing. in contrast, if they view it as positive challenge that they have the resources for, they will have a profile of quicker recovery, as summarized elsewhere . thus the appraisal of the stressor codetermines the physiological response. it is not just stress responses to major events that matter. our frequent daily stress responses have cumulative effects: the tendency to have slower recovery of negative mood or greater loss of positive mood after a daily stressor predicts inflammation and long term disease and mortality mroczek et al., ; piazza et al., ; sin et al., ) . short term manageable stressors, such as physical or cognitive challenge that can promote growth, learning and development can lead to protective responses. an example of this is found in studies of the experience corps. exposing elderly retired people who are often isolated to a job mentoring at-risk youth in schools is often viewed as stressful but leads to feeling more purpose in life. in men, it has been linked to better health and increases in hippocampal volume gruenewald et al., ; varma et al., ) . in the case of coping with chronic stressors, most people (around %) recover to baseline levels of well being after a loss or disaster (galatzer-levy et al., ) . resilience may develop over time, leading to more mastery, purpose, faith, self esteem, and thus more resilient responses to future stressors. in contrast, toxic exposures accumulate over a lifetime, promoting "stress-acceleration of aging" processes. chronic stressors for decades, multiple shorter term exposures over years, and stressors embedded early in life, can all have toxic effects when there is insufficient resources to cope, and nor opportunities to fully recover. developmental factors are critical for understanding when stressors can be hormetic vs. toxic. we do not know precise developmental trajectories for differential effects of toxic stress on mental and physical health and even less is known about hormesis across the human lifespan. with aging, there is a decrease in both the reproductive and anabolic hormones that are part of a salutary acute stress response (epel et al., ) , and also a reduction in aspects of molecular hormesis, such as a lower heat shock response to stressors (calabrese et al., ; epel, ) . we know most about the developmental impact of toxic stressors. while there are myriad individual patterns of exposures, traumatic stress or material deprivation have larger effects early in life than when they occur at later periods; early life adversity is predictive of a range of poor outcomes, including poor mental health, health behaviors, biomarkers of aging, and earlier disease onset (deighton et al., ; hughes et al., ) although plasticity is still possible (mcewen & morrison, ) . given the sensitive period j o u r n a l p r e -p r o o f of pregnancy, it is not surprising there is evidence of transgenerational effects of stress and pregnancy complications on systems regulating aging like telomeres and epigenetics (epel, ; girchenko et al., ; ross et al., ) . a careful meta-analysis of the effects of early adversity points to psychological threat, rather than material deprivation, as the factor underlying accelerated biological aging such as early puberty, telomere shortening, and brain development (colich et al., ) . for trauma and abuse, the earlier in life, prenatal and pre-pubertal, when the brain is most rapidly developing, the larger the imprint of lifelong effects on mental and physical health (agorastos et al., ) . there are many examples of early adversity with accelerated biomarkers of aging in children: in prepubertal children, early life adversity leads to greater inflammatory acute stress response, and basal inflammation several years later (slopen et al., ) . in prepubescent youth, exposure to violence is associated prospectively with telomere shortening (shalev et al., ) , and telomere shortness in early childhood predicts carotid artery thickness several years later, and during puberty (barraclough et al., ; skilton et al., ) . early adversity may accelerate aging in part through inducing early puberty which in turn is linked to earlier onset of metabolic disease (gur et al., ; sun et al., ) . early adversity may also initiate a trajectory of early aging through lower reserve capacity such as low optimism and higher stressful events in adulthood (surachman et al., ) . early adversity also predicts poor health behaviors such as sedentariness, smoking and substance use in youth (wiehn et al., ) and these habits appear to persist long into adulthood . the pillars of mammalian aging, represent fundamental and related pathways such as genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, metabolic pathways such as deregulated nutrient sensing, mitochondrial j o u r n a l p r e -p r o o f dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication, macromolecular damage, chronic low-grade inflammation, and adaptation to stress (kennedy et al., ; lópez-otín et al., ) . other hallmarks of cellular aging are being identified in the brain (mattson & arumugam, ) . molecular pathways are often not closely related to each other, pointing to the use of algorithms, for better prediction of outcomes, described below. several of these basic mechanisms in immune cells have been associated with aspects of social stress, including systemic inflammation and shorter telomeres (epel et al., ; kiecolt-glaser et al., ; miller et al., ) poor mitochondrial function (picard et al., ) , and accelerated epigenetic aging (park et al., ; wolf et al., ) . these associations with lifespan stress demonstrate there is no closed system of intrinsic aging, and even at these most molecular levels our aging rate is influenced by our life exposures. we cannot rule out the possibility that some of these observations are from transgenerational effects. a new practical approach already used in humans is to measure a panel of biomarkers of aging that reflect cumulative damage across regulatory systems (e.g., metabolic, immune, stress related), and reducing this a composite measure. the first of these measures was allostatic load (seeman et al., ) , and there are newer algorithm measures like 'pace of aging' (belsky, caspi, et al., ) , and lack of normal covariation among regulatory systems (belsky, huffman, et al., ) . these measures serve as a barometer of biological aging across the lifespan, linked to early experience, and may be useful to examine the effect of interventions (moffitt, this issue). so far, the markers used have been chosen out of convenience of availability, but there is exciting potential to develop further translational measures based more directly on the basic mechanisms of aging. this admittedly requires high intensity collaboration between basic and clinical scientists (eg, assessments of mtor activity, senescent cells, mitochondrial functioning). . speed of recovery as a novel measure of latent aging at any age. geroscience recognizes that physiological adaptation to stress stands out as a common phenotype of aging j o u r n a l p r e -p r o o f across model systems of aging. stress resilience, and its impairment, is partly an outcome of the social hallmarks of aging, and a common underlying process that in part regulates the cellular hallmarks of aging. snapshot one-time measures of aging based on blood have inherent limitations in that they do not directly test how a person responds to an acute stressor. recovery from challenge is a critical measure of stress resilience that may be important, as it assesses the latent homeostatic capacity of a system. speed of recovery is thought of as intrinsic homeostatic capacity, a latent capacity that reflects biological aging. recovery is such an important marker of aging that it is central to the emerging areas of 'physiological geroscience" and "translational geroscience." naturalistically, acute events often precede a rapid decline in function, reflecting lack of stress resilience. for example, % of new disabilities develop after an acute accident or illness and hospitalization (gill et al., ) . there are many examples of paradigms measuring recovery that have validated the importance of using a challenge, and measuring functional or biological recovery from the challenge. frailty is a measure of advanced biological age that reflects loss of stress resilience due to age related decline in physiological reserve (hoogendijk et al., ) . however, frailty is a final common pathway, one that is probably not reversible. stress resilience interventions will need to target people earlier in life long before frailty sets in. in contrast physiological resilience, which refers to ability to bounce back from a stressor, is measurable at any age (whitson et al., (whitson et al., , . stress resilience depends in part on the pre-existing level of reserve capacity, the positive protective factors of an organism, as well as the immediate adaptive psychological response to stressors (cognitive appraisals). in the stress literature, reserve capacity has been defined as j o u r n a l p r e -p r o o f combination of personal resources such as optimism and sense of control, and social factors such as social support. high psychosocial reserve capacity appears to buffer those from low ses backgrounds from developing cardiovascular disease (matthews et al., ) . in geroscience, reserve capacity refers to a broader set of resources of or buffers, social, psychological or physiological including cognitive function (e.g., high iq), physiological (e.g., aerobic fitness, sleep), and psychological assets (e.g., high optimism or positive affect). high reserve capacity increases the likelihood that one will have a hormetic protective response to a stressor. as shown in figure , after diverse types of stressors (eg, chemical, physiological or psychological), an organism reacts and recovers to baseline with different speeds and this is moderated by baseline reserve capacity. for example, in response to a hip replacement surgery, the biggest predictor of good recovery was reporting good physical function at baseline (colón-emeric et al., ) . in response to general anesthesia, predictors of protection from dementia and other cognitive outcomes was measures of cognitive reserve such as education and vocabulary ability (cizginer et al., ) . indices of reserve capacity in functional abilities (such as ability to stand, gait speed, level of fitness), and glucose-insulin response to a glucose load, predict time to mortality, as reviewed elsewhere (seals & melov, ) . one of the most well-developed areas of reserve capacity comes from examination of individual dispositions of temperament, typically called personality traits. there are many psychological assets in adulthood that are associated with both better recovery from stressors, and with health and mortality. these include optimism, positive affect, mindfulness, coping with stress with cognitive reappraisal or active coping, high presence of social support or seeking support, purpose in life, and quality relationships. many of these assets have been associated with indices of good health, such as self-reported health and higher heart rate variability (carnevali et al., ) . these positive assets are both shaped by genetics and life experience. will promote further positive responses to future stressors, and may help slow the rate of aging in humans, as shown in figure . how can we best translate hormetic interventions to humans? there are many potential interventions that may improve stress resilience, listed in table . lifestyle interventions, such as exercise, caloric restriction, intermittent fasting, challenging cognitive activities, and response to phytochemicals in vegetables and fruits, are thought to work in part through hormesis (mattson, b; radak et al., ) . at least one group is pilot testing a cocktail of stressors in humans to examine rejuvenation effects, using intermittent cold, heat, fasting and hypoxia, together with phytochemicals (pruimboom et al., ) . the hypoxic preconditioning effect demonstrates protection of neurons and cardiac cells, and is a potential area of translation (li et al., ). an interesting novel intervention inducing acute stress (through exposure to intermittent hypoxia and cold) appears to improve immune response to endotoxin at least in a small initial study, with a replication underway (kox et al., ) . at ucsf we are testing a similar protocol to see if it improves autonomic and neuroendocrine response profiles (including a quicker recovery from acute stress). another dramatic way to increase stress resilience is to enter periods of fasting, or fasting mimicking with low calories. in rodents, this leads to stress resistance and regeneration and rejuvenation processes through hormesis, in part by down regulating gh, igf- . mtor, and pka signaling (longo, ; rangan et al., ) . reserve capacity is built during formative developmental experiences, such as level of education, attachment relationships, and stress exposures that shape the neural architecture of stress responding, narratives of optimism, and foster positive challenge mindsets. one can build reserve capacity by increasing physiological buffers (fitness, or antioxidant diet), or psychological stress resilience, through psychological trainings that might decreasing chronic stress arousal and bolster one's mental filter so they j o u r n a l p r e -p r o o f habitually perceive less threat. interventions that build psychological positive assets like optimism, mastery, and purpose in life need to be further developed and refined. mind-body interventions have a strong empirical base for improving self-reported wellbeing (creswell, ) with mixed effects on basal inflammation (bower & irwin, ) . mindfulness training may lead to changes in heart rate variability and telomere biology, although the evidence again varies by population and study (rådmark et al., ) (conklin et al., ) and appears stronger with clinical samples-those with high stress or early disease. there is emerging evidence that mind-body interventions improve physiological acute stress reactivity, changing stress appraisals and physiology to more of a positive challenge profile with a strong peak and faster recovery (daubenmier et al., ; lindsay et al., ) health behaviors regulate healthspan. the social hallmarks of aging shape health behaviors from an early age, which track throughout life. adverse health behaviors, such as diet, physical activity, sleep, and smoking are shaped by social stress. chronic stress both biologically drives toxic nutrition choices (sugar, fast food), impairs sleep, and promotes addictions, an indirect pathway in stress-acceleration of aging. the converse is also true, health behaviors lead to stress-slowing of aging. seventh day adventist who practice lifelong positive health behaviors, and lack the adverse behaviors of substance use, tend to have optimal longevity, living at least four years longer than the average us life expectancy and thus being the only blue zone in the us (fraser & shavlik, ) . exercise is the prototypical hormetic intervention. it increases the odds of healthy aging by % (daskalopoulou et al., ) . the mechanisms at the cellular level are becoming well explicated, as it can enhance mitochondrial health, telomere biology glucose, v max, oxidative stress, no) and upregulation stress resistance pathways, such as autophagy, and creating the opportunity for a long healthspan for all (health equity) requires improving economic and social factors. social factors are intrinsic to aging, our rate of aging depends on our social context and conditions. material deprivation and poor neighborhood quality confer psychological stress and risk of poor mental and physiological health (brisson et al., ) . for example, food insecurity is associated with over two fold risk of clinical anxiety or depression in adults, and confers even higher risk in college students (arenas et al., ; leung et al., ) . we now have a better understanding of how social threats lead to toxic stress. the primary motivational forces shaping human behavior are seeking safety and connection with others, and avoiding danger and anxiety. our mind is constantly seeking cues for safety or danger, even when we are not aware of this, and these social signals are transduced to biological signals, including patterns of autonomic activity and gene expression that are linked to inflammation. it is thought that exposure to or perception of frequent social threats (such as social rejection, discrimination, violence, and lack of safety) creates higher chronic systemic inflammation and sympathetic arousal, even while sleeping, and greater risk of affective disorders (brosschot et al., ; o'donovan et al., ; slavich, ; slavich et al., ) . conversely, social support, and social capital including perceived safety in neighborhoods, may be stress buffering, and are often associated with less inflammation and longer telomeres (brown et al., ; rentscher et al., ; thames et al., ) (geronimus et al., ; m. park et al., ) . social support and social networks can bolster healthspan interventions: our stress, emotional and physiological, is contagious to close others (carnevali et al., ; engert et al., ) , and conversely positive emotion and positive health behaviors are also socially influenced (christakis & fowler, ; kim et al., ) . the geroscience interventions that may work in mice will not be useful if they cannot be translated well to humans, taking into account our need for support and the significant challenges we have with adherence to exercise and other lifestyle changes. poor behaviors can override effects of protective pharmaceuticals. a common example of this is that people still develop diabetes while taking metformin due to overeating a western junk food diet. improving health behaviors can best be prioritized and implemented in the context where basic social needs are met. creating a supportive built environment and positive social environment are critical to promoting long-term behavior change. the science of behavior change, including the nih initiative focusing on this (nielsen et al., ) , has dramatically raised the sophistication of the research in this area, using the experimental medicine model to identify and manipulate the behavioral and social factors that facilitate adherence to health behaviors. behavioral or health interventions that work beyond the individual level, that can decrease loneliness and improve support will be more successful. the covid- pandemic demonstrates well the role of social factors in resilience to mental health disorders and infection. the pandemic led to dramatic increases in mental health disorders in the us and other countries (xiong et al., ) but this was not equally distributed. those with low education, income, minority status, loneliness, or low social support have significantly higher rates of mental health disorders from pandemic stress (arafa et al., ; holingue et al., ; palgi et al., ) . these vulnerable groups also tend to have higher rates of covid severity (adhikari et al., ; webb hooper et al., ) . any policies that improve social equity are also 'stress reducing' health policies that may contribute to healthspan, and can be incorporated into the geroscience agenda. geroscience is now more important than ever, both to our aging global demography but also to the health challenges we face going forward. in our new era we have dramatically increasing temperature extremes, wildfires and small particle pollution, and new zoonotic viruses to contend with intermittently. thus reducing social disparities, improving stress resilience and bolstering immune function have become critical public health goals. the vulnerability to covid-complications, while still largely unknown beyond older age and pre-existing diseases, clearly depends on ability of the immune system to respond robustly. the relevance of immune senescence in covid-lethality has stimulated many hypotheses about geroscience-related prevention and treatment (barzilai et al., ; salimi & hamlyn, ; sargiacomo et al., ) . while vaccination is essential for traditional prevention, it is not a universal solution: the elderly have poorer antibody responses to vaccination, there are many strains of the current virus, and there are expected to be many proliferations of future viral strains novel to the human body, due to climate change. therefore, geroscience interventions have unique universal importance across time. pharmacological interventions have been suggested for covid such as rapalogs, senoytics, nicotinamide adenine dinucleotide nad+, and metformin for anti-inflammation, telomere stability, or to boost vaccination response (omran & almaliki, ) . those with diabetes appear to benefit from metformin, which has hormetic properties, to prevent covid-related mortality (luo et al., ) . beyond pharmacological treatments, it is likely some of the interventions for boosting stress resilience in table may enhance resistance to viral infections, from common cold to novel viruses. the malleable lifestyle behaviors like fitness, nutrition, sleep quality, and stress reduction, are important ways to reduce insulin resistance and comorbidities, and thus may help prevent immune senescence and covid complications. one pathway through which stress resilience interventions could impact immunity is through stabilizing telomere length. short telomeres predict greater vulnerability to rhinovirus infection, acute respiratory syndrome disorders, and mortality from sepsis (cohen et al., ; liu et al., ) . chronic psychological stress shortens telomeres in animal studies and impairs viral immunity (cohen et al., (cohen et al., , . short telomeres indicate lower ability to mount a robust replicative t cell response, and this may be a critical or even fatal j o u r n a l p r e -p r o o f limitation in the face of covid related lymphopenia (aviv, ) . in short, covid- presents a potent example of the potential for using indices of aging as predictors of disease and targets of intervention. the goal of geroscience is to slow aging to improve healthspan. in the next generation of research, we will benefit greatly from incorporating the large malleable factors that impact human aging-biobehavioral and social factors. the nia's intervention and testing program, a multi-institutional infrastructure to study biological agents for healthspan in animals is a model that can be extended to human trials that takes into account the social and behavioral factors (moffitt, this issue). the social hallmarks of aging shape rate of aging, in part through toxic stress processes. the understanding of toxic stress and hormetic stress as factors shaping aging will have implications for interventions. stress resilience, the ability to recover quickly and turn on rejuvenative processes, is an important dynamic endophenotype of healthy aging. it remains to be seen how much resilience is merely a characteristic of healthy aging or a causal factor, although much evidence reviewed here suggests it is at least partly causal. a better understanding of how to measure stress resilience, and to promote stress resilience at the cellular, physiological and psychosocial levels will lead to important gains in slowing aging. the science of stress is an integral part of geroscience, and offers insights on how to harness stress for optimal longevity, and implications for how to conduct the most effective interventions incorporating these stress processes as both target mechanisms and outcomes. by having an integrative paradigm that can be examined across levels, we can reduce the gap between physiological stress research in model organisms and human research on stress, resilience and adaptation. there are many ways to measure biological aging in humans that can serve as a barometer of change for interventions. this includes cellular level markers, multi-system composites, and ways of examining dynamic stress resilience, as reviewed. this j o u r n a l p r e -p r o o f can include recovery from a medical event in the elderly, but also recovery to standardized challenges, and to naturalistic stressors. geroscience offers an exciting opportunity for high feasibility impact interventions. this integrative paradigm can shape the next generation of researchers. the training models need to change to bridge the many fields as outlined by pioneers in geroscience (newman et al., ) . models which are focused on pharmacological interventions must expand to be inclusive of both social and behavioral interventions, the current 'big levers.' lastly, this field, like all of science, needs to actively encourage and support young investigators from diverse and underprivileged backgrounds to enter this important and growing field which has the potential to minimize socioeconomic and ethnic/racial health disparities not just for equity but for the improved science that results with diverse life experiences and perspectives. frameworks for proof of concept trials related to loss of physiological resilience have been initially outlined (justice et al., ) , and there are many geroscience trials in the field. these human trials can draw on the rich insights from decades of biobehavioral basic and intervention research. the science of behavior change initiative at nih is supporting the development of more effective behavioral and social interventions using the experimental medicine model using the same attention and rigor as pharmaceutical studies. by working across disciplines, with an understanding the role of lifespan experiences, and complexity of human environments, the geroscience framework has tremendous potential for breakthrough innovations in increasing healthspan. there has been a proliferation of terms from related disciplines that overlap and are differentiated here. the discipline most often using the term is noted, but these terms could be used to describe all levels of analysis, including cellular, physiological/organ systems, or psychological, behavioral and social processes. resilience has also been applied ast the systems level, to organizations, communities, societies, and ecosystems. how systems change in response to stressors, typically referring to level of hermetic or a protective adaptive responses in cell such as heat shock protein increases. (mattson) . mild stressors induce adaptive capacities that protect an organism for a short while from future stressors and may improve the physiological state of that organisms. preconditioning is a case of hormesis where exposure to a chemical agent leads to a - % stronger adaptive response to subsequent exposures, across cell types and stressors (calabrese, ) . in psychology, the tem 'stress inoculation' is used in a similar way. in terms of psychological stressors, under-exposure to the typical daily and major life events can lead to lack of development of stress buffering resources, and poor ability to quickly recover from stressors. biologically the lack of acute stressors prevents the intermittent episodes of cellular 'housecleaning' activities that slow aging. ideal exposure to sufficient numbers of manageable challenges throughout life stimulate cognitive growth, coping skills, and emotion regulation skills, as well as the need for supportive social networks. biologically, ideal exposure to acute stress can have hormetic effects, leading to rejuvenescence-functioning that is enhanced (or "younger") compared to baseline. overexposure to stress without sufficient resources (toxic stress) can lead to maladaptive neural pathways of overresponding to stress, depression, and stress related acceleration of aging from cells to regulatory systems. this figure is adapted from francheschi et al, . in response to acute stressors, individuals have a kinetic trajectory of responses across psychological and physiological regulatory systems that lead to reactivity and recovery profiles. resilient stress responses (typically rapid high peak and rapid recovery) often have hormetic effects at the cellular or systemic levels. high levels of reserve capacity predict more rapid recovery, and this may lead to a positive feedback loop promoting higher reserve capacity. level of stress resilience is multiply determined by the social context and individual reserve capacity. 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the host contribute to microbial pathogenesis. to address this impediment to studies of host–microorganism interactions, we propose a new theoretical approach to understanding microbial pathogenesis, known as the 'damage-response' framework. the 'damage-response' framework of microbial pathogenesis is based on three tenets , (box ). first, that microbial pathogenesis is the outcome of an interaction between a host and a microorganism, and is attributable to neither the microorganism nor the host alone. second, that the pathological outcome of the hostmicroorganism interaction is determined by the amount of damage to the host. third, that damage to the host can result from microbial factors and/or the host response. these tenets form a scaffold -or framework -on which a formal theory can be built and tested. (glossary terms within this article are defined as applied in the damage-response framework - .) it is likely that relationships have existed between hosts and microorganisms for as long as there have been interactions between host and microbial cells. indeed, ancient host-microorganism interactions, in which bacteria were incorporated into a primordial host as organelles, are likely to have resulted in the evolution of eukaryotes , . the outcome of many hostmicroorganism interactions can be either beneficial or detrimental to the microorganism, to the host, or to both the microorganism and the host (table ) . mutualism and commensalism are examples of interactions that are beneficial to both the host and the microorganism. in the case of microorganisms that replicate within their hosts, detrimental outcomes result in the inability to replicate further and/or death. although microbial replication can cause host damage, and possibly disease, host damage and/or disease are not essential for microbial survival. furthermore, microbial viability is not a requirement for microbial pathogenesis. for example, cysticercosis -a devastating neurological disease that is caused by the host inflammatory response to the cestode taenia solium -can be precipitated by the death of the parasite, such that anti-helminthic therapy is considered to be detrimental in certain clinical situations . host-microorganism interactions that result in the clearance and/or control of a microorganism without the development of clinically relevant host damage represent a basis for the development of vaccines and immune-response-based therapies for infectious diseases. however, host-induced cell and/or tissue damage can also produce detrimental outcomes, which can result in disease or death -although certain manifestations of host damage represent the outcome of a successful immune response to microbial infection. some interactions are highly complex and illustrate the difficulty that can be associated with discriminating between a host and a microorganism. for example, the ingestion of certain microorganisms by amoebae (the host) can be beneficial to the amoebae,as the microorganisms are a source of food, and detrimental the late twentieth century witnessed the emergence of numerous infectious diseases that are caused by microorganisms that rarely cause disease in normal, healthy immunocompetent hosts. the emergence of these diseases shows that the existing concepts of pathogenicity and virulence do not take into account the fact that both the microorganism and the host contribute to microbial pathogenesis. to address this impediment to studies of host-microorganism interactions, we propose a new theoretical approach to understanding microbial pathogenesis, known as the 'damage-response' framework. host an entity in which microorganisms reside and/or replicate; an entity in which microbial pathogenesis occurs. damage disruptions in the normal homeostatic mechanisms of a host that alter the functioning of cells, tissues or organs; for microorganisms, disruptions in the normal mechanisms that enable host entry, replication and/or the ability to establish residence in a host. infectious diseases that are caused by microorganisms that have been referred to as 'weak' , 'non'-pathogens or those that are 'less virulent'. similarly, the concept of microbial 'opportunism' was devised to explain the emergence of previously rare infectious diseases in individuals with impaired immunity , . however, the classification of such microorganisms as 'opportunistic' was often inadequate and misleading, as some so-called 'opportunistic' microorganisms can also cause disease in normal hosts . host failure, which is a 'host-centred' view of microbial pathogenesis, has also been used to explain the occurrence of diseases that are caused by microorganisms that do not fit koch's postulates because they lack virulence determinants. these microorganisms do not cause disease in all hosts and so cannot be explained by 'microorganism-centred' views. examples of such diseases are catheter-associated candidiasis, staphylococcus epidermidis endocarditis and pneumocystis carinii pneumonia. the study of microbial pathogenesis has its intellectual and scientific origins in microbiology and immunology -two fields that are largely 'microorganism-centred' and 'host-centred' , respectively. indeed, an inherent bias towards either the host or the microorganism is often central to the thinking and investigative approaches of immunologists and microbiologists, respectively.'microorganism-centred' investigators tend to test the influence of microbial manipulations (mutants) on virulence by holding the host variables constant -as exemplified by the frequent use of immunologically naive and genetically inbred hosts. the capacity of a defined characteristic, or mutation, to confer virulence in a host is required to fulfil molecular koch's postulates . by contrast, the approach of 'hostcentred' investigators, such as immunologists, generally involves manipulation of the host as the microbial variables are held constant. this is exemplified by studies that attempt to establish that a host component is essential for host defence by comparing the outcome of infection with the relevant microorganism in both normal (wild-type) and deficient (knockout) mice. notably, the field of vaccinology has elements of both views, as evidenced by 'microorganism-centred' approaches with 'host-centred' thinking -a distinction that can place it at odds with its parental discipline of immunology . although 'microorganism-centred' and 'hostcentred' views of microbial pathogenesis have each provided valuable insights into microbial pathogenesis, neither simulates the reality in which each hostmicroorganism interaction is unique and occurs against a background of constant changes in both immune function and microbial fitness. it might not always be possible to account for both 'microorganism-centred' and 'host-centred' views in experimental design. however, consideration of the possible contributions of both the host and the microorganism to host damage could focus studies of microbial pathogenesis around a common principle, and has the potential to unify the field of microbial pathogenesis and the allied disciplines of immunology and vaccinology. to the ingested microorganism. however, the ingestion of certain microorganisms, such as legionella pneumophila or cryptococcus neoformans, by the amoeba acanthamoebae castellanii can result in death of the host -a process with striking similarities to the interaction of these microorganisms with macrophages in mammalian hosts , . the same amoebae, however, can be microorganisms in human hosts and cause amoebic keratitis. therefore, the distinction between host and microorganism is not invariant, yet it is relevant to understanding the outcome of certain host-microorganism interactions. despite being the outcome of an interaction, microbial pathogenesis is often viewed as a 'microorganismcentred' process. there are also proponents of the view that pathogenesis is 'host-centred'. although 'microorganism-centred' advocates recognize the importance of the host, they generally attribute the capacity for pathogenicity and virulence to the activity and functions of microbial gene products and/or microbial replication. inherent in this view, is the belief that microorganisms possess certain attributes that make them pathogenic, and that microorganisms that do not possess these attributes are not pathogenic (non-pathogens). 'microorganism-centred' views have their intellectual origin in the discovery of virulins at the turn of the twentieth century , are strongly supported by the discovery of virulence genes and pathogenicity islands, and are common in the field of bacterial pathogenesis , . the existence of microbial factors that confer virulence, such as capsular polysaccharide or toxins, gives credence to the concept that individual gene products can determine pathogenicity. such microbial determinants validate molecular koch's postulates . however, 'microorganism-centred' views fail to account for the pathogenicity of common microorganisms, such as candida albicans and staphylococcus aureus, as the strains of these microorganisms that are isolated from symptomatic or asymptomatic individuals are often indistinguishable. 'host-centred' views of microbial pathogenesis subscribe to the concept that a failure of host defence mechanisms results in susceptibility to certain microorganisms and their ability to establish themselves in protected niches.'host-centred' views are based on the fact that not all individuals in a population become ill after infection with a pathogenic microorganism. these views find support in the observation that immunocompromised hosts have a markedly increased incidence of box | basic tenets of the damage-response framework • microbial pathogenesis is an outcome of an interaction between a host and a microorganism. • the host-relevant outcome of the host-microorganism interaction is determined by the amount of damage to the host. • host damage can result from microbial factors and/or the host response. as illustrated by the discovery that p. carinii is a fungus after many years of being considered a protozoan parasite . classifications based on the perceived capacity of a microorganism to cause disease are also inadequate, because changes in host immune function, ecology and/or behaviour can render them obsolete. for example, candida spp. were considered essentially non-pathogenic until the mid- s, when the introduction of antimicrobial chemotherapy and corticosteroids resulted in the recognition that they could cause disease in certain hosts . similarly, immunization can render a pathogenic microorganism a 'nonpathogen', as host immunity abrogates the ability of the relevant microorganism to cause disease. for example, variola virus is not pathogenic in individuals immunized with vaccinia virus. the central tenets of the 'damage-response' framework -that the outcome of microbial pathogenesis is the result of a host-microorganism interaction, and that the relevant outcome of this interaction is host damage -provide the basis for a new pathogen-classification scheme. this scheme is based on damage-response curves, which depict the host-microorganism interaction (fig. ). it consists of six different parabolic curves that represent the amount of host damage as a function of the intensity and degree of the host response. each type of curve represents a type or a 'class' of pathogen (fig. ) . in the absence of a host response, damage is the result of the ability of the microorganism to induce damage. however, it is likely that most, if not all, hosts generate some degree of response to microbial infection and that many mechanisms that produce this response have the capacity to induce host damage. the use of host damage as the common principle to classify pathogens allows them to be grouped according to common pathogenic outcomes. microorganisms that cause similar types of diseases can be grouped together -despite their differences at the level of phylogeny, growth characteristics or other criteria that are used for pathogen classification. the pathogens that are grouped in a single class might not cause similar diseases, but what they have in common is the extent to which the damage or disease they cause is a function of the host immune response. therefore, the pathogen classes convey the relative ability of a microorganism to cause disease based on the immune status of in the damage-response framework, a pathogen is defined as a microorganism that is capable of causing damage to a host , . this definition allows the terms that have been used to define microorganisms that do, and do not cause disease, such as commensal, saprophyte, non-pathogen, opportunist and primary pathogen, to be dispensed with. this is a less ambiguous definition of a pathogen than that used previously, as the outcome or possible outcomes of damage are used to define the pathogen, and it dispenses with the need for modification or qualification to encompass microorganisms that cause disease rarely, or only in certain hosts. in the past, the meaning of the word 'virulence' has also caused confusion . the damageresponse framework defines virulence as the relative capacity of a microorganism to cause damage to a host . the word 'relative' is included because virulence is frequently measured in comparison with another microorganism or a variant of the same microorganism. however, when a more complete understanding of quantitative measures of damage is available, it should be possible to dispense with the word 'relative'. the damage-response framework definitions of pathogen and virulence underscore the concept that only in a susceptible host is a microorganism a pathogen, and virulence can be expressed . consequently, neither the characteristics of a pathogen, nor virulence, can be considered independent microbial variables. at present, microbial classifications are largely based on phylogenetic groupings (for example, bacteria, viruses, parasites and fungi), the perceived capacity to cause disease (for example, primary pathogens, saprophytes, opportunistic pathogens or commensals) or, for bacteria, growth or other identifying characteristics (for example, whether it is an anaerobe, aerobe or facultative microorganism, or its appearance under gramstain). these classifications are confusing, because, in addition to being problematic and often inadequate, they are also overlapping. phylogenetic groupings are not very useful for pathogen classification because most of the members of any group are not pathogenic in a host. for example, of the more than , fungal species, only about are pathogenic for humans, and these are often genetically distant from each other. phylogenetic classifications are also subject to change, • the phenotype or property that is under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species. • specific inactivation of the gene or genes that are associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence. • reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity. downwards and to the left, deteriorations in host immunity can shift them upwards, and exuberant immune responses, such as those elicited by some vaccines, can shift them upwards and to the right. finally, the damage-response curves can help in the classification of new pathogens and diseases. for example, based on the available information, the new coronavirus associated with severe acute respiratory syndrome (sars) could be classified as a class -, -, -or -like pathogen, depending on the information that is obtained from subsequent studies. pathogens in these classes all cause host damage in the setting of strong immune responses, and sars has been associated with lung damage in the setting of apparently robust immune responses. indeed, the proposed system is flexible and allows pathogenic microorganisms to be regrouped as new information becomes available. for example, saccharomyces cerevisiae (bakers' yeast) was considered to be non-pathogenic until it was associated with disease in certain patients with severe immune impairment , which would have led it to be categorized as a class pathogen. however, recent evidence indicates that certain mutations in s. cerevisiae increase its virulence in normal mice by altering its surface properties to elicit a potent pro-inflammatory response that damages tissues and causes disease . consequently, it might be more appropriate to characterize s. cerevisiae as a class or pathogen in mice. microorganism-mediated damage in a host can result from a variety of mechanisms, ranging from replication of the pathogen, the production of toxic substances and subversion of the normal host homeostatic and/or immune mechanisms ( (table ) . notably, each also has a polysaccharide capsule that is the key determinant of their capacity to induce host damage, and each causes a similar clinical syndrome. p. carinii is a class pathogen because it does not cause disease in hosts with normal immunity, and aspergillus fumigatus is a class pathogen because it causes disease in individuals with either weak or strong immunity. in the case of individuals with strong immunity, disease results from an exuberant response to aspergillus antigens that damages the host through hypersensitivity reactions. the use of host damage to classify the outcome of a host-microorganism interaction acknowledges and accounts for the contribution of the host immune response to microbial pathogenicity and virulence. therefore, the 'damage-response' framework can be used to characterize host-microorganism interactions that are either detrimental or beneficial to the host. a benefit to the host is indicated by the damage-response curve extending below the x-axis. the framework is a flexible system in that it can accommodate changes in both host and microorganism. for example, therapeutic interventions can shift the damage-response curves the arrow indicates that the position of the curve is variable, and depends on the particular host-microorganism interaction. the y-axis denotes host damage as a function of the host response. in this scheme, host damage can occur throughout the host response, but is magnified at both extremes. the host response is represented by a continuum from 'weak' to 'strong'. 'weak' and 'strong' are terms that can encompass both quantitative and qualitative characteristics of the host response and are used as the best available terms to denote the spectrum of host response as more precise terms are limiting. weak responses are those that are insufficient, poor or inappropriate -that is, they are not strong enough to benefit the host. strong responses are those that are excessive, overly robust or inappropriate -that is, they are too strong and can damage the host. when a threshold amount of damage is reached, the host can become symptomatic and if damage is severe, death can ensue. green, yellow and purple represent health, disease and severe disease, respectively. from the immune response of normal individuals to these microorganisms. for example, the chemokine and cytokine mediator response to s. pneumoniae is responsible for the clinical manifestations of meningitis. a long-lasting inflammatory response to microbial components can result in cell death and the replacement of viable cells and parenchyma with fibrosis. similarly, chronic inflammation can lead to amyloid production, as is observed in patients who have had tuberculosis (tb), and antigen-antibody complexes that form in the course of the immune responsesuch as those that develop in staphylococcal endocarditis or rickettsial diseases -can form deposits in vascular tissues and other organs, which trigger inflammation, organ damage and dysfunction. antigenic mimicry can evoke immune responses that destroy cells and tissue -as exemplified during the pathogenesis of acute rheumatic fever and the development of haemolytic anaemia in the course of mycoplasma pneumonia. host-microorganism contact is followed by two main outcomes, namely, the elimination of the microorganism from the host or infection, which can be defined as the acquisition of a microorganism by a host . following infection by the microorganism, four main outcomes, or states, are possible: commensalism, colonization, persistence (or latency) and disease (fig. ) . these states are a consequence of the outcome of the amount of host damage that results from hostmicroorganism interactions over time, and are generally continuous, such that when damage exceeds a threshold amount, another state becomes relevant . for example, the state of colonization becomes the state of disease when a critical amount of host damage has occurred; the state of disease can become the state of latency; and the state of latency can become the state of disease, again depending on the amount of damage resulting from the host-microorganism interaction. these definitions make no assumptions about the length of time that a state predominates, but instead indicate that as time progresses, different outcomes of infection are determined by the nature and degree of damage that results from the host-microorganism interaction. the role of the host response in the outcome of microbial infection is twofold. first, it defines the threshold that distinguishes host damage from clinical disease, and second, it represents a factor that mediates transitions between the different states. at present, the inability to precisely define both damage and the threshold between damage and disease, makes the distinction between certain states difficult. for example, distinguishing commensalism from colonization is not possible when the latter is accompanied by little or no damage , . in contrast to microorganism-based classification schemes, the damage-response framework is based on the concept that the fundamental difference between pathogenic and non-pathogenic microorganisms is a function of the host response to the microorganism. an effective immune response can abrogate the factor is entirely responsible for host damage -for example, a toxin that causes damage irrespective of the host response because toxin action is so rapid and/or the amount of toxin is insufficient to trigger an immune response. previously, we have proposed that toxin-producing microorganisms are a variant of class where the curve is flat at both ends , but here we suggest that this type of interaction might be unique and warrants a separate panel. as shown here, the damage-response classification scheme is flexible and makes it possible to postulate the existence of pathogens for which there are no known examples at present. such pathogens could be recognized in the future as 'emerging' pathogens as shown in c and d. c | the class curve is extended below the x-axis. such a theoretical microorganism would be a commensal in the setting of intermediate host responses, but pathogenic in hosts with either weak or strong responses. d | the inverted parabola represents a putative host-microorganism interaction that induces damage over a narrow and limited range of responses, but not in the presence of either strong or weak host responses. one example of such a phenomenon would be an antibody response to a hypothetical microorganism, whereby host damage is caused by antigen-antibody complexes. although we cannot think of a specific microorganism that fits this description at this time, examples of this type of host damage are the host-microorganism interactions characterized by the herxheimer reaction following treatment of syphilis, the similar reaction that can occur after the initiation of therapy for pneumocystis carinii pneumonia, and serum sickness following the injection of foreign protein. this we make no apologies, as the field of microbial pathogenesis seeks to understand how microorganisms cause disease, an outcome that can only occur in a susceptible host. the only components of the damage-response framework are microorganisms that can cause disease and hosts. similarly, the outcomes of a host-microorganism interaction after initial contact are simplified to infection, commensalism, colonization, persistence (latency) and disease. the use of damage as the common classification for microbial pathogenicity and virulence simplifies the lexicon of microbial pathogenesis and makes it possible to discard ambiguous terms, such as commensal, saprophyte, opportunist, exposure and carriage , , , . the damage-response framework was proposed based on clinical and experimental observations of the outcome of host-microorganism interactions. despite being supported by a large body of observational evidence, it should also be subjected to experimental validation and/or refutation. to accomplish this, it will be necessary to develop better quantitative and qualitative measures of the immune response and host damage, as, at present, the information needed to validate or refute the damage-response framework is unavailable. the damage-response framework plots damage as a function of the immune response and uses the qualifiers 'strong' and 'weak' to denote quantitative and qualitative characteristics of the immune response. we appreciate that these terms are vague and, as such, that they might seem to oversimplify the complex, diverse and multidimensional nature of the host response -which includes both humoral and cellular components of the innate and adaptive immune systems. pathogenicity of a microorganism. even highly virulent toxigenic microorganisms are not virulent in hosts that have been immunized with toxoid vaccines, such as tetanus toxoid. therefore, although the existence of microbial virulence factors is incontrovertible, for many microorganisms it is the ability of these factors to override the mechanisms of host defence that makes them virulence factors. the damage-response framework is neither microorganism-centred nor host-centred. it does, however, state that the outcome of host-microorganism interactions in a host is the defining aspect of microbial pathogenesis. in this regard, the damage-response framework might be considered to be biased towards the host. however, rather than holding the host responsible for pathogenesis, the damage-response framework places the context of microbial pathogenesis within the host. for colonization a state of host-microorganism interaction that leads to a variable amount of host damage, from minimal to great, thereby reflecting host immune responses that have the capacity to eliminate the microorganism or to promote the development of another state. latency a state of host-microorganism interaction in which a microorganism persists in a host and can be associated with damage that can be evident at the cellular or tissue level, but is not associated with disease. a microbial component that can damage a host. the shortcomings of the terms 'strong' and 'weak' are evident if the case of p. carinii pneumonia is considered. this disease occurs almost exclusively in severely immunocompromised individuals and, consequently, p. carinii was classified as a class pathogen. however, clinical experience has shown improved outcomes in patients that are treated with corticosteroids, which indicates that lung damage in aids patients with p. carinii pneumonia is mediated largely by the residual immune system. therefore, p. carinii pneumonia occurs in individuals with impaired ('weak') immune responses, but damage is likely to be immune-mediated. if treated as singular parameters, measurements of the immune response that are available at present, such as the amount and type of antibody response, delayed-type hypersensitivity, lymphocyte proliferation, cytokine levels or immunoglobulin e levels, are too limited and one-dimensional to be useful quantitatively on the x-axis of the damage-response curves. even in combination, these parameters are not, at present, useful as we lack the knowledge to devise formulas that could take their relative contributions into account. similarly, measures of damage, such as fever, organ dysfunction and cytokine levels in serum, are too insensitive to provide an accurate measure of the amount and quality of damage that results from the host-microorganism interaction. damage, like the immune response, is the result of multiple events and their contributions from cellular, tissue and organ toxicity. for example, it has been shown that the interaction of c. albicans with different host effector cell receptors can result in different inflammatory profiles, which, in turn, translate into differences in virulence . however, at present, we cannot predict the point at which subclinical damage becomes clinical disease or, indeed, the level of disease that results in the death of the organism. therefore, the correct parameters for plotting on the x-and y-axes should be functions of multiple variables that include the contribution of multiple components of the immune system and damage, respectively. the damage-response framework proposes classifications, statements and predictions that are, in principle, amenable to experimental testing. we anticipate that, in the future, accurate measurements of host damage and the immune response will be available, which will allow the generation of experimental curves to which mathematical functions can then be fitted to formally describe the host-microorganism interaction. any effort to investigate the validity of the damageresponse framework is likely to stimulate research into the relationship between host damage and the immune response. such endeavours will provide new scientific insights, irrespective of whether the framework is ultimately validated or falsified. we anticipate several important uses of the damageresponse framework. first, as has already been alluded to, efforts to validate or refute the framework will undoubtedly foster research into improved measures of damage and the immune response. second, the framework can lead to predictions that might enhance the development and predictability the host-microorganism interaction can be depicted by plotting host damage as a function of time. panels a-d show how plotting damage versus time can be used to denote the states of the host-microorganism interaction for four different pathogens. infection represents the acquisition of the microorganism by the host and is followed by the states of commensalism, colonization, latency and disease, depending on the amount of damage to the host , . these plots highlight the fact that for certain pathogens there is continuity between the various states. the colours green, yellow and purple denote health, disease and severe disease, respectively, and the relevant states for each host-microorganism interaction are highlighted in bold. www.nature.com/reviews/micro r e v i e w s blood donors . recently, several countries have been affected by coronavirus-associated sars . if disease in sars is found to be associated with a large viral burden, it might be anticipated that the process involves viralmediated damage and that antiviral therapy could be helpful therapeutically. however, if disease is found to be associated with inflammation and a paucity or absence of virus in tissues, then the pathogenic process might reflect immune-mediated damage for which antiinflammatory therapy could be helpful. initial reports that corticosteroid therapy might be helpful are consistent with the possibility that host-mediated damage is responsible for the disease. fourth, the damage-response framework promises to be a useful educational tool as it avoids the 'bug parade' that students of microbial pathogenesis find so unsatisfactory, and instead provides a more straightforward classification of pathogenic microorganisms that integrates the contributions of both host and pathogen. finally, the damage-response framework should provide a mechanism that brings together, under one umbrella, the different areas of microbial pathogenesis that, at present, are isolated from one another, such as viral and bacterial pathogenesis. such an approach would foster collaboration at all the interfaces between microbiology and immunology, and ultimately advance our understanding of infectious diseases. of rational vaccine design. consider tb, a disease for which there has been a major effort to develop a vaccine and/or immune-based therapy. the causative agent of tb, mycobacterium tuberculosis, is a class pathogen that induces damage at the extremes of the immune response. in immunocompetent individuals, damage reflects a robust, often helper-t-cell- -like response, whereas in immunocompromised individuals it reflects an insufficient inflammatory response. as the basis of host damage differs, depending on the immune status of the individual, the damage-response framework would predict that the use of different types of vaccines, vaccine antigens or vaccination strategies might be necessary to prevent tb in immunocompetent and immunocompromised individuals. similarly, immune therapies could be directed towards reducing the inflammatory damage in immunocompetent hosts and enhancing the immune function in immunocompromised hosts. third, the damage-response framework has the potential to characterize new and emerging infectious diseases, thereby facilitating a more rapid and focused response by the public authorities and research community. two recent emerging infectious diseases exemplify this point. the recognition that the disease-to-infection ratio for west nile virus in endemic areas is very low, led to the successful experimental use of passive antibody therapy with immunoglobulins from asymptomatic introduces the 'damage-response' framework and proposes a new classification for pathogens based on their ability to damage the host as a function of the host response expands the 'damage-response' framework to incorporate the variable of time and proposes that the outcome of the host-microorganism interaction can be reduced to four states that differ in the amount of damage incurred by the host as a function of time what is a pathogen? discusses limitations of the existing concepts of virulence and problems with definitions of virulence factors highlights how the damage-response framework can be used to classify the host-microorganism interaction in clinical practice on the definition of virulence the origin of plant and animal cells symbiosis and evolution principles and practice of infectious diseases legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages common themes in microbial pathogenicity revisited. microbiol pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution landmark paper that proposed a rigorous approach to establishing whether a gene was involved in microbial virulence the role of opportunistic bacteria in human disease history of opportunistic infection in the immunocompromised host an informative story of how the concept of microbial opportunism evolved in clinical practice pathogenicity and virulence: another view an excellent discussion of the limitations of koch's postulates exploiting the redundancy of the immune system: vaccines can mediate protection by eliciting 'unnatural' immunity ribosomal rna shows pneumocystis carinii to be a member of the fungi antimicrobial therapy severe acute respiratory syndrome (sars): breath-taking progress saccharomyces cerevisiae bacteremia a saccharomyces cerevisiae mutant with increased virulence parasitealtered behaviour: is the effect of toxoplasma gondii on rattus norvegicus specific? dendritic cells and receptors: a host perspective of fungal virulence prophylactic and therapeutic efficacy of human intravenous immunoglobulin in treating west nile virus infection in mice colonization with caga-positive helicobacter pylori strains inversely associated with reflux esophagitis and barrett's esophagus the following terms in this article are linked online to: infectious disease information: http://www.cdc.gov/ncidod/diseases/index.htm candidiasis | cryptococcus neoformans | cysticercosis | legionella pneumophila | meningitis | mycoplasma pneumonia | pneumocystis carinii pneumonia | syphilis | tuberculosis access to this interactive links box is free online. key: cord- - wennun authors: altmann, daniel m title: adaptive immunity to sars-cov- date: - - journal: oxf open immunol doi: . /oxfimm/iqaa sha: doc_id: cord_uid: wennun the majority of severe acute respiratory syndrome coronavirus (sars-cov- exposed individuals mount an antibody response within around -weeks and spike antigen-binding responses correlate well with functional virus neutralization. a minority makes little detectable antibody, generally those with either very mild/asymptomatic disease or those with severe/lethal infection. however, in general, antibody titre correlates with viral load and duration of exposure. there is evidence for cross-reactivity with the other human coronaviruses, though the functional impact of this is as yet unclear. therapeutic use of neutralizing monoclonal antibodies offers potential for clinical use. while there is evidence for neutralizing antibody as a correlate of protection, some cases indicate the potential for full recovery in the absence of antibody. studies of t-cell immunity following acute infection show cd and cd responses to epitopes across diverse viral antigens, possible cross-reactivity with epitopes from the common cold human coronaviruses and large-scale activation. however, in severe cases, there is evidence for t-cell lymphopaenia as well as expression of exhaustion markers. analysis of serum biomarkers of disease severity implicates a hyperinflammatory contribution to pathogenesis, though this has not been mechanistically delineated beyond a likely role of raised il- , considered a therapeutic target. despite rapid progress, there remain pressing unknowns. it seems likely that immune memory to sars-cov- may be relatively short lived, but this will need longitudinal investigation. also, this is a disease of highly variable presentation and time course, with some progressing to protracted, chronic symptoms, which are not understood. the contribution of immunopathological mechanisms to tissue damage, whether in the lung, kidney, heart or blood vessels, is unclear. the immunology underlying the differential susceptibility between the very young and the very old is unresolved, a question with ramifications for vaccine roll-out. the greatest challenge relates to rapid generation, testing and manufacture of vaccines that are immunogenic, protective (at least from symptomatic disease) and safe—a challenge that looks achievable. infection, immunology research has been placed in the spotlight, with intense curiosity and scrutiny about many specific aspects of the immune response to this viral infection: when does immunity develop, what are the correlates of protection, what is the temporal relationship between immunity and infectivity, do all develop protective immunity and can reinfection occur, what part is played by immunopathology in pathological damage to the lungs and other organs? on top of this has been impatience for updates on progress in rapid resolution of the translational challenges posed by global roll-out of reliable antibody serodiagnostics and of safe reliable vaccines [ ] [ ] [ ] [ ] . immunology has never had to grapple with questions of this enormity under such time pressure. among the countless manifestations of the 'new normal' has been an overturning of conventions for publishing so as to address the pressure for data updates in real time: the tendency has been for data to emerge as soon as it is generated, on social media, then within days or weeks posted on repositories such as biorxiv as a non-peer-reviewed preprint, then subsequently snapped up for full publications in prestigious journals. there have been many consequences of this publishing revolution. there has been a vibrant, refreshing foreshortening of the publication timeline. this is a field that had developed norms whereby big papers necessitated the pooled work of perhaps a score of scientists over years of funded research, submitting a manuscript for laboured, iterative, peer-review stretching over - months, so that the full cycle from concept, to funding, to research, to publication might be years plus. in the 'new normal', some of the highest profile papers have used standard, pre-existing technologies such as multiparameter flow cytometry panels and rnaseq pipelines to describe and define immune parameters in patients hospitalized in january and february, the papers reporting them appearing in march and april. while there may indeed be a price for reduced rigour in peer review, many might argue that the scrutiny of a scientific peer group via social media has gone some way to substituting a proxy arbiter of quality control. with these points in mind, my aim here has been to present an overview of some of the key knowns and unknowns of sars-cov- adaptive immunity, relying both on preprints and on published findings. my focus has been to some extent informed by the recurrently posed questions that have clearly been of concern, whether in media interviews or policy discussions. few in the immunology research community had prior, handson, experience with the immunology of coronaviruses, and one of the challenges has been to convey the notion that, as for any host defence programme, once you drill down beyond textbook generalities of viral immunity, the devil is in the detail, and that is what current research must resolve. the spread of a fatal respiratory syndrome focused on initial cases who had visited wuhan seafood market in china and was first reported in december [ , ] . within a little over weeks of the initial case, genomic sequence for a novel coronavirus was published in early january [ ] . it was initially termed -ncov and then, sars-cov- . in terms of known human infections, it is phylogenetically close to sars-cov and is believed to be a new zoonotic transfer, probably from bats, although it is still unknown whether there was an intermediate species, such as pangolin [ ] . interestingly, a recently sequenced betacoronavirus from bats in yunnan province, termed rmyn , has % sequence identity with sars-cov- , but with critical changes in receptor-binding domain (rbd) of the spike protein, meaning it probably lacks the key feature of binding to human angiotensin-converting enzyme (ace ) [ ] . the coronaviruses have single-stranded rna genomes encoding non-structural proteins as well as structural proteins: spike, envelope, membrane and nucleocapsid. while host immunity may be revealed against any part of the viral proteome, much initial immunology research has focused on immunity to the spike antigen; this is driven by the knowledge that the interaction between the rbd within the spike antigen and human ace is critical for viral entry and infectivity, and that antibodies against spike can be protective through neutralization. in this regard, many initial clues came from extrapolating from the immunology of the closely related infections caused by sars-cov and mers [ ] [ ] [ ] [ ] . the genomic sequence was rapidly shared and published, facilitating design of pcr and antibody-based diagnostics [ ] . an initial challenge in design of antibody tests, especially in the face of limited supply of positive control serum samples for testing, was to validate binding that would be specific to this virus [ ] . the confounder of cross-reactivity with sars or mers antibodies is a concern, while prior exposure to the common cold hcov viruses is considerably more widespread in human populations, though the genomic sequences show far lower conservation [ ] . most test strategies have relied on recombinant spike antigen. some use the nucleoprotein, though this is more conserved across coronaviruses and therefore more prone to detect crossreactive binding. while lab assays were validated and cohort antibody data accrued [ ] [ ] [ ] [ ] [ ] , there was a considerable public health imperative to scale-up antibody tests for patient screening and for seroprevalence studies [ ] . clearly, there is a range of approaches for antibody tests, covering lateral flow devices optimized to give a yes/no binary answer for antigen-binding, elisa-based approaches, and then functional neutralization assays based either on pseudotype virus or on live virus assayed under bsl conditions. rolling out reliable antibody testing at scale and at speed proved extremely challenging and has only recently been resolved, based largely on elisa approaches. by analogy to studies on sars and mers, most exposed, symptomatic individuals would be predicted to show an antibody response to spike antigen [ ] [ ] [ ] [ ] . furthermore, when tested for functional virus neutralization, this has tended to correlate well with total antigen-binding antibody by elisa [ ] . data sets have now been shared for spike antibody in a large number of sars-cov- patient cohorts. a number of general points emerge from this: patients show a wide range of antibody titres, absence or near absence of antibody is associated with very mild infection, with fatal outcome or with immunodeficiency; the time course for appearance of igm, igg and iga antibodies follows conventional kinetics over the initial - days of infection, the appearance of detectable antibody roughly contemporaneous with the disappearance of infectious virus from nasopharyngeal swabs; despite differential disease susceptibility between children, adults and the elderly, this is not reflected in simple quantitative differences of antibody titre [ ] [ ] [ ] [ ] [ ] ] . some data indicate that cumulative viral load is correlated with antibody titre, raising concerns that those with very mild exposure may have more marginal or undetectable antibody responses, as was seen in sars and mers. however, while initial antibody studies were skewed to analysis of those 'tip of the iceberg' cases severe enough to be hospitalized, more data sets are now available for milder exposures such as healthcare workers, reassuringly showing significant antibody responses [ ] . chung and colleagues used a systems serology approach to investigate functional correlates of antibody responses across age groups and disease profile [ ] . systems serology harnesses the power of machine learning with data sets from multiple assays of antibody functionality including avidity and fc receptor binding to generate correlative signatures [ ] . they identified a convalescence biosignature associated with igg , fccr binding and c q engagement, as well as an influence of hlaii polymorphisms. there has been much speculation as to whether differential immune repertoires help to inform the differential susceptibility of the very young and the very old. this study offers the perhaps counterintuitive suggestion that children may benefit from an igm-dominated signature, unlike the class-switched igg and iga signatures of the elderly. differences across the lifespan may also relate to recent exposure to epitope-cross-reactive hcov common cold viruses, though the functional extent of any such cross-reactivity and associated protection has been a source of controversy. a recent study by ng et al. finds evidence for cross-reactivity between hcov and sars-cov- spike and nucleoprotein, even to the extent of functional neutralization [ ] . since many key questions about durability of the antibody response and about correlates of protection have been hard to address in this short timeframe, there has been value in recourse to the coronavirus immunology literature, especially in relation to sars and mers [ ] [ ] [ ] [ ] . this suggests a consensus of around weeks to igg seroconversion, lower responses in asymptomatic or mild infection, variably poor durability of antibody response beyond or years and neutralizing antibody as a likely correlate of protection. certainly in follow-up of mers patient, a significant minority showed no detectable antibody at months. more studies will be needed to ascertain whether the greater igg repertoire of older individuals, including an hcov crossreactive repertoire, may contribute to enhanced pathogenesis through antibody-dependent enhancement [ ] . many teams have moved rapidly to express neutralizing human monoclonals characterize the binding site structural biology and evaluate potential translational use as therapeutics [ ] [ ] [ ] , following the rationale trialled in recent years for infections including hiv, ebola and c. diff [ ] [ ] [ ] . in some reported cohorts, there has been a minority of patients who have made a full recovery, seemingly without generating any detectable antibody response [ ] . there are also reports of people unable to make any b-cell response at all due to agammaglobulinaemia who can also make a full recovery [ ] . whatever else, this suggests that neutralizing antibodies are not strictly required for recovery and other parts of the specific response, such as t cells, may serve to offer sufficient protection. the nuances of these emerging data sets have significant ramifications for clinical management of patients with inflammatory and autoimmune diseases across many specialties, weighing up the profile of their different disease-modifying therapy protocols to establish which are most likely to be safe. meanwhile, following many false starts, seroprevalence data sets are starting to appear from diverse urban locations around the globe. some care is needed in collating these data as it can sometimes be hard to ascertain the precise sampling location and procedure, sample size, detection assay used or indeed whether derived from actual antibody tests or from predictive models. while results obviously look very different in different affected centres, seroprevalence levels seem typically to be in the - % range, far short of the % plus needed for herd immunity, thus the urgent imperative for effective vaccines [ ] . experience from t cell studies in sars immune donors suggests that strong cd and cd immunity may endure for a number of years [ ] . initial analysis of immune subsets in acute covid- largely focused on hospitalized patients with severe infection, though studies of the response during milder infection are now appearing [ , [ ] [ ] [ ] [ ] [ ] [ ] . analysis of the hospitalized cohorts shows a picture of large scale t-cell activation, especially cd cells, along with t cell lymphopaenia as a correlate of severity, and expression of exhaustion markers such as pd- and tim- [ , ] . cd and cd t-cell immune responses can be detected to diverse regions of the viral proteome including the nucleoprotein, spike/rbd and the main protease [ ] [ ] [ ] . antiviral t-cell immunity correlates with neutralizing antibody titres [ ] . peptide epitope mapping of antiviral t-cell responses has been taken to support a case for the possibility of cross-reactive protection by memory t cells recognizing epitopes shared with hcov sequences [ ] . thieme et al. recently evaluated cd and cd responses against spike, membrane and nucleocapsid antigens, comparing between moderate, severe and critical disease. all three antigens contained epitopes, though the strongest response was to the membrane protein. the strongest responses, often polyfunctional, were seen in severe cases [ ] . should we be devoting extensive attention to the details of t-cell immunity if the data suggest that neutralizing antibody titre is itself a candidate correlate of protection? the answer is resoundingly in the affirmative, for several reasons. as we debate one of the biggest concerns around the fragile durability of coronavirus antibody responses, t-cell memory may be more enduring than b cell. extrapolating from progress in influenza vaccinology, it is likely that the most efficacious vaccine approaches need to look to strong recognition by both b-cell and t-cell receptors [ ] . whether in the context of natural or vaccine-induced t-cell immunity, there is a need for some scrutiny of the elicited cytokine profile. for example, in sars infection, th cytokine responses can be a cause of lung immunopathology [ ] . another recent study reported a deep immune profiling analysis, comparing parameters in patients compared to convalescent individuals or controls with respect to around immune parameters and clinical parameters [ ] . the comprehensiveness of this analysis is useful for its ability to reinforce a number of observations suggested by smaller studies. the study reiterates the observations of others in relation to lymphopaenia, t-cell exhaustion, decreased tregs and cd t cells, and the notion of development of neutralizing antibody as a likely correlate of protection. analysis of the parameters by tsne-enabled delineation of distinct, clinically related, immune phenotypes including an activated immune profile associated with expanded b-cell plasmablasts and activated cd t cells that are positive for ki , hla-dr, cd , cd , pd- , icos and fas. back to back papers from barouch and colleagues using a macaque infection and challenge model show the benefits of relevant animal models for clarifying some of the unknowns, although, with the caveat that short-term rechallenge at weeks is a poor proxy for understanding longer-term protection. the studies nevertheless show that either viral infection or dna vaccination results in non-sterilizing protection from symptomatic disease and that neutralizing antibody may indeed be the best correlate of protection [ , ] . from some of the earliest reports of serum biomarkers for severity of disease in patients who require ventilation, there has been evidence of a signature that includes inflammatory markers such as high ferritin, crp, d-dimer and il- [ ] . consistent reports of raised il- led to a series of clinical trials with the therapeutic monoclonal, tocilizumab, in what appear to be promising trials [ ] . another recent study highlights a biosignature of cxcl , ccl and il- receptor antagonist associated with high viral load, impaired lung function, lung injury and lethal outcome [ ] . we perhaps need some caution in overuse of the rather too simple term, 'cytokine storm', lest it blinds us to considering the aetiology and detail of what looks a rather unusual signature, worthy of investigation. which cells have produced the response and to what specific viral stimulus? certainly, this is not particularly reminiscent of a classic superantigen septic shock, to which it is sometimes related [ ] and, for example, many classic t-cell-derived cytokines are not a feature here. furthermore, there have been a number of reports of a kawasaki-like disease in children, now numbered at several hundred cases [ ] , with a disease now provisionally termed, 'paediatric inflammatory multisystem syndrome temporally associated with sars-cov- ' (pims-ts) [ ] . kawasaki disease itself still lacks a fully defined immunopathogenesis-it is considered an infection-triggered inflammatory cascade associated with the il- b pathway [ ] , and responding to therapeutics including ivig. despite rapid progress, there are rather urgent unknowns. this is an infection that affects different populations, whether in relation to age group or ethnicity, extremely differently, with old age by far the largest risk factor for severe outcome. while immunology has been able to offer hints as to the differential response that may underpin these differences, we still lack clarity. we are only starting to grapple with the detail of the diversity of cell types and tissues that can be infected by sars-cov- . understanding the nuances of infection, immunity and immunopathology in the lung, kidney and heart will be important. note that ace expression is relatively widespread across different cell-types, and we are only at the beginning of understanding the ramifications of this beyond the upper respiratory tract. while much has been learnt about immune correlates of infection, data so far are rather heavily weighted to analysis of severe, hospitalized patients. this leaves a vital knowledge gap to fill about the nature of immune recognition and immune memory of the many millions of individuals infected by the virus, either asymptomatically or symptomatically but managed in the home. such individuals comprise the vast majority of the exposed population, and there is an urgent imperative to build the narrative as to the extent and features of their adaptive immune response. perhaps the most critical aspect of this is the durability of that response, of concern with respect to mitigation of further waves. by analogy to the other human coronaviruses, the worrying prediction is that immunity may not commonly be durable beyond a few years. till now, all resources have necessarily been devoted to the firefighting of the response to the acute pandemic. attention is gradually shifting to the need for a more granular understanding of the interplay between host immunity and damaging immunopathology, whether in lung disease, in the pathway leading to thrombosis, or at other sites. in the future, considerable healthcare resource will need to be allocated to covid- follow-up clinics. the indications already are that, both among those who were severely affected and those that were not, there may a need to treat those living with long-term consequences of the infection, from lung fibrosis or bronchiectasis, to renal and cardiac complications and to chronic systemic, post-viral syndromes. the real test of how far the fast track immunology pipelines have progressed will be whether effective vaccines can be widely delivered in the near future. while there has been much optimism about the likelihood or even, inevitability, of phase i/ii trials shifting promptly into wide-scale production and roll-out during , the ups and downs of many other initiatives over the past decade caution against blithe assertions. there has at times been an unhelpful tendency to flip-flop between extremes of unrealistic optimism ('definitely by september') and unrealistic pessimism ('we must accept then there may never be a vaccine'). as with any vaccine programme in history, there is a multitude of possibilities between these. experience to date with sars-cov- suggests that this may not prove to be an infection that throws up insurmountable confounders to vaccine design-approaches that can safely and durably elicit neutralizing antibody look likely to work. the effort already has been unprecedented, spanning every known vaccinology platform, from adjuvanted conjugate vaccines, to attenuated or inactivated virus, to rna and dna vaccines and recombinant adenovirus approaches. while they are all well-established approaches with strong credentials and track records in a research setting, many have never before crossed the finish line to full manufacture and licensure. each approach comes with its own nuanced strengths and weaknesses in terms of magnitude of response, durability of response, number of boosts likely to be required, ability to stimulate different aspects of mucosal or systemic immunity, bias in terms of b cells, t cells and t-cell subset polarization, safety profile, ease of manufacture and supply chain/storage issues. these are important questions that have thus far received little attention in the discussions framed as a simple race to the finish line. it will also be important to educate policymakers and the public about the nature and limits of clinical trials to avoid erosion of confidence through unrealistic expectations: it seems unlikely that any of the vaccines will elicit sterilizing or lifelong immunity, but ones that significantly mitigate the most severe disease manifestations would be 'good enough'. on the contrary, under this intense scrutiny, any adopted candidates that are seen to be less good than this carry the risk of collateral damage for all other vaccines at a time of global health vulnerability under pressure from vaccine hesitancy. this pandemic merits a response from the immunology community that promotes the 'best answer', which may not be the 'first'. for this purpose, it is invaluable to foster coordinated endeavours through bodies such as cepi, gates foundation and wellcome, as an antidote to parochial 'vaccine nationalism' that risks the piecemeal development of rival, regional vaccines, which may impact both on control of infection and on equity of access. this is a global infection that crosses all borders-the specific language or nationality of the vaccine(s) is an irrelevance. serology assays to manage covid- labs scramble to produce new coronavirus diagnostics sars-cov- vaccines: status report developing covid- vaccines at pandemic speed early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical features of patients infected with novel coronavirus in wuhan, china a new coronavirus associated with human respiratory disease in china unveiling the origin and transmission of -ncov a novel bat coronavirus closely related to sars-cov- contains natural insertions at the s /s cleavage site of the spike protein detection of sars-cov- -specific humoral and cellular immunity in covid- convalescent individuals neutralizing antibodies responses to sars-cov- in covid- inpatients and convalescent patients neutralizing antibody responses to sars-cov- in a covid- recovered patient cohort and their implications a serological assay to detect sars-cov- seroconversion in humans pre-existing and de novo humoral immunity to sars-cov- in humans national covid testing scientific advisory panel. antibody testing for covid- : a report from the national covid scientific advisory panel. longitudinally profiling neutralizing antibody responses to sars coronavirus with pseudotypes antibody responses against sars coronavirus are correlated with disease outcome of infected individuals potent and persistent antibody responses against the receptor-binding domain of sars-cov spike protein in recovered patients a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease serologic responses to sars-cov- infection among hospital staff with mild disease in eastern france distinct systems serology features in children, elderly and covid patients prospects from systems serology research cross-reactive antibody response between sars-cov- and sars-cov infections human neutralizing antibodies elicited by sars-cov- infection a human neutralizing antibody targets the receptor binding site of sars-cov- a human monoclonal antibody blocking sars-cov- infection broadly neutralizing antibodies for hiv prevention safety, tolerability, pharmacokinetics, and immunogenicity of the therapeutic monoclonal antibody mab targeting ebola virus glycoprotein (vrc ): an open-label phase study antibody responses to sars-cov- in patients with covid- what policy makers need to know about covid- protective immunity t cell responses to whole sars coronavirus in humans breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid- reduction and functional exhaustion of t cells in patients with coronavirus disease (covid- ) targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals the sars-cov- t-cell immunity is directed against the spike, membrane, and nucleocapsid protein and associated with covid- severity different pattern of pre-existing sasr-cov- specific t cell immunity in sarsrecovered and uninfected individuals deep immune profiling of covid- patients reveals patient heterogeneity and distinct immunotypes with implications for therapeutic interventions novel bivalent viral-vectored vaccines induce potent humoral and cellular immune responses conferring protection against stringent influenza a virus challenge immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus sars-cov- infection protects against rechallenge in rhesus macaques dna vaccine protection against sars-cov- in rhesus macaques interleukin- in covid- : a systematic review and meta-analysis exuberant elevation of ip- , mcp- and il- ra during sars-cov- infection is associated with disease severity and fatal outcome the immunology of sepsis an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study kawasaki-like disease: emerging complication during the covid- pandemic dissecting kawasaki disease: a state-of-the-art review key: cord- -uxsjyezo authors: hedges, jodi f.; jutila, mark a. title: harnessing γδ t cells as natural immune modulators date: - - journal: mucosal vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: uxsjyezo there has been renewed interest in harnessing the power of the immune system as a countermeasure against infectious pathogens and cancers. one immune cell target in the development of these approaches is the γδ t cell. these cells are involved in innate and adaptive immune responses against infectious agents and cancers, and they migrate to, and reside in, mucosal tissues. γδ t cells exhibit a broad array of natural (or constitutive) and induced effector functions, including antigen presentation, that can be fine-tuned depending on their stimulation. they express unique antigen receptors as well as nonantigen, innate immune type surface receptors that can be targeted. in this chapter, we will review the biology and the basis for use of γδ t cells as a therapeutic target. we will then summarize novel plant- and microbe-derived materials that enhance γδ t cell activity in animal models and humans that can be used as a new strategy for mucosal vaccine development. lymphocytes are important in both innate and adaptive immune responses. innate lymphocytes represent a heterogeneous group of cells that include cells lacking receptors for antigen, such as the group of innate lymphoid cells, of which natural killer (nk) cells are the prototypical example. innate lymphocytes expressing antigen receptors include b b cells, natural killer t (nkt) cells, and γδ t cells. while innate lymphocytes are relatively rare in circulation and in lymphoid tissues, they are found in mucosal surfaces that represent portals of entry into the body [ ] . in this chapter, we will focus on some of our laboratory's work on one of the major antigen-specific subsets of innate lymphocytes: γδ t cells. γδ t cells have important roles in both innate and adaptive immune responses, wound healing, and tissue homeostasis. there are many outstanding reviews of the biology and function of γδ t cells. a select few relevant to the topic of this chapter are listed in table . . briefly, γδ t cells express unique t cell receptors (tcrs) that recognize self and foreign antigens in the absence of the requirement for presentation by major histocompatibility complex (mhc) class i or class ii molecules. this feature leads to a broad range of innate responses against pathogens, as well as recognition of stressed or tumor cells. subsets of γδ t cells are defined by restricted tcr gene usage in addition to expression of various surface molecules and preprogrammed functional responses imprinted prior to their egress from the thymus. γδ t cells also express myriad innate receptors, such as toll-like receptors (tlrs), scavenger receptors, and lectin receptors, such as dectin- , that can directly sense infectious agents. these receptors, along with cytokine receptors, fine-tune sensing and response of γδ t cells adapting to the tissue microenvironment. tcr stimulation leads to a variety of functional responses, such as cytolysis, cytokine production, regulatory effects, and even phagocytosis and antigen presentation, that depend on the activation of receptors and coreceptors. γδ t cells respond rapidly to external signals, leading to early cytokine responses in a variety of disease settings. furthermore, they are uniquely positioned at virtually all portals of entry into the body where this type of innate immune response is critical. indeed, γδ t cells, like other innate lymphocytes, are found at all mucosal surfaces and make up a large fraction of the intraepithelial lymphocyte population. they are also recruited to sites of inflammation, tumor growth, or other tissue insults. in addition to the γδ tcr, γδ t cells express a variety of non-tcr receptors that affect their function. γδ t cells express the nk c-type lectin-like receptors, such as nkg d, which recognize cellular stress proteins resulting in cellular activation [ , ] . they also express tumor necrosis factor (tnf) receptor family molecules cd , cd , and cd [ ] . cd is a costimulatory receptor to the tcr [ ] , and cd is also expressed on tcr-stimulated tumor-reactive γδ t cells [ ] . cd (of the ig superfamily) is also a γδ tcr coreceptor. the aryl hydrocarbon receptor (ahr), generally known for its role in homeostasis for mucosal t cells, is also expressed by mouse γδ t cells that produce innate interleukin (il- ) [ ] , as well as the mouse skin γδ t cell subset [ ] . γδ t cells also express various cytokine receptors that contribute to their activation (il- r, il- r, il- r, etc.) and-fine tune their functional responses. the expression of pathogenassociated molecular pattern receptors has been detected on γδ t cells. these include another lectin receptor, dectin- , a receptor for fungal, plant, and bacterial-derived polysaccharides [ , ] ; the tlrs [ , ] ; cd [ ] ; scavenger receptors [ ] ; and nod-like receptors [ ] . though not a focus of this chapter, γδ t cells also express a variety of receptors that downregulate their function. examples include killer cell immunoglobulin-like receptor and leukocyte immunoglobulin-like receptor, b and t lymphocyte attenuator, and programmed cell death receptor, which are regulatory receptors that suppress the function and/or proliferation of the cells [ ] . γδ t cells are an ancient immune cell lineage, found in all jawed vertebrates. phylogenetic evidence suggests that they are the progenitors of both αβ t cells and b cells [ ] . they predate adaptive immunity, so it is not surprising that they retain many innate functions similar to those of monocytes and macrophages. zebrafish γδ t cells both are phagocytic and can present antigen, in addition to their expression of cd [ ] . we characterized transcript expression in subsets of bovine γδ t cells [ À ] . the primary outcome of these studies was the recognition of multiple transcripts similar to those found in monocyte and macrophage cells, indicating their innate function. as part of these studies, we detected b-lymphocyte-induced maturation protein (blimp- ) transcripts in bovine γδ t cells [ ] . blimp- , also known as prdi-bf , is a key regulator in the differentiation of hematopoietic cells into myeloid or b cells [ ] ; therefore, its detection in a t cell subset was notable at the time. we recognized this significance and further confirmed the expression of transcripts in resting bovine γδ t cells and not αβ t cells [ ] . more recent findings have further confirmed the innate function of γδ t cells in the appropriate contexts. transcript analyses in bovine γδ t cells also suggested expression of transcripts encoding solute carrier a (slc a , also denoted natural resistance-associated macrophage protein , or nramp- ) in these cells [ ] . slc a is a divalent metal transporter that is thought to be expressed only in myeloid and macrophage cells; it is important in effective responses against intracellular bacterial infections [ À ]. slc a enhances signaling and activation in macrophages [ ] . we defined protein expression and a similar function in activation in bovine and human γδ t cells and nk cells. expression of slc a was strongly correlated to the activation and, in particular, the expression of interferon gamma (ifnγ) in these cells [ ] . thus slc a is an additional monocyte/ macrophage protein that is also expressed in γδ t cells with functional relevance. another similarity to myeloid cells is the ability of γδ t cells in a number of species to process and present antigen. effective antigen presentation is required for the initiation of adaptive immunity and is studied primarily in conventional antigen-presenting cells (apcs), such as dendritic cells (dcs), activated macrophages, and b cells. γδ t cells express an array of surface receptors, such as scavenger receptors, cd b, and cd , that facilitate uptake of particulate antigens [ , ] . subsets also express mhc class ii and necessary coreceptors for effective antigen presentation to cd t cells [ À ] . antigen uptake and presentation to cd t cells were first shown for bovine γδ t cells [ ] . it was also shown that mhc class ii expression and antigen presentation is enhanced in bovine wc γδ t cells during viral infection [ ] . similar functions were described for porcine, human, and mouse γδ t cells [ À ] . γδ t cells in contact with bacteria can transition from cytokine-producing cells to phagocytic apcs, demonstrating their functional plasticity [ ] . the phagocytic capacity of γδ t cells is augmented by opsonization [ , ] . combined, these studies show that subsets of γδ t cells in various species can be induced to present antigens via mhc class ii. clearly, γδ t cells have a unique role in innate immunity that is similar in some respects to that of monocytes and macrophages, and further is involved in the subsequent initiation of antigendependent acquired immunity. ligation of receptors expressed on the γδ t cell can lead to potent cytolytic responses vii. new and novel approaches for mucosal vaccine development against stressed, infected, and malignant cells [ À ], though γδ t cells can be permissive to growth of some tumors [ ] . ligation of tcr, in combination with other receptors such as nkg d and cytokine receptors such as the il- receptor, enhances and directs cytotoxic responses along with cytokine production [ , ] . cytotoxicity is a function of γδ t cells that is conserved across species [ À ]. for example, granzyme b, perforin, and fasl are expressed in wc γδ t cells from bovine peripheral blood mononuclear cells (pbmcs), with fasl expression increasing upon activation of these cells [ , ] . perforin expression is also found in bison γδ t cells [ ] . perforin and granzyme, fasl-fas, and the tnf-related apoptosis-inducing ligand pathway are also features of human γδ t cells [ , ] . the cytotoxic activity of γδ t cells likely plays an important role in multiple species for optimal immune responses by these cells to a subset of malignant and infected cells. another important functional response of γδ t cells is their regulation of the tissue environment through cytokine generation. these cytokines include those that drive inflammatory responses and contribute to downstream adaptive immune responses as well as cytokines that affect epithelial cell health and tissue homeostasis. although the number of cytokines produced by γδ t cells is large, a few, such as il- , ifnγ, and the tissue cytokines keratinocyte growth factor (kgf) and insulin-like growth factor (igf), are of particular importance in the function of subsets of these cells. il- and ifnγ are potent activators of cells of the myeloid lineage and contribute to downstream inflammatory responses. in mice, γδ t cells are a major source of innate il- early in response to infection [ , ] . two populations of γδ t cells contribute to the il- response. one is referred to as "natural" il- -producing cells, which acquire effector function prior to egress from the thymus [ ] . these cells are found in mucosal tissues and are thought to be early responders to infectious insult. another population is referred to as "induced," and these cells rapidly acquire effector function after egress from the thymus and in response to antigen and cytokine in the periphery [ ] . some reports suggest that although human and large animal γδ t cells produce il- (induced phenotype), they may not be a major early source of this cytokine in these species [ , ] . although they are clearly protective in most instances and are thought to be important to the early innate immune response, dysregulation of il- production leading to excessive il- can also be pathogenic [ ] . kgf and igf are also produced by tissue γδ t cells and are important in maintaining epithelial cell health and effective wound repair responses [ À ]. though defined as important in tissue homeostasis, these responses are also important for host defense, since health of the epithelial cell barrier contributes to protection against various pathogens and the creation of homeostatic environment for commensal microbiota. γδ t cells have been shown to respond to and participate in host defense responses in a variety of infectious diseases, including viral, bacterial, and parasite-induced disease, many at the mucosal surface [ , ] . recently, γδ t cells have been found to be important for protection against emerging viruses such as chikungunya and west nile virus [ , ] . in hiv infection, the peripheral subset of human γδ t (vδ ) cells is severely depleted and does not completely recover, even in patients who have had successful antiretroviral treatment. this deficit may increase the likelihood for secondary infections and could be a critical target for new immunotherapies for hiv patients [ ] . γδ t cells are clearly important in antibacterial immunity as a source of early ifnγ and il- [ , , , ] . as human γδ t cells are preprogrammed for recognition of bacterial phosphoantigens, they are particularly important in protection from mycobacterium and legionella infections [ , ] . human γδ t cells expand during salmonella enterica serovar typhimurium (st) infection of the intestinal mucosa [ ] and are a source of early ifnγ [ , ] . bovine γδ t cells also respond to oral st infection [ ] . γδ t cells also play a critical role in protection against infection with brucella sp., which are facultative intracellular bacteria [ ] . this appears to be primarily through production of ifnγ, and was found in mice, cattle, and sheep [ , ] . however, our results showed no contribution of mouse γδ t cells to infection with another emerging intracellular pathogen, coxiella burnetii (unpublished results). following mucosal infection but not peripheral infection, mouse γδ t cells were also found to have a role in downstream memory immune responses to listeria infection [ ] . thus, γδ t cells play an important role in response against many different bacterial infections. this suggests that their specific stimulation may contribute to protection and may potentially replace or at least reduce the need for antibiotics and could be considered as a new target for future vaccine development. γδ t cells also play protective roles in parasite infections. they respond to and are protective following initial infection with the malaria plasmodium falciparum, owing to recognition of phosphoantigens produced by the parasite. however, upon subsequent infection, the numbers of γδ t cells drop, similar to the situation with long-term hiv infection. nonetheless, higher numbers of functional vδ t cells are correlated with greater protection from reinfection with plasmodium and also increased symptoms upon infection, as they are sources of ifnγ and tnf-α [ ] . similarly, the first instance of bovine il- -producing cells was demonstrated and protects against a related parasite [ ] . indeed, in most instances of protection from pathogens, γδ t cells are similarly protective in humans and other animals [ ] . common features across species provide a rationale for the use of various animal models to test the role and importance of γδ t cells in disease settings of relevance to humans, which will lead to the creation of strategic platforms for γδ t-cell-targeted vaccine development. γδ t cells are characterized by a unique and specific tissue location, rapid response to external signals and insults, and the existence of preprogrammed and induced effector subsets. combined with the ability to expand these cells in vitro and their critical roles in a variety of infectious and cancerous disease settings, γδ t cells have been the target for new immunotherapeutics [ , À , , ] . in humans, both tcr and tlr agonists have been studied for their effects on enhancing γδ t cell function. prenyl phosphates and bisphosphonates that directly or indirectly drive expansion and cytokine production in a major subset of circulating γδ t cells have been pursued for treatment of certain tumors and infections [ ] . two approaches have been used. in the first approach, γδ t cells are expanded to large numbers in vitro and then adoptively transferred to patients. in the second approach, these agonists are given directly to the patient, inducing responses in vivo. the in vivo responses of γδ t cells to these agonists are impressive, leading to significant expansion in tissues, such as the lung and production of immune cytokines [ ] . of note, though originally pursued for cancer treatments, the potential application of phosphoantigen stimulation of γδ t cells in infectious disease was recently demonstrated in mycobacterium tuberculosis infection in primates [ ] . the application of these therapeutic approaches to stimulate γδ t cells is limited to humans and nonhuman primates, since γδ t cell responses to the prenyl phosphates are restricted to primate cells. other therapeutic approaches to increase γδ t cell activity have focused on other receptors, such as tlrs and scavenger receptors [ , ] . our recent endeavor has been to expand the number of materials that enhance the activity of γδ t cells in multiple species. this was achieved by screening various natural product libraries and other sources of natural products, including nutritional supplements. they were assessed for their capacity to upregulate il- receptor expression on primary γδ t cells, thereby enhancing responses to il- in the absence of antigen [ , , À ] . follow-up functional assays examined their cell type specificity, induced cytokine responses, and benefit in various infectious disease models [ , ] . two classes of plant products-polyphenols and polysaccharides-and one example of a microbial product that stimulate these cells, which came from these studies, are summarized below. a class of plant polyphenol called oligomeric procyanidins (opcs) produced by apples, grapes, and some other plants was determined to be a potent priming agent for γδ t cells. several studies suggest that ingestion of plant and berry compounds containing polyphenols expand human γδ t cells in vivo [ À ]. our study showed that opcs from apple peel prime human, mouse, and bovine γδ t cells, and nk cells in some instances [ ] , for enhanced responses to secondary signals provided by cytokines and antigens. other groups also found that opcs expand mouse γδ t cells in vivo [ ] and stimulate goat γδ t cells [ ] . opcmediated γδ t cell responses increase the expression of activation markers, but the cells do not actively proliferate in the absence of a secondary signal, such as cytokine or tcr engagement [ ] . opc treatment also induces production of a restricted number of cytokines, many of which act on myeloid cells, such as colony-stimulating factors (csfs) and chemokines such as il- , and various tissue growth factors [ ] . one of the consequences of opc treatment of bovine and human γδ t cells is a significant extension of the stability of csf and chemokine transcripts [ ] . the ability to extend the functional lifetime of these transcripts enables γδ t cells to more rapidly and robustly produce certain cytokines in response to secondary signals. importantly, opcs show bioactivity when ingested and are safe over a range of doses in all species tested [ , , ] . such supplements increase γδ t cells in the periphery or in tissues [ , ] . following oral delivery of very large doses of the opcs in mice, a significant reduction of inflammation was seen in dextran sulfate sodium (dss)induced colitis [ ] . the anti-inflammatory effects are independent of γδ t cells and require αβ t cells. interestingly, in the absence of αβ t cells, a rag-protein-dependent population of cells, likely γδ t cells, is responsible for a robust but noninflammatory cytokine response in opc treated mice in the dss model [ ] . consistent with this observation, opc ingestion in some mice was shown to induce increased levels of g-csf in circulation without obvious deleterious inflammation (unpublished results). induced g-csf is normally considered a proinflammatory response, but it can also contribute to protective immune support in certain instances. clearly, we have much to learn about the myriad effects of ingestion of opcs on γδ t cells and other immune cells (e.g., αβ t cells) in vivo. we expect that these potent plant chemicals (e.g., opc) and their derived products may be a safe novel immunotherapeutic and immunomodulator in some settings. our study has also identified unique polysaccharides from various plants that are potent agonists for γδ t cells and other cells of the immune system. the first source of polysaccharide agonist was funtumia elastica bark (yamoa). yamoa polysaccharides activate γδ t as well other immune cells, such as monocytes, and, when given in vivo, enhance protection from infection [ ] . optimal activation or priming of γδ t cells by these polysaccharides requires monocytes or macrophages in a mixed in vitro culture. following our initial characterization of the yamoa polysaccharides, similar activity was defined in extracts from other plants, including tansy (unpublished), juniper (unpublished), and, most recently, açai [ , , ] . many of the polysaccharide preparations being tested, except for those generated from açai, were positive in the limulus amebocyte lysate assay for lipopolysaccharides [ ] . açai polysaccharide responses are conserved in γδ t cells across species, including humans, cattle, and mice [ ] . monocytes and macrophages are also activated by the polysaccharides and are required for optimal responses by the γδ t cell. instillation of açai polysaccharides into the lungs of mice induces dose-dependent il- production, accumulation of myeloid cells, and activation of local dcs and macrophages [ ] . it was subsequently shown that prophylactic or therapeutic nasal administration of açai polysaccharides significantly enhances host innate defense responses against the intracellular bacterial pathogens francisella tularensis and burkholderia pseudomallei [ ] . protection could also be achieved following oral delivery, although responses were more variable. mechanism of action studies showed that açai polysaccharides enhance ifnγ expression by γδ t cells and nk cells following f. tularensis and b. pseudomallei infections. inhibition of ifnγ blocked the protective effect of the polysaccharides [ ] . thus, the stimulation of γδ t cells, as well as other innate immune cells, by açai or similar plant agonists and subsequent type t helper cell-associated responses, could have therapeutic applications in bacterial infections. since açai is a commonly ingested dietary supplement and has shown therapeutic benefit following oral delivery [ ] , we examined the effects of these agonists in two additional intestinal models. dysbiosis is a condition usually induced by antibiotic use in which the normal flora is disrupted. this state can lead to increased susceptibility to infection and colitis [ ] . mice with dysbiosis were treated with açai polysaccharides to assess whether these polysaccharides could aid in recovery from this susceptible state. when cytokine expression in mesenteric lymph nodes (mlns) and spleen cells were measured, the feeding of açai polysaccharides induced expression of il- in supernatant fluids from cultured mln and spleen cells from the treated mice. il- was also detected in the serum of the mice [ ] . expression of ifnγ was also increased in spleen cells from açai polysaccharide-fed mice, similar to the previous finding using nasal administration [ ] . no adverse effects were noted in the açaitreated mice. in a model of chemically induced colitis, mice that were fed açai had a reduced deleterious inflammatory response in the gut [ ] . considering that there are no adverse effects following açai ingestion, this polysaccharide could represent a safe and novel approach to stimulating γδ t cells and other innate cells, potentially to promote their innate protective and homeostatic functions at the mucosal surface. our next study aimed to examine potential receptors involved in the sensing and responses to the açai polysaccharides by immune cells. some of responses were lost in mice lacking functional tlr or the innate adaptor protein myd . however, neutrophils were still recruited into the peritoneum of these mice following intraperitoneal injection of açai [ ] . the role of the β-glucan receptor dectin- was particularly investigated, since il- is produced by immune cells following ingestion of β-glucans [ ] . our result demonstrated that açai polysaccharides contain appropriate linkages for recognition by dectin- using an inhibition elisa against β-glucan [ ] . furthermore, açai polysaccharides specifically block binding of anti-dectin- antibodies to immune cells in a flow cytometry based assay. thus, açai polysaccharides bind to multiple innate immune cell receptors, contributing to unique effects of innate and likely downstream adaptive immune responses. açai polysaccharides can be considered as a new mucosal immunomodulator molecule for the regulation of antigen-specific immune response and inflammation. activation-based screening assays resulted in the detection of robust agonist activity for γδ t cells in multiple microbial extracts (unpublished results). one such agonist was determined to be amphotericin b (amb), produced by streptomyces nodosus. amb is a commonly used antifungal drug that has previously been shown to stimulate innate immune cells [ À ] . amb induces expression of cytokines in macrophages, mediated by tlr recognition [ À ] . amb treatment of bovine pbmcs leads to increased expression of il- r selectively on γδ t cells, activation of bovine monocytes and nk cells, and enhanced ifnγ from nk cells [ ] . addition of il- to these cultures induces a robust, antigen-independent proliferation of the treated γδ t cells [ ] . the agonist activity of amb is not restricted to cattle, in that similar effects are seen on expression of activation markers and proliferation of γδ t cells in humans and mice as well [ ] . thus the response is highly conserved. in a separate study, amb was shown to increase ifnγ production in mouse lung cells following in vitro infection and costimulation by avirulent c. burnetii bacteria [ ] . amb also enhances antibody responses against ovalbumin when used as an immunizing adjuvant [ ] . thus amb has potential both to enhance innate and acquired responses to infection and to function as a vaccine adjuvant. since bovine γδ t cells and nk cells respond to amb at very low, nontoxic doses, our next experiment aimed to test it in an in vivo model of infectious enterocolitis. calves were given one intravenous injection of approximately . À . mg/kg amb or saline hours prior to st infection by the oral route. ambtreated calves had lower fevers, had overall reduced morbidity, and shed less bacteria into the environment in comparison to control calves [ ] . thus amb protected from disease severity and reduced the level of shed bacteria. the result suggested that amb could be used as a potent immunomodulatory molecule to enhance disease resistance against st in calves. our efforts are continuing to assess the immune protective effects of amb on very young calves, which are highly prone to infection. when bovine calves are less than a weekold, they have a variable colostrum status, and they experience a broad spectrum of natural scouring and respiratory maladies in their first week to months of life. these symptoms are typically caused by rotavirus, coronavirus, cryptosporidium, or a combination of virus and parasite infections. regardless of the cause, the calves are treated with a hydration therapy. if signs of a secondary bacterial infection become apparent, antibiotics are administered. the calves were likely preexposed to a variety of pathogens; this would explain the early disease that occurs when they are housed indoors in clean facilities. with years of data on these occurrences of natural illness in our facilities, our study was directed to test whether early minimal treatments with amb could potentially be used as a broad-spectrum prophylactic immunomodulator. a dose of . mg/kg injected intravenously as previously described [ ] was used in the study. this is approximately -fold less than the doses given to patients for antifungal treatment and was determined to be nontoxic in calves. there were two treated groups (n per group). one group received a single injection of amb on the day of arrival at our facility (amb x ). a second group received this initial dose on the day of their arrival and a second dose after days (amb x ). thus, for the first days, there were calves treated with one dose of amb. health condition was assessed by evaluating each animal's subjective appearance and attitude, appetite, temperature, pulse and respirations, fecal consistency, and treatments on a scale of À . health condition was assessed for all calves twice daily and was compared to calves acquired in the same months in a -year span before and after this experiment that did not receive any treatment. in a given period, the study tallied the number of days the calves had perfect health scores (scores of ). the calves that received one injection of amb had improved health assessments in their first days in comparison to calves that received no treatment (fig. . a) . the period was then extended to the first days. in this case, the untreated calves were compared to the amb x and amb x groups. whereas one dose of amb appeared to benefit in the short term (in the first days), the amb x treatment had no lasting effect. in contrast, calves treated with amb x had longer-lasting positive benefit (fig. . b) . these data suggest that minimal early doses of an innate immune stimulant could benefit the health of livestock for extended periods. this is especially important for cattle that are subject to repeated infections early in life. it also provides proof of principle that broad-spectrum because of their position in the body and their capacity for varied, appropriate responses depending on the environmental signals, γδ t cells are an optimal target for novel immunotherapeutic and vaccine development. some tcr and tlr agonists that can stimulate γδ t cells have already been used extensively for new cancer treatments. ample data suggest that the cells might also be specifically stimulated to protect from infectious and inflammatory disease. considering the growing concerns about the use and overuse of antibiotics, it is critical that such novel approaches to counter infectious agents be pursued. γδ t cells in homeostasis and host defence of epithelial barrier tissues γδ•t cells: first line of defense and beyond antigen recognition by γδ t cells determining γδ versus αβ t cell development comparative biology of γδ t cell function in humans, mice, and domestic animals response of γδ t cells to plant-derived tannins zú ñ iga-pflü cker jc. enforcement of γδ-lineage commitment by the pre t-cell receptor in precursors with weak γδ-tcr signals five layers of receptor signaling in γδ t-cell differentiation and activation human γδ t-cell subsets and their involvement in tumor immunity γδ t cells in cancer immunotherapy γδ t cells in cancer the emerging protumor role of γδ t lymphocytes: implications for cancer immunotherapy regulatory and effector functions of gamma delta (γδ) t cells and their therapeutic potential in adoptive cellular therapy for cancer complex role of γδ t-cellderived cytokines and growth factors in cancer γδ t cell effector functions: a blend of innate programming and acquired plasticity antigen independent priming: a transitional response of bovine γδ t cells to infection gammadelta t cells: an alternative type of professional apc modulation of γδ t cell responses by tlr ligands insights into the relationship between toll like receptors and gamma delta t cell responses the natural and the inducible: interleukin (il)- -producing γδ t cells il- γδ t cells as kick-starters of inflammation differential responsiveness of innate-like il- and ifn-γ-producing γδ t cells to homeostatic cytokines il- -producing γδ t cells switch migratory patterns between resting and activated states functions of skin-resident γδ t cells homing and function of human skin γδ t cells and nk cells: relevance for tumor surveillance γδ t cells exhibit multifunctional and protective memory in intestinal tissues in vitro expansion of gamma delta t cells with anti-myeloma cell activity by phosphostim and il- in patients with multiple myeloma key features of gamma-delta t-cell subsets in human diseases and their immunotherapeutic implications prospects for chimeric antigen receptor (car) γδ t cells: a potential game changer for adoptive t cell cancer immunotherapy activation of nk cells and t cells by nkg d, a receptor for stress-inducible mica activation of v gamma v delta t cells by nkg d cutting edge: adaptive versus innate receptor signals selectively control the pool sizes of murine ifn-γ or il- producing γδ t cells upon infection activating and propagating polyclonal gamma delta t cells with broad specificity for malignancies interleukin- -producing γδ t cells selectively expand in response to pathogen products and environmental signals aryl hydrocarbon receptor is critical for homeostasis of invariant γδ t cells in the murine epidermis fungal [beta]-glucans and mammalian immunity pattern recognition receptors: doubling up for the innate immune response γδ t cells respond directly to pathogen associated molecular patterns lta recognition by bovine {gamma}{delta} t cells involves cd scavenger receptor cysteine-rich domains and of wc are receptors for the wc counter receptor the distinct response of γδ t cells to the nod agonist, muramyl dipeptide the evolution of vertebrate antigen receptors: a phylogenetic approach characterization of γδ t cells from zebrafish provides insights into their important role in adaptive humoral immunity differential mrna expression in circulating γδ t lymphocyte subsets defines unique tissue-specific functions serial analysis of gene expression in vii. new and novel approaches for mucosal vaccine development references circulating γδ t cell subsets defines distinct immunoregulatory phenotypes and unexpected gene expression profiles transcriptional profiling of gamma delta t cells blimp- : trigger for differentiation of myeloid lineage haplotype mapping and sequence analysis of the mouse nramp gene predict susceptibility to infection with intracellular parasites the ity/lsh/bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the nramp gene expression of the human nramp gene in professional primary phagocytes: studies in blood cells and in hl- promyelocytic leukemia nramp- expression modulates protein-tyrosine phosphatase activity in macrophages: impact on host cell signalling and functions slc a is expressed by innate lymphocytes and augments their activation human γδ t cells: a lymphoid lineage cell capable of professional phagocytosis comparative gene expression by wc {gamma}{delta} and cd {alpha}{beta} t lymphocytes, which respond to anaplasma marginale, demonstrates higher expression of chemokines and other myeloid cell-associated genes by wc {gamma}{delta} t cells differential expression of bovine mhc class ii antigens identified by monoclonal antibodies gammadelta t cells present antigen to cd alphabeta t cells porcine γδ t cells: possible roles on the innate and adaptive immune responses following virus infection rapid and transient activation of γδ t cells to ifn-γ production, nk celllike killing, and antigen processing during acute virus infection a subpopulation of circulating porcine γδ t cells can act as professional antigen presenting cells professional antigen-presentation function by human gammadelta t cells mouse γδ t cells are capable of expressing mhc class ii molecules, and of functioning as antigen-presenting cells coli promotes human vγ vδ t cell transition from cytokine-producing bactericidal effectors to professional phagocytic killers in a tcrdependent manner proliferative and cytolytic responses of human gamma delta t cells display a distinct specificity pattern inhibitory mhc class i receptors on gammadelta t cells in tumour immunity and autoimmunity defining the nature of human γδ t cells: a biographical sketch of the highly empathetic six-of-the-best: unique contributions of [gamma][delta] t cells to immunology il- r and tcr signaling drives the generation of neonatal vgamma vdelta t cells expressing high levels of cytotoxic mediators and producing ifn-gamma and il- activated γδ t cells express the natural cytotoxicity receptor natural killer p and show cytotoxic activity against myeloma cells sheep perforin: identification and expression by gammadelta t cells from pregnant sheep uterine epithelium acquisition of non-mhc restricted cytotoxic function by il activated thymocytes with an "immature" antigenic phenotype a murine thymocyte clone expressing γδ t cell receptor mediates natural killer-like cytolytic function and th -like lymphokine production functional and phenotypic characterization of wc gamma/delta t cells isolated from babesia bovis-stimulated t cell lines gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus role of chicken il- on gammadelta t-cells and eimeria acervulina-induced changes in intestinal il- mrna expression and gammadelta t-cells comparison of gene expression by cocultured wc γδ and cd αβ t cells exhibiting a recall response to bacterial antigen wc γδ t cells indirectly regulate chemokine production during mycobacterium bovis infection in scid-bo mice cd /perforin /wc γδ t cells, not cd αβ t cells, infiltrate vasculitis lesions of american bison (bison bison) with experimental sheep-associated malignant catarrhal fever synovial t cells by borrelia-reactive fas-ligand high γδ t cells in lyme sensitization of human osteosarcoma cells to vγ vδ t-cell-mediated cytotoxicity by zoledronate il- production is dominated by {gamma}{delta} t cells rather than cd t cells during mycobacterium tuberculosis infection resident v{delta} {gamma}{delta} t cells control early infiltration of neutrophils after escherichia coli infection via il- production identification of cd γδ t cells as fetal thymus-derived naturally occurring il- producers γδ t cells recognize a microbial encoded b cell antigen to initiate a rapid antigen-specific interleukin- response modulation of inflammation through il- production by γδ t cells: mandatory in the mouse, dispensable in humans? high fat diet exacerbates murine psoriatic dermatitis by increasing the number of il- -producing γδ t cells protection of the intestinal mucosa by intraepithelial gamma delta t cells a role for skin gammadelta t cells in wound repair il- enhances activation and igf- production of dendritic epidermal t cells to promote wound healing in diabetic mice dendritic epidermal t cells regulate skin homeostasis through local production of insulin-like growth factor γδ-t cells: an unpolished sword in human anti-infection immunity gamma-delta t cells play a protective role in chikungunya virus-induced disease role of natural killer and gammadelta t cells in west nile virus infection γδ t cells in hiv disease: past, present, and future gamma interferon production by bovine gamma delta t cells following stimulation with mycobacterial mycolylarabinogalactan peptidoglycan escherichia coli produces phosphoantigens activating human gamma delta t cells comparative gamma delta t cell immunology: a focus on mycobacterial disease in cattle role of innate t cells in antibacterial immunity predominant activation and expansion of v gamma -bearing γδ t cells in vivo as well as in vitro in salmonella infection th and th -inducing cytokines in salmonella infection early interferon-γ production in human lymphocyte subsets in response to nontyphoidal salmonella demonstrates inherent capacity in innate cells mucosal lymphaticderived γδ t cells respond early to experimental salmonella enterocolits by increasing expression of il- rα murine and bovine γδ t cells enhance innate immunity against brucella abortus infections acute infection by conjunctival route with brucella melitensis induces igg cells and ifn-γ producing cells in peripheral and mucosal lymph nodes in sheep vδ t cell response to malaria correlates with protection from infection but is attenuated with repeated exposure two distinct populations of bovine il- t-cells can be induced and wc il- γδ t-cells are effective killers of protozoan parasites phosphoantigen/il expansion and differentiation of vγ vδ t cells increase resistance to tuberculosis in nonhuman primates adoptive transfer of phosphoantigen-specific γδ t cell subset attenuates mycobacterium tuberculosis infection in nonhuman primates select plant tannins induce il- rα up-regulation and augment cell division in γδ t cells polysaccharides derived from yamoat (funtumia elastica) affect innate immunity in part by priming γδ t cells polysaccharides isolated from acai fruit induce innate immune responses amphotericin b stimulates γδ t and nk cells, and enhances protection from salmonella infection immunity and antioxidant capacity in humans is enhanced by consumption of a dried, encapsulated fruit and vegetable juice concentrate bioactive food components that enhance {gamma}{delta} t cell function may play a role in cancer prevention consumption of cranberry polyphenols enhances human gammadelta-t cell proliferation and reduces the number of symptoms associated with colds and influenza: a randomized, placebocontrolled intervention study dietary unripe apple polyphenol inhibits the development of food allergies in murine models condensed tannins from botswanan forage plants are effective priming agents of γδ t cells in ruminants contribution of transcript stability to a conserved procyanidin-induced cytokine response in gammadelta t cells intestinal immune system of young rats influenced by cocoa-enriched diet grape consumption supports immunity in animals and humans apple polyphenols require t cells to ameliorate dextran sulfate sodium-induced colitis and dampen proinflammatory cytokine expression immunomodulatory activity of acidic polysaccharides isolated from tanacetum vulgare l botanical polysaccharides: macrophage immunomodulation and therapeutic potential methods of endotoxin removal from biological preparations: a review nasal acai polysaccharides potentiate innate immunity to protect against pulmonary francisella tularensis and burkholderia pseudomallei infections a mouse model of clostridium difficile associated disease adjuvant materials that enhance bovine γδ t cell responses )-β-d-glucans from oat activate nuclear factor-κb in intestinal leukocytes and enterocytes from mice amphotericin b alters the affinity and functional activity of the oligopeptide chemotactic factor receptor on human polymorphonuclear leukocytes effect of amphotericin b on natural killer cell activity in vitro the relationship between adjuvant and mitogenic effects of amphotericin methyl ester pharmacologic modulation of interleukin- expression by amphotericin b-stimulated human mononuclear cells infusion-related toxicity of three different amphotericin b formulations and its relation to cytokine plasma levels amphotericin b activation of human genes encoding for cytokines we acknowledge support from the agriculture and key: cord- -g rmzsv authors: wynn, james l.; wong, hector r. title: pathophysiology of neonatal sepsis date: - - journal: fetal and neonatal physiology doi: . /b - - - - . - sha: doc_id: cord_uid: g rmzsv nan a successful immune response is critically necessary to eradicate infectious challenges and prevent dissemination of the infection in the host. however, if inflammation is not limited and becomes generalized, it can result in the constellation of signs and symptoms of a systemic inflammatory response syndrome (sirs). if the infection is not contained, the spread of the pathogen from its local origin through the blood may result in systemic endothelial activation and precipitate sepsis, severe sepsis, and septic shock. progression of sepsis to shock may lead to multiple organ dysfunction syndrome (mods) and ultimately death. host immunity is divided into innate and adaptive immune systems for purposes of discussion and teaching but there is a great deal of interaction between the two systems. innate immunity is rapid, largely nonspecific, and composed of barriers, phagocytic cells, the complement system, and other soluble components of inflammation. after breech of a barrier, cellular elements of the innate immune response are the first line of defense against the development and progression of infection. adaptive immunity, which is antigen specific, is long lived, and often takes several days to develop, provides immunologic specificity and memory. these systems work together to protect the host from pathogenic challenge but may also precipitate host injury through aberrant responses. the outcome of infection is dependent on at least four major factors: ( ) the pathogen, ( ) the pathogen load, ( ) the site of infection, and ( ) the host response. less is known about the host response in neonates compared with adults for a number of reasons, the principal one being a highly variable definition of disease. our understanding of the pathophysiology of sepsis is largely from investigations in adult populations, including both humans and animals. there is clear evidence from both preclinical models of sepsis and humans that neonates manifest different host immune responses as compared with adults. [ ] [ ] [ ] [ ] even in comparison with children, neonates manifest a unique host immune response to septic shock. thus neonatal-specific clinical investigations, particularly in very preterm infants, are required to improve both survival and long-term outcomes for these populations. a better understanding of the pathophysiology will uncover new opportunities for interventional studies ultimately aimed at improving outcomes. to this end, in this chapter we explore the pathophysiology of sepsis in the neonate, with special attention paid to the immunobiology of sepsis. adult and pediatric intensivists currently use generally accepted definitions for sepsis for goal-based therapeutic interventions. [ ] [ ] [ ] [ ] these definitions are critical to facilitate epidemiologic studies, to accurately determine disease prevalence, to select patients for clinical trials, and ultimately to improve the delivery of care. the generally accepted pediatric definition for sepsis, established in , was intended for all children (< years old), including term neonates (≥ weeks' completed gestation). preterm neonates (< weeks' completed gestation) were specifically excluded from the pediatric generally accepted definitions, and neonatal-perinatal subspecialists were not represented among the pediatric consensus experts. to investigate whether the pediatric generally accepted definitions for sirs and sepsis applied to term infants, hofer and colleagues retrospectively examined term neonates and found that the generally accepted definitions applied to only % of cases of culturepositive early-onset sepsis. neonatal sepsis has been inconsistently defined on the basis of a variety of clinical and laboratory criteria, which makes the study of this condition very difficult. diagnostic challenges and uncertain disease epidemiology necessarily result from a variable definition of disease. the lack of a generally accepted definition for neonatal sepsis remains a significant hindrance towards improving outcomes and accurately describing disease pathophysiology. thus working definitions for the sepsis continuum, specific for preterm and term neonates, are needed to provide a uniform basis for clinicians and researchers to study and diagnose severe sepsis. the addition of immune biomarker-based staging of disease to clinical sign staging is highly likely to increase the accuracy of patient classification for future multicenter clinical trials that will test novel interventions. sepsis or serious infection within the first weeks of life kills more than million newborns globally every year. , the incidence of neonatal sepsis is variable (from less than % to more than % of live births) on the basis of gestational age and time of onset (early-onset sepsis [< hours after birth] or late-onset sepsis [≥ hours after birth]). [ ] [ ] [ ] [ ] [ ] [ ] [ ] preterm neonates have the greatest sepsis incidence and mortality rates among all agegroups [ ] [ ] [ ] [ ] [ ] [ ] (figure - ) . risk factors for developing sepsis in neonates, particularly the very premature, have been well described. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] prematurity, low birth weight (especially infants weighing less than , g), male sex, a maternal vaginal culture positive for group b streptococcus (gbs), prolonged rupture of membranes, maternal intrapartum fever, and chorioamnionitis are strongly associated with an increased risk for early-onset sepsis. chorioamnionitis is associated with the greatest risk for subsequent clinical or culture-proven sepsis. recent studies demonstrate the risk for sepsis in newborn infants born to women with clinical chorioamnionitis is strongly dependent on gestational age, with minimal risk in neonates aged weeks or older and greater risk with increasing degrees of prematurity. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the risk for neonatal sepsis conferred by maternal gbs colonization is significantly reduced with adequate intrapartum antibiotic prophylaxis. section xxvi pathophysiology of neonatal diseases ated with sepsis-like syndromes (e.g., echovirus, enterovirus, parechovirus, coxsackie virus, adenovirus, parainfluenza virus, rhinovirus, and coronavirus). , [ ] [ ] [ ] physical barriers, including skin and mucosal surfaces, are the first point of contact between the host and potential pathogens. thus a successful immune defense in addition to epithelial barrier function is critical to prevent the development of local infection. multiple immune elements are present to prevent attachment and propagation of pathogens while simultaneously permitting the presence of commensal organisms required for homeostasis. vernix enhances skin barrier function in late-preterm and term neonates. vernix is a complex material comprising water ( . %), lipids ( . %), and proteins ( . %) produced by fetal sebaceous glands during the last trimester and is largely absent in preterm neonates born before weeks' gestation. vernix provides a barrier to water loss, improves temperature control, and serves as a shield containing antioxidants and innate immune factors such as antimicrobial proteins and peptides (apps). the apps on the surface of the newborn's skin (and replete in the amniotic fluid [ ] [ ] [ ] ) are capable of killing/inactivating common neonatal pathogens, including gbs, e. coli, and candida species. erythema toxicum is an immune-mediated manifestation that results from bacterial colonization of the skin occurring shortly after birth. , this common cutaneous immune response is less common in preterm infants than in term infants, highlighting the impact of developmental age on host immune capabilities. in contrast to the moist mucosal surfaces of the respiratory and gastrointestinal (gi) tracts, the skin is arid, which further reduces the chances for microbial invasion. the outermost layer of the skin, the stratum corneum, prevents microbial invasion, maintains temperature, and reduces the risk for dehydration through prevention of transcutaneous water loss. the immature and incompletely developed stratum corneum of preterm newborns takes at least to weeks after birth to become fully functional and may take up to weeks in the extremely preterm neonate, significantly increasing the risk for barrier dysfunction. disruption of the cutaneous barrier by trauma (e.g., placement of an intravenous catheter or heel stick) or chemical burn allows microorganisms to enter the subcutaneous tissue, increasing the likelihood of their establishing a local infection ( figure - ). the likelihood of a microbial breach of the cutaneous barrier rises in the presence of intravenous catheters, which are essential for critical care. emollients, aimed at enhancing the barrier function of preterm newborn skin, increase the risk for nosocomial infection and their use is not recommended. mucosal barriers contain multiple components that serve to prevent infection, including acidic ph, mucus, cilia, proteolytic enzymes, apps, opsonins such as surfactant proteins, sentinel immune cells such as macrophages, dendritic cells, polymorphonuclear neutrophils (pmns), and t cells, as well as commensal organisms. like the skin, the gi mucosa is quickly colonized after birth and contains a significant repository of microorganisms. [ ] [ ] [ ] gi barrier integrity, paramount for prevention of spread of microorganisms out of the intestinal compartment, is dependent on the interaction between commensal organisms and host epithelium. interleukin (il)- , produced by type intestinal innate lymphoid cells in the presence of the microbiota, drives granulocytosis and may protect the neonatal host from infectious challenge. a loss of intestinal barrier integrity likely plays a role in the development of necrotizing enterocolitis (nec) and late-onset sepsis. , prolonged antibiotic treatment, hypoxia, and remote infection are factors known to despite the efficacy of this intervention, the incidence of invasive gbs disease in african american neonates is still more than twice that in white babies, and the incidence of escherichia coli sepsis may be rising in very-low-birth-weight (vlbw) neonates. vaginal delivery in the presence of maternal active primary herpes simplex virus significantly increases the risk for a neonatal herpes simplex virus infection, which has a fulminant course and high mortality. [ ] [ ] [ ] preexisting maternal immunodeficiency or sepsis also increases the risk for sepsis in the neonate. in addition, care practices after birth, such as intubation, mechanical ventilation, and placement of central venous lines, increase the risk for the development of sepsis. a number of pathogens have been associated with sepsis in the neonatal period. the predominant cause is bacterial; however, certain viral infections are associated with a fulminant course and significant mortality. [ ] [ ] [ ] in a large (n = , ), multicenter study of vlbw infants (< g), gram-positive organisms accounted for % of pathogens causing early-onset sepsis and % of those causing late-onset sepsis. in contrast, gram-negative organisms were responsible for % of early-onset sepsis and % of late-onset sepsis. candida species accounted for % of cases of early-onset sepsis and % of cases of late-onset sepsis. infection by gram-negative organisms, particularly pseudomonas species, carries a higher risk for fulminant course and death than infection by other pathogen groups. , , , [ ] [ ] [ ] gram-positive causes of sepsis are dominated by gbs and coagulase-negative staphylococci (cons). , although the high mortality rate for gbs has been well described (especially among infants born prematurely), mortality rates associated with cons are significantly lower. , fungi may also be associated with fulminant neonatal sepsis and predominantly affect vlbw infants. , , independent predictors of in-hospital neonatal mortality after late-onset sepsis were pseudomonas infection (adjusted odds ratio [or], . ; % confidence interval [ci], . % to . %) and fungemia (or, . ; % ci, . % to . %). the limited sensitivity of current methods to identify causative organisms is partially due to an inability to take a large sample of blood from newborn infants with suspected sepsis. blood culture-negative ("clinical") sepsis is estimated to occur at a nearly -fold greater rate than blood culture-positive sepsis. in some of these infants, sepsis may also be due to novel viral pathogens associ- y e a r s - y e a r s - y e a r s - y e a r s - y e a r s - y e a r s - y e a r s the production of mucus and mucociliary clearance of pathogens and debris. premature neonates have relatively more goblet cells than do maturer neonates, leading to a decrease in mucociliary clearance. respiratory mucosal function can be impaired by surfactant and saliva deficiency, altered mucus production, and mechanical ventilation. ventilation is associated with decreased mucociliary clearance, airway irritation, and parenchymal lung injury (see figure - ). intubation is also associated with the progressive accumulation of colonizing bacteria and bacterial endotoxin in respiratory fluids, with concomitant mobilization of endotoxin-modulating apps to the airway. neonates with surfactant deficiency lack apps such as surfactant proteins a and d, which are also absent in commercially available surfactant preparations. there is an age-dependent maturation in the ability of respiratory epithelium to elaborate apps (cathelicidin and β-defensins), such that the respiratory epithelium of preterm newborns mounts a deficient app response. disrupt or injure the neonatal intestinal barrier (see figure - ). [ ] [ ] [ ] under these circumstances, the gut may become the motor of systemic inflammation. mechanistically, paneth cells and intestinal lymphoid cells may release excessive amounts of il- , which, in turn, plays a critical role in the development of sirs. many interventions aimed at reducing the frequency of sepsis in neonates via enhancement of mucosal barrier integrity have been evaluated. neither probiotics nor glutamine supplementation has reduced the incidence of neonatal sepsis. in contrast, human milk feeding is associated with a reduction in the risk for sepsis and nec , and is strongly encouraged, especially in preterm infants. respiratory mucosa is defended in utero by amniotic fluid and pulmonary apps, surfactant proteins a and d, alveolar macrophages, and pmns, among other immune elements. the surface and submucosal gland epithelium of the conducting airways is a constitutive primary participant in innate immunity through c the cell surface and in endosomes, whereas rlrs and nlrs detect pathogens only intracellularly. the discovery that tlr was integral for a robust lipopolysaccharide (lps)-mediated inflammatory response after gram-negative sepsis may be why tlrs have been more thoroughly investigated in the setting of sepsis than other prrs. each of the known tlrs in humans, present on and within multiple cell types, recognizes extracellular and intracellular pathogens via specific pamps. , multiple tlrs may be activated in concert by intact or partial microorganisms and in turn activate multiple second-messenger pathways simultaneously. , lps is the prototypic mediator of systemic inflammation and generates many of the clinical findings of sepsis and septic shock, including mods and death. lps signals through tlr in conjunction with the adaptor proteins cd and myeloid differentiation factor . in adults a reduction in mortality and improvement in hemodynamics were demonstrated when the level of serum lps was reduced. the level of lps is elevated in blood from infected neonates and those with nec even in the absence of gram-negative bacteremia. high levels of circulating endotoxin found during sepsis and nec are associated with multiorgan failure, thrombocytopenia, neutropenia, and death. administration of anti-lps antibodies to a small number of these deficiencies as well as those related to cellular function in combination with invasive procedures lead to a reduction in respiratory barrier function that increases the risk for sepsis. once the local barrier function has been compromised, pathogen recognition by local immune sentinel cells is the first step towards the development of an immune response (figure - ) . elegant sensing mechanisms have evolved to facilitate detection of potentially pathogenic microorganisms. multiple classes of pathogen recognition receptors (prrs) have been discovered that serve as detectors of pathogen-associated molecular patterns (pamps), including cell wall and membrane components, flagellum, nucleic acids, and carbohydrates. a litany of prr classes have been discovered, including the toll-like receptors (tlrs), nod-like receptors (nlrs), retinoic acid-inducible protein i like receptors (rlrs), peptidoglycan recognition proteins, β -integrins, and c-type lectin receptors. the tlrs, β integrins, and c-type lectin receptors detect pathogens both on neonates with sepsis (n = ) with serum endotoxin present reduced the time to recovery but not mortality as compared with the values in placebo-treated neonates. reduction of serum lps levels by exchange transfusion in infected neonates (n = ) was associated with improved survival. bacterial cell wall components (such as lipoteichoic acid) signal primarily through tlr , tlr , and tlr , flagellin signals through tlr , and cpg double-stranded dna signals through tlr . common viral pamps such as double-stranded rna or single-stranded rna signal through tlr , and tlr and tlr , respectively. agonist-tlr binding results in a signaling cascade of intracellular second-messenger proteins ultimately leading to production of cytokines and chemokines, as well as activation of other antimicrobial effector mechanisms. signaling through tlrs typically leads to the production of nuclear factor κb (nf-κb)-dependent inflammatory cytokines and chemokines, whereas signaling through toll/il- receptor-domain-containing adapter inducing interferon (ifn)-β (trif) induces production of type i ifns, as well as nf-κb-related inflammatory cytokines. in neonates of all gestational ages, up-regulation of tlr and tlr messenger rna (mrna) occurs during gram-positive and gram-negative infection, respectively. dysregulation or overexpression of tlr is involved in the development of nec in experimental animal models, implicating the importance of tlrs in the initial immune response to pathogens and their role in neonatal sepsis. other important intracellular prrs include nlrs and rlrs. for nlrs, multiple cytosolic proteins are able to act as pamp sensors (e.g., nlrp , nlrp , and nlrc ) and coalesce with adaptor proteins and procaspase to form a multimeric protein complex termed the inflammasome. the formation of the inflammasome results in the conversion of procaspase to active caspase , which cleaves the inactive precursor proteins il- β and il- to their active forms. rlrs are cytoplasmic rna helicases that, like tlr , sense double-stranded rna of viral origin and induce type i ifn production and nf-κb activation. to date, the impact of rlr and nlr signaling has not been specifically examined in neonates with sepsis. in addition to its roles in leukocyte function (adhesion, phagocytosis, migration, and activation) and complement binding, complement receptor (cr , also known as mac- and cd b-cd ) functions as a pathogen sensor on the surface of phagocytes. cr binds lps, as well as a broad range of other microbial products, in cooperation with or independently of cd , leading to up-regulation of inducible nitric oxide (no) synthase and no production. diminished expression of l-selectin and cr on stimulated neonatal pmns impairs activation and accumulation at sites of inflammation. , , decreased expression of l-selectin and cr persists for at least the first month of life in term infants, possibly contributing to an increased risk for infection. the expression of cr (cd b) may be reduced further in preterm neonates as compared with term neonates. in umbilical cord blood from neonates of less than weeks' gestation, pmn cr content was similar to levels found in patients with type leukocyte adhesion deficiency (failure to express cd ). , thus decreased leukocyte cr surface expression increases the likelihood of suboptimal pathogen detection and cellular activation, particularly in the preterm neonate. c-type lectin receptors are prrs that recognize bacterial, viral, fungal, and parasitic carbohydrate moieties. c-type lectin receptors may be expressed on the cell surface (e.g., macrophage mannose receptor, mincle receptor, dectin , and dectin ) or secreted as soluble proteins (e.g., mannose-binding lectin [mbl], (which is also named mannan-binding protein or mannanbinding lectin) as one of the acute-phase reactants. once bound to its carbohydrate ligand, mbl initiates activation of complement via the lectin pathway to promote opsonization and phagocytic clearance of pathogens. plasma mbl concentrations are low at birth (especially in preterm infants) but rise steadily throughout infancy and childhood. low levels of mbl are associated with the increased incidence of sepsis in neonates. [ ] [ ] [ ] in addition to decreased concentrations at birth, certain genetic polymorphisms of mbl (namely, mbl ), have been associated with an increased risk for infection in some, but not all, studies. - m-ficolin activates the complement system in a manner similar to mbl and its level is elevated in neonates with sepsis. prr stimulation results in rapid inflammatory mediator transcription and translation directed at cellular activation and clearance of pathogenic organisms (see figure - ). during sepsis and septic shock, multiple proinflammatory cytokines have been identified, including il- β, il- , il- (cxcl ), il- , il- , ifn-γ, and tumor necrosis factor (tnf)-α. compared with adults with sepsis, neonates with sepsis produce less il- β, tnf-α, ifnγ, and il- . [ ] [ ] [ ] [ ] [ ] [ ] the decreased cytokine production is due in part to decreased production of important intracellular mediators of tlr signaling, including myeloid differentiation factor , ifn regulatory factor , and p , which exhibit gestational age-specific decrements. recent studies have demonstrated impaired inflammasome activation and mature il- β production by neonatal mononuclear cells. , in a comprehensive study (> analytes) of serum from neonates evaluated for late-onset sepsis, il- emerged as a predictive biomarker to differentiate infected neonates from uninfected neonates. il- reduces pmn apoptosis, drives ifn-γ production, and induces production of tnf-α, il- β, and cxcl . il- primes pmns for degranulation with production of reactive oxygen intermediates on subsequent stimulation. dysregulation of many of these functions linked to il- are seen in sepsis and septic shock. increased il- levels have been demonstrated in premature neonates with brain injury and also in an experimental model of nec, [ ] [ ] [ ] highlighting activation pathways common with those in ischemia and inflammation. excessive levels of il- β, tnf-α, il- , cxcl , il- , and il- , such as those seen with advanced-stage nec, severe sepsis, or septic shock, correlate with poor survival. , [ ] [ ] [ ] [ ] altered cytokine levels (increased il- and il- levels and decreased ccl levels) may identify those neonates at highest risk for the development of sepsisassociated disseminated intravascular coagulation (dic). proinflammatory cytokine production leads to activation of endothelial cells, including increased expression of cell adhesion molecules that facilitate leukocyte recruitment and diapedesis ( figure - ) . up-regulation of cell adhesion molecules (soluble intercellular adhesion molecule, vascular cell adhesion molecule, l-selectin, p-selectin, e-selectins, and cd b-cd ) during sepsis facilitates rolling and extravascular migration of leukocytes. [ ] [ ] [ ] [ ] decreased production of l-selectin and expression of c in pmns and monocytes derived from neonates may impair accumulation at sites of inflammation. , chemokine gradients produced by endothelial cells and local macrophages are necessary for effective and specific leukocyte attraction and accumulation (see figure - ). without adequate leukocyte recruitment, there is increased risk for propagation from a localized to a systemic infection. although poor cellular chemotaxis in the neonate has been observed, it is not likely a result of reduced serum concentrations of chemokines as baseline levels are similar in preterm and term neonates as compared with adults. suboptimal cellular chemotaxis may be related to other mechanisms, such as poor complement receptor translocation. the role of hmgb- and rage signaling in human neonates with sepsis has not been well characterized but has been shown to be involved in the pathophysiology of nec in an experimental model. significantly lower soluble rage levels were found in human fetuses that mounted robust inflammatory responses and hmgb- levels correlated significantly with the levels of il- and s β calcium-binding protein in the fetal circulation. other specific damage-associated molecular patterns, including heat shock proteins and uric acid, may also stimulate tlrs, regulate pmn function, and serve as immune adjuvants. heat shock protein production in infected neonates has not been evaluated but polymorphisms in heat shock proteins increase the risk for acute renal failure in preterm neonates. the levels of heat shock proteins are significantly elevated in infected adults and children. elevated heat shock protein and heat shock protein level measured within hours of pediatric intensive care unit admission were associated with septic shock and there was a strong trend towards increased mortality. , uric acid can increase cytokine production, pmn recruitment, and dendritic cell stimulation and may also serve as an antioxidant. the level of uric acid is reduced in the serum of neonates with sepsis as compared with control neonates. in addition to facilitating leukocyte attraction, proinflammatory stimuli result in production of vasoactive substances that decrease or increase vascular tone and alter vascular permeability (see figure - ). these include platelet-activating factor, thromboxane, leukotrienes, no, histamine, bradykinin, up-regulation after stimulation, deficiencies in another downstream signaling process, or inhibition by bacterial products. the levels of a wide variety of chemokines are increased during sepsis, including cxcl (ip- ), ccl (rantes), ccl (monocyte chemoattractant protein ), ccl (macrophage inflammatory protein α), and cxcl . the levels of other chemoattractive molecules also increase with sepsis, including complement proteins c a and c a, apps, including cathelicidins and defensins, and components of invading bacteria themselves. , the importance of chemoattractive substances in the pathogenesis of severe sepsis is highlighted by studies showing that cxcl can be used as a stratifying factor for survival in children, and c a is implicated in sepsis-associated organ dysfunction in adults. chemokine investigations in infected neonates revealed that cxcl is a sensitive early marker of infection, and low ccl levels may predict development of dic. damage-associated molecular patterns (or alarmins), such as intracellular proteins or mediators released by dying or damaged cells, may also active prrs. for example, the damage-associated molecular pattern high-mobility group box (hmgb- ) is involved in the progression of sepsis to septic shock in adults. , macrophages or endothelial cells stimulated with lps or tnf-α produce hmgb- , which signals through tlr , tlr , and receptor for advanced glycation end products (rage). hmgb- results in cytokine production, activation of coagulation, and pmn recruitment. , hmgb- mediates disruption of epithelial junctions within the gut via the induction of reactive nitrogen intermediates, leading to increased bacterial a twin study which assessed the frequency of infections among monoygotic and dizygotic prematurely born twins concluded that . % (p = . ) of the variance in susceptibility to lateonset sepsis was due to genetic factors alone. the impact of genetics in the host response is also underscored by the increased risk for death from infection seen with african american race or male sex among low-birth-weight infants. an ethnically unique single nucleotide polymorphism in the tlr promoter region was significantly associated with gram-negative bacterial infections in preterm infants. several recent studies in newborn infants have demonstrated an association between small variations in dna, specifically single nucleotide polymorphisms, and infection development and outcomes. , [ ] [ ] [ ] [ ] [ ] because tlrs play an essential role in recognition and response to pathogens, alterations in their expression, structure, signaling pathways, and function can have consequences for host defense. polymorphisms or mutations in tlrs are associated with increased risk for infection in adults [ ] [ ] [ ] [ ] and children , , but are less well characterized in neonates. after confounders had been controlled for, the presence of a tlr single nucleotide polymorphism was associated with a three-fold increase in the risk for gram-negative infections in vlbw infants. polymorphisms in the tlr , tlr , il , and pla g a (which encodes pla ) genes were associated with the development of neonatal sepsis. modifications in expression or function of costimulatory molecules necessary for tlr activation are also associated with an increased risk for infection. for example, the levels of lpsbinding protein (lbp; which binds intravascular lps) and the lps coreceptor cd are both increased during neonatal sepsis. , , furthermore, genetic variations in these proteins have been associated with increased risk for sepsis in adults. [ ] [ ] [ ] genetic polymorphisms in myeloid differentiation factor , a small protein involved in lps signaling through tlr , increase the risk for organ dysfunction and sepsis in adults but the significance in neonates is unknown. polymorphisms in post-tlr activation intracellular signaling molecules, including myeloid differentiation factor , il- receptor-associated kinase , and nf-κb essential modulator, are associated with invasive bacterial infection in older populations. additional genetic polymorphisms in intracellular second-messenger inflammatory signaling systems with impact on neonatal sepsis risk and progression are likely to be uncovered with the implementation of biobanking and mining of stored samples. mutations have been identified in nlrs that are involved in the pathogenesis of crohn's disease (nod ) and neonatalonset multisystem inflammatory disease (nlrp ). rlr mutations have been identified but have unknown clinical significance. no mutations in specific domains of nlrs have been found in neonates with sepsis or nec. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the importance of nlrs in listeria monocytogenes infections in neonates is unknown. complement is an important component of early innate immunity that facilitates killing of bacteria through opsonization and direct microbicidal activity. complement components also possess chemotactic or anaphylactic activity that increases leukocyte aggregation and local vascular permeability. furthermore, complement reciprocally activates a number of other important processes, such as coagulation, proinflammatory and prostaglandins. , these substances are produced predominantly by host endothelium and mast cells. activated pmns produce phospholipase a (pla ), the level of which is increased in the serum of neonates with sepsis and leads to generation of vasoactive substances, including prostaglandins and leukotriene. thromboxane produced by activated platelets and endothelin produced by activated endothelium are potent vasoconstrictors that participate in the development of pulmonary hypertension. [ ] [ ] [ ] [ ] overproduction of cytokines and vasoactive substances is associated with circulatory alterations and organ failure seen in severe sepsis and septic shock ( figure - ) . , [ ] [ ] [ ] [ ] if the pathogen is not contained locally and inflammatory homeostasis is not restored, sirs may develop, and lead to mods and death (see figure - ). the traditional paradigm for understanding the host response to sepsis consists of an intense proinflammatory response, or sirs, temporally followed by a compensatory antiinflammatory response syndrome. this paradigm has been challenged by the failure of multiple antiinflammatory strategies to improve sepsis outcomes in adults. new data in adults and children demonstrate simultaneous proinflammatory/antiinflammatory responses where the magnitude of either response may determine outcome. , near simultaneous increases in antiinflammatory cytokine production (transforming growth factor β, il- , il- , il- , and il- ) occur in neonates during infection, countering the actions of proinflammatory cytokines. , , these mediators blunt the activation and recruitment of phagocytic cells, reduce fever, modify coagulation factor expression, and decrease production of reactive oxygen and nitrogen intermediates, no, and other vasoactive mediators. [ ] [ ] [ ] [ ] [ ] [ ] soluble cytokine and receptor antagonists produced during sepsis also modulate proinflammatory mediator action. elevation of the levels of tnf receptor (which regulates the concentration of tnf-α), soluble il- receptor, soluble il- , and il- receptor antagonist have been documented in neonatal sepsis with resolution after effective treatment. , , the role of these regulatory cytokine inhibitors in the immune response to neonatal sepsis and septic shock has been incompletely characterized. soluble rage competes with cell-bound rage for the binding of hmgb- and other rage ligands. soluble rage has antiinflammatory effects and its level is elevated in adults during sepsis. furthermore, soluble rage improved survival and reduced inflammation when given to infected adult rodents. serum soluble triggering receptor expressed on myeloid cells may reduce inflammatory signaling for triggering receptor expressed on myeloid cells , and predict mortality in preterm neonates. micrornas may regulate inflammation at the level of gene expression via several putative mechanisms. several pilot studies in rodents and humans have demonstrated regulatory functions for microrna in neonates. [ ] [ ] [ ] [ ] [ ] [ ] the impact of regulatory micrornas and their effects on the host inflammatory response in neonates with sepsis are unclear. endogenous cortisol is induced by proinflammatory cytokines and attenuates the intensity of sirs associated with severe sepsis and septic shock. the use of cortisol in adults with sepsis has been controversial. , cortisol production in newborn infants is significantly increased early in shock. however, very preterm neonates may have relative adrenal insufficiency that may contribute to hemodynamic instability and hypotension. cortisol replacement may be critical in these infants and deserves further study. it is important to note, however, that in children with septic shock, adjunctive corticosteroid therapy is associated with repression of gene programs corresponding to the adaptive immune system. complement-mediated activation of leukocytes during sepsis occurs via up-regulated cell surface receptors (complement receptor [cd ] and cr ). , c b and c a facilitate opsonization (primarily c b), redistribute blood flow, and increase inflammation, platelet aggregation, and release of reactive oxygen intermediates (primarily c a). , c a-mediated local leukocyte activation also results in increased cytokine production with subsequent up-regulation of adhesion molecules on vascular endothelium and increased cell recruitment to the site of infection. data in adults link elevated c a levels with multiple facets of sepsis-associated disease, such as dic, cardiac dysfunction, increased proinflammatory cytokine levels, sirs, apoptosis of adrenal medullary cells leading to adrenal cytokine production, and leukocyte activation. contrary to its name, the alternative pathway is the primary mechanism of amplification of complement activation after c convertase assembly (which cleaves c to c a and c b). dysregulation of complement activation may contribute to adverse effects in individuals with severe sepsis or septic shock. neonates, particularly the very premature, exhibit decreased basal levels of complement proteins and function for both the alternative pathway and the classical pathway. , moreover, as compared with adults, neonates exhibit gestational age-related degrees of depressed complement-mediated opsonic capabilities. as such, complement-mediated opsonization is poor in premature neonates and limited in term neonates. nization and pathogen clearance may help explain the lack of efficacy of intravenous immunoglobulin to prevent sepsis or death from sepsis in neonates. [ ] [ ] [ ] [ ] [ ] [ ] [ ] apps are the most phylogenetically ancient means of innate immune defense against microbial invasion. present in nearly every organism, including bacteria, plants, insects, nonmammalian vertebrates, and mammals, these small, often cationic peptides are capable of killing microbes of multiple types, including viruses, bacteria, parasites, and fungi, largely by disruption of the pathogen membrane. constitutive expression of apps occurs in humans on barrier areas with consistent microbial exposure such as skin and mucosa. after microbial stimulation, both release of preformed apps and inducible expression are thought to contribute to early host defense. importantly, there is no evidence for the development of microbial resistance to apps that target fundamental components of the microbial cell wall. some apps can bind and neutralize microbial components such as endotoxin, precluding engagement with tlrs and other prrs, and diminish inflammation. many apps can potentially reduce the intensity of the inflammatory response associated with the presence of bacterial toxins. [ ] [ ] [ ] because endotoxemia is an important contributor to neonatal mods and death with sepsis and nec, lps-binding/blocking strategies, including use of synthetic apps, may have a significant positive impact on outcomes. , bactericidal/permeability-increasing protein (bpi) is a -kda protein present in the respiratory tract, pmn primary granules, and plasma. bpi exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria. plasma bpi concentrations were higher in critically ill children with sepsis syndrome or organ system failure than in critically ill children without sepsis syndrome or organ system failure, and bpi levels positively correlated with the pediatric risk for death score. pmns from term neonates are deficient in bpi, potentially contributing to the increased risk for infection. whereas term neonates demonstrate up-regulation of plasma bpi during infection, premature neonates showed a decreased ability to mobilize bpi on stimulation, which may contribute to their risk for infection with gram-negative bacteria. polymorphisms in bpi increase the risk for gram-negative sepsis in children, but the impact of these polymorphisms in neonates is unknown. compared with pmns from adults, pmns from term neonates produce similar quantities of defensins but reduced quantities of bpi and elastase. , , recombinant bpi (rbpi ) treatment was associated with improved functional outcome, reduced amputation, but no difference in mortality in a multicenter study of children with severe systemic meningococcal disease. lactoferrin is the major whey protein in mammalian milk (in particularly high concentrations in colostrum) and is important in innate immune host defenses. lactoferrin is present in tears and saliva and has antimicrobial activity both via binding iron and by direct membrane disruption activity via a portion of its amino-terminal lactoferricin. lactoferrin is also an alarmin (e.g., hmgb- or il- ), capable of activating leukocytes, binding endotoxin, and modifying the host response by acting as a transcription factor that regulates mrna decay. , bovine lactoferrin has been shown to reduce the incidence of bacterial and fungal sepsis , and nec in preterm infants. lysozyme is present in tears, tracheal aspirates, skin, and pmn primary and secondary granules and contributes to degradation of peptidoglycan in bacterial cell walls. secretory pla can destroy gram-positive bacteria through hydrolysis of their membrane lipids. pmn elastase is a serine protease released by activated pmns with microbicidal function and is believed to play a role in the inflammatory damage seen with pmn recruitment, particularly in the lung. , cathelicidin and the insufficiency, and pmn dysfunction. septic shock in adult humans was associated with extensive complement activation, c-reactive protein-dependent loss of c a receptor on neutrophils, and the appearance of circulating c a receptor in serum, which correlated with a poor outcome. deficiencies in c a receptor found in term neonates as compared with adults may limit the ability to respond to c a and therefore increase the likelihood of infection. the expression of c a receptor on preterm pmns is unknown. the extent to which c a or other complement proteins play a role in the development of disease in septic neonates remains to be determined. complement regulatory proteins modify the effects of complement and prevent potential damage due to overactivation. in particular, cd blocks c polymerization and target lysis, cd destabilizes cd and c and c convertases, and cd accelerates the deactivation of c b. dysregulation of complement activation can lead to a vicious activation cycle that results in excessive cellular stimulation, cytokine production, endothelial cell activation, and local tissue damage promulgating sirs and septic shock (see figure - ). in addition to the initial inflammatory response including complement activation, molecular detection of pamps promotes il- β and il- production, which in turn increases the production of multiple other innate proteins that possess valuable immune function and serve to reduce pathogen load. acutephase reactant proteins, produced predominantly in the liver, include c-reactive protein (opsonin), serum amyloid a (cellular recruitment), lactoferrin (reduces the level of available iron/ antimicrobial peptide lactoferricin), procalcitonin (unknown function), haptoglobin, fibronectin (opsonic function), pentraxin (binds c q and activates the classical complement pathway), mbl, and lps-binding protein. , , , [ ] [ ] [ ] [ ] [ ] [ ] acutephase reactant proteins have been studied in neonates with sepsis primarily to assess them for diagnostic utility rather than immunologic function. in particular, elevated plasma concentrations of c-reactive protein and lps-binding protein are often associated with early-onset sepsis. , the levels of il- and c-reactive protein were significantly higher in preterm infants who did not survive sepsis, pneumonia, or nec. a lack of sustained increase in the production of c-reactive protein and serum amyloid a during sepsis has also been associated with a fulminant course. the fetus receives antibodies from the mother via active placental transfer, with a significant increase beginning around weeks' gestation. as a result of a shorter period of gestation, preterm neonates have lower igg subclass levels as compared with term neonates, particularly igg and igg subclasses. preterm neonates ( to weeks' gestation) with low igg levels (serum total igg levels below mg/dl at birth) were at increased risk for development of late-onset sepsis but not death compared to those with levels above mg/dl. however, igg titers and opsonic activity to cons were not predictive of late-onset cons sepsis. reliance on other means of innate immune defense likely provides the premature neonate with alternative microbial control mechanisms. despite the presence of maternally derived immunoglobulin and acute-phase reactant proteins, neonates exhibit impaired opsonizing activity compared with adults, which likely increases the risk for progression of infection. complement plays a critical role in immunoglobulin-mediated opsonization and effector cell phagocytosis. although immunoglobulin has many putative beneficial immunologic functions, most of these have not been demonstrated or examined in preterm infants. the dependence on complement for effective immunoglobulin-based opso-thrombocytopenia in neonates, which is attributed to reduced megakaryopoiesis in the setting of consumption with clot formation. decreased platelet function in preterm neonates with sepsis further increases the risk for bleeding. in extremely low-birth-weight infants, platelets are hyporeactive for the first few days after birth, complicating the ability of the immune system to contain a microbiologic threat and increasing the risk for hemorrhage. clotting can lead to propagation of inflammation via thrombin-induced production of platelet-activating factor. pmns activated by platelet-activating factor or platelet tlr may then contribute to further endothelial injury and dysfunction, leading to the development of a vicious clottinginflammation-clotting cycle. activated platelets may be consumed in clot formation and/or may also be removed from the circulation by the liver, potentially resulting in thrombocytopenia, particularly during gram-negative and fungal infections. , , systemic activation of coagulation is associated with consumption of clotting factors and increased risk for bleeding, prolonged proinflammatory responses, and dic. , , this finding is consistent with the elevated serum levels of il- and high frequency of dic seen with disseminated herpes simplex virus infection. in adult mice, protease-activated receptor plays a major role in orchestrating the interplay between coagulation and inflammation. protease-activated receptor may modify the endothelial response during neonatal sepsis and thus is a target for therapeutic intervention. recent studies have shown the critical importance of vascular endothelial activation in the early recognition and containment of microbial invasion. in transgenic mice, it was shown that pulmonary endothelial cells sense blood-borne bacteria and their products, whereas alveolar macrophages patrol the air spaces. these data illustrate the role of endothelium and help to explain in part the occurrence of acute respiratory distress syndrome (ards) and persistent pulmonary hypertension of the newborn associated with severe sepsis in the absence of a primary pulmonary infectious focus. expression of tlrs allows endothelium to become activated in the presence of microbial components, leading to production of cytokines, chemokines, and adhesion molecules (e.g., vascular cell adhesion molecule, intercellular cell adhesion molecule, l-selectin, p-selectin, and e-selectin). these substances are all necessary to attract immune cells (primarily pmns) to the site of infection and to facilitate pathogen containment. [ ] [ ] [ ] [ ] vasoactive substances released from activated leukocytes, platelets, and endothelial cells include platelet-activating factor, thromboxanes, leukotrienes, no, histamine, bradykinin, and prostaglandins. , the balance of no and endothelin , a vasoconstrictor, may be disrupted with endothelial damage, favoring the constrictive effects of endothelin and leading to ischemia and injury. this phenomenon may explain in part why no inhibitors increased mortality in adults with septic shock. stimulated endothelium can be a doubleedged sword, however, because excessive activation can lead to systemic overproduction of cytokines and vasoactive substances (including no). endothelial cell apoptosis, detachment from the lamina, and alterations in vascular tone combine to promote capillary leak, leading to hypovolemia, shock, and organ failure , , (see figure - ). release of myeloperoxidase from pmns may also injure surrounding endothelium. activated or damaged endothelium establishes a prothrombotic environment that can result in local microvascular occlusion or progress to dic. the glucocorticoid receptor is the target for cortisol, the primary endogenous glucocorticoid in humans, produced in the zona fasciculata of the adrenal glands. endothelial glucocorticoid defensins are other apps that possess antimicrobial properties. cathelicidin is present in the amniotic fluid, vernix, skin, saliva, respiratory tract, and leukocytes. α-defensins are cysteine-rich -kda peptides found in amniotic fluid, vernix, spleen, cornea, thymus, paneth cells, and leukocytes. β-defensins are found in the skin, gi tract, urinary system, reproductive organs (placenta, uterus, testes, kidney), respiratory tract, breast milk, mammary gland, and thymus. in addition to microbicidal action, apps have a wide range of immunomodulatory effects on multiple cell types from both the innate immune system and the adaptive immune system. , , these immunomodulatory effects include altered cytokine and chemokine production, improved cellular chemotaxis and recruitment, improved cell function (maturation, activation, phagocytosis, reactive oxygen intermediate production), enhancement of wound healing (neovascularization, mitogenesis), and decreased apoptosis. the cytosolic granules of pmn are rich in apps, including α-defensins, lactoferrin, lysozyme, cathelicidin, soluble pla , and bpi. gestational age-related decreases in the umbilical cord blood concentration of several apps (cathelicidin, bpi, calprotectin, soluble pla , α-defensins) in comparison with maternal serum levels have been drescibed. plasma app deficiencies may contribute to the increased risk for infection associated with prematurity, and their absence may increase the risk for endotoxemia. compared with term neonates, preterm neonates showed lower human β-defensin levels in umbilical cord blood. up-regulation of apps (defensins) occurs in blood of infected adults and children (defensins, lactoferrin). the effect of sepsis on the production of plasma apps in neonates has not been investigated in detail. the development of a procoagulant state in the surrounding microvasculature allows the trapping of invading pathogens and prevents further dissemination (see figure - ). in general, the intrinsic pathway amplifies coagulation after initiation by the extrinsic pathway. reduced levels of vitamin k-dependent factors (factors ii, vii, ix, and x), reduced thrombin generation, reduced consumption of platelets with formation of microthrombi, and reduced levels of counterregulatory elements (inhibitors) increase the risk for bleeding in infants and children. during sepsis, a microvascular procoagulant state develops via stimulation of phagocytes, platelets, and endothelium, resulting in expression of tissue factor. , tissue factormediated activation of the coagulation cascade results in activation of thrombin-antithrombin complex, plasminogen activator inhibitor type , and plasmin-α -antiplasmin complex, as well as inactivation of protein s and depletion of the anticoagulant proteins antithrombin iii and protein c. [ ] [ ] [ ] decreased activated protein c levels were associated with increased risk for death from sepsis in preterm neonates. a randomized controlled trial of activated protein c revealed no change in mortality among pediatric patients with sepsis, but term infants younger than days old experienced increased bleeding. the coagulation cascade is intimately tied to inflammation and complement activation. cytokine production increases expression of endothelial tissue plasminogen activator inhibitor type . plasminogen activator inhibitor type inhibits fibrinolysis by inhibiting the conversion of plasminogen to plasmin, which in turn is important for the breakdown of fibrin. deposition of fibrin in small vessels leads to inadequate tissue perfusion and organ failure. increased plasminogen activator inhibitor type excessive local inflammation and tissue damage. high early levels of circulating free pmn-derived dna produced by nets are associated with mods and death. nets contain destructive proteases capable of killing bacteria even after the pmn has died. formation of nets is reduced in pmns from preterm neonates and nearly absent in term neonates but may occur with sustained cellular stimulation. net formation may result in collateral damage to surrounding tissues when the target microbe is too large to be effectively phagocytosed (e.g., fungal hyphae). the contribution of net production to detrimental outcomes in infected neonates is unknown but excessive net formation with collateral tissue injury may contribute to the poor outcomes seen in preterm neonates with fungal infections. rapid depletion of bone marrow pmn reserves during infection, particularly in neonates, can lead to neutropenia, with consequent impaired antimicrobial defenses and significantly increased risk for death. in a multivariate analysis, neutropenia and metabolic acidosis were associated with fatal neonatal sepsis. neutropenia is particularly common in gram-negative sepsis in neonates. release of immature pmn forms (bands), which exhibit greater dysfunction than mature pmns, may further predispose to adverse outcomes. murine neonates with experimental sepsis exhibit delayed emergency myelopoiesis (a process by which the host repopulates peripheral myeloid cells lost early during sepsis), that is independent of trif and myeloid differentiation factor . interventions aimed at addressing reduced pmn numbers in neonates have included provision of mature pmns and prophylaxis or treatment with colonystimulating factors (granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor). despite strong biologic plausibility, these interventions have been unsuccessful at reducing the neonatal infectious burden. [ ] [ ] [ ] in a metaanalysis, treatment with colony-stimulating factor therapy (granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor) in a subgroup (n = ) of neutropenic neonates (absolute neutrophil count less than /µl) with culture-positive sepsis (largely gram-negative and gbs) significantly reduced the risk for death (relative risk, . ; % ci, . to . ). therefore, stimulation of granulopoiesis may be beneficial under these specific circumstances, although further studies focused on this subpopulation and outcomes are needed. irreversible aggregation and accumulation of newborn pmns in the vascular space after stimulation leads to decreased diapedesis, rapid depletion of bone marrow reserves, vascular crowding, and increased likelihood of microvascular occlusion. neonatal pmn deformation compared with adult pmn deformation is reduced at the baseline, which increases the risk for occlusion. furthermore, low blood pressure/flow states seen during septic shock further exacerbate existing microvascular ischemia. in combination, these deficiencies increase the propensity for systemic spread of infection, and set the stage for microvascular occlusion. many other cells besides pmns are involved in the development of an immune response to infection. monocytes, macrophages, and dendritic cells amplify cellular recruitment through production of inflammatory mediators, activation of endothelium, phagocytosis and killing of pathogens, and antigen presentation to t and b cells of the adaptive immune system. the primary functions of monocytes are the synthesis of crucial inflammatory proteins and antigen presentation to naïve cd + t cells. the patterns of cytokine production can promote the differentiation of naïve cd + t cells into distinct subtypes of t cells that serve important roles in the clearance of pathogens. for example, t-helper (t h ) cells are produced from naïve cd + t cells after exposure to ifn-γ and il- , and support cellmediated immunity against intracellular pathogens through production of ifn-γ, tnf-α, and lymphotoxin. t-helper (t h ) cells receptor is a critical negative regulator of inducible no synthase expression and nf-κb activation, demonstrating a protective role of the endothelium during sepsis. studies have revealed a potential role of plasma angiopoietin during pediatric septic shock. the level of angiopoietin , which protects against vascular leak, was reduced, whereas the level of angiopoietin , which promotes vascular permeability, was elevated, highlighting a novel potential therapeutic opportunity to reduce endorgan injury. the roles for endothelial glucocorticoid receptor and angiopoietin in neonatal sepsis are unknown. the role of endothelium activation during sepsis and septic shock in neonates, particularly in premature neonates, has been less well investigated. toxins from gbs have been shown to damage pulmonary endothelium and likely participate in pulmonary complications associated with gbs pneumonia such as ards and the development of persistent pulmonary hypertension of the newborn. the levels of the adhesion molecules e-selectin and p-selectin, expressed and secreted by activated endothelium, are increased in the serum of neonates with sepsis and likely reflect significant endothelial activation. endothelial tlr activation impaired intestinal perfusion in an experimental model of nec, via endothelial no synthasenitrite-no signaling. the pmn is the primary effector of innate immune cellular defense. endothelial cells produce activating cytokines and chemokine gradients that recruit circulating pmns to the site of infection. expression of cell adhesion molecules by pmns and endothelium allows cells to roll and extravasate into surrounding tissues. activated pmns phagocytose and kill pathogens via oxygen-dependent and oxygen-independent mechanisms. il- β is produced by activated pmns largely via an nlrp -asccaspase -dependent* mechanism that amplifies the recruitment of additional pmns from the bone marrow to the site of infection. activated pmns may release reactive nitrogen species, reactive oxygen species, and proteolytic enzymes via activation of membrane-associated nadph oxidase. these reactive intermediates and enzymes can lead to destruction of nonphagocytosed bacteria but can also cause local tissue destruction, including neonatal endothelial and lung injury, as well as surfactant inactivation, , and thus play a role in progression from sepsis to mods. neonatal pmns exhibit quantitative and qualitative deficits as compared with adult pmns. , respiratory burst activity is suppressed in pmns during neonatal sepsis and may contribute to poor microbicidal activity. [ ] [ ] [ ] compared with adult pmns, neonatal pmns exhibit delayed apoptosis, , as well as sustained capacity for activation (cd b up-regulation) and cytotoxic function (reactive oxygen intermediate production) that contributes to tissue damage. reduced apoptosis with prolonged survival of pmns may result in improved bacterial clearance but may also paradoxically increase the risk for sustained pmn-mediated tissue damage. increased serum pmn elastase, urokinase plasminogen activator, and urokinase plasminogen activator receptor levels are found at the time of presentation in infected neonates. with pmn death, dna (chromatin), histones, and apps are expelled into the environment and serve to trap bacteria (neutrophil extracellular traps [nets]). the formation of nets can occur after activation of platelet tlr and may lead to *asc, apoptosis-associated speck-like protein containing a carboxyterminal card. eosinophils phagocytose antigen-antibody complexes and release cytokines, chemokines, cytotoxic molecules, apps, and other substances (prostaglandins, thromboxanes, leukotrienes) when stimulated. eosinophilia is commonly observed in neonates with sepsis due to candida sp. and bacteria, and is seen in infants with nec. in infants of less than weeks' gestation, eosinophilia (absolute eosinophil count more than /mm ) may predict bacterial sepsis. eosinophilia in premature infants is not associated with production of ige. studies have demonstrated an integral role for eosinophils in adult intestinal integrity and revealed a novel innate bactericidal nonphagocytic function via extracellular catapulting of mitochondrial dna nets with associated bound toxic proteins. the precise role of eosinophils in the neonatal immune response to sepsis and in maintenance of intestinal integrity has yet to be determined. mast cells play a role in the response to pathogen invasion as a part of the innate cellular immune system via production of histamines (which promote vasodilation and up-regulation of p-selectin), cytokines, pmn recruitment, bacterial phagocytosis, and antigen presentation. , mast cell involvement was demonstrated in infants with erythema toxicum, where mast cell recruitment, degranulation, and expression of apps occurs. adult rodents deficient in mast cells exhibit impaired pmn influx, impaired clearance of enteric organisms, and decreased sepsis survival. mast cell production of histamine likely contributes to the vasodilation associated with sepsis and septic shock. like eosinophils and pmns, mast cells are capable of killing bacteria via generation of extracellular traps in adults. this means of immune protection has not been investigated in neonates. mast cells may also alter adaptive immune function by patterning the t h immunophenotype seen in the neonate and therefore contribute to the increased risk for infection. immature dendritic cells exposed to histamine during maturation (with lps) exhibit altered t-cell polarizing activity with predominance towards the t h phenotype via increased production of il- and decreased production of il- . furthermore, mast cells from neonates were shown to secrete significantly more histamine after stimulation as compared with adults, which may contribute to the development of shock. the role of natural killer (nk) cells in neonatal bacterial sepsis is incompletely defined. nk cell numbers increase with increasing gestational age, furthermore, a reduced percentage of nk cells present at birth may be a risk factor for late-onset sepsis in preterm infants. it is noteworthy that the numbers of circulating nk cells are not significantly different in neonates with or without infection , ; however, the numbers of circulating nk cells are decreased in newborn infants with shock. the mechanisms used by nk cells to destroy bacteria include secretion of apps (defensins), direct contact and lysis, antibody-dependent cellular cytotoxicity, and ifn-γ production. in neonates with bacterial sepsis, nk cells are activated, as evidenced by up-regulation of cd . , despite activation, nk cell cytotoxicity is deficient in infants with sepsis and recurrent infections. , although neonatal macrophages exhibit impaired baseline activation in response to ifn-γ, nk cell-mediated production of ifn-γ can enhance their phagocytic capability. further studies are necessary to more clearly define the role of nk cells in neonatal bacterial sepsis. cd + ter + (erythroid) cells may contribute to the increased susceptibility of the neonate to infection by reducing the inflammatory response associated with bacterial colonization of the gut. for example, ex vivo tnf-α production by stimulated adult effector cells was reduced in the presence of murine neonatal splenic cd + erythroid cells via an arginase -dependnent mechanism. the cd + erythroid population represents a large portion of murine fetal liver, neonatal spleen/bone marrow, and adult bone marrow. [ ] [ ] [ ] furthermore, the murine neonatal spleen contains large numbers of colony-forming progenitor cells for to weeks after birth. of note and in stark contrast arise in the presence of il- and il- , produce il- , il- , and il- , down-regulate t h responses, and support humoral immunity, as well as defense against extracellular parasites. a third subset of t h cells, t-helper cells, are generated in the presence of transforming growth factor β, il- , il- , and il- . these cells produce il- and il- , which are important for defense against extracellular bacteria and fungi. neonatal mononuclear cells exhibit a bias away from t h cell-polarizing activity because of increased il- and low tnf-α production. this may be beneficial because of mobilization of antiinfective proteins/ peptides that serve to protect the newborn during microbial colonization and development of immune tolerance. the adverse consequence is a reduced ability to respond to infection with microorganisms; particularly intracellular pathogens such as listeria sp. and mycobacteria. preterm infants (< weeks) may have greater attenuation of tnf-α and il- secretion compared with term infants and adults. there is decreased monocytic recruitment to areas of inflammation during sepsis because of decreased chemotactic ability. although the levels of peripheral monocytes decrease early during sepsis (between nd hours), secondary to extravasation and differentiation into macrophages, sepsis-related elevation of macrophage colony-stimulating factor results in a late increase in the number of peripheral monocytes (> hours). in addition to altered cytokine production and suboptimal recruitment, monocyte phagocytic function is reduced during sepsis. antigen presentation to naïve cd + t cells is an important immune function performed by monocytes. the decreased antigen-presenting function in monocytes from newborn infants is in part due to decreased mhc class molecule expression and decreased expression of costimulatory molecules, including cd and cd . monocytes leave the bloodstream, enter the tissues, and differentiate into macrophages and dendritic cells. monocytes and macrophages are closely related to pmns (common myeloid progenitor) and can kill pathogens by similar means. circulating monocytes differentiate into macrophages after exposure to maturing cytokines, and exit the bloodstream into tissues. important substances produced by stimulated monocytes/macrophages include complement components, cytokines (both proinflammatory and antiinflammatory), coagulation factors, and extracellular matrix proteins. located just below epithelial borders, macrophages encounter pathogens immediately after entry. macrophages are avidly phagocytic and generate apps to reduce bacterial burden, such as lactoferrin, defensins, transferrin, and lysozyme. in addition, macrophages play an important role in the amplification of the immune response through the production of cytokines and chemokines, as well as in antigen presentation to naïve cd + t cells. macrophages are poorly responsive to several tlr agonists. dendritic cells are antigen-presenting cells that function as a liaison between the innate immune system and the adaptive immune system through induction of antigen-specific t cellmediated immunity. dendritic cells from newborn infants exhibit a reduced antigen-presenting function when compared with adult cells and require increased stimulation for activation. evaluations of neonatal dendritic cell function suggest a tendency towards poor up-regulation of costimulatory molecules (cd /cd ) and activation markers (cd ), poor stimulation of t-cell proliferation, and a tendency towards the induction of immune tolerance. although preterm and term infants and adults have similar numbers of "plasmacytoid" dendritic cells in their blood, the capacity to produce ifn-α on tlr challenge was significantly decreased in preterm neonates and may increase susceptibility to viral infections. dendritic cells in umbilical cord blood can effectively induce cytotoxic lymphocyte responses. depletion of dendritic cells has been reported in adult animals and adult patients with sepsis; their role in the immune response to neonatal sepsis is not well characterized. pmn and γδ t cells. these data show that neonatal t cells are activated and are capable of playing a role in the host response to bacterial sepsis in vivo. neonatal lymphocyte function is skewed towards t h responses, setting the stage for immune tolerance (t h ) rather than immune priming for infection (t h ). newborn infants must overcome that immune modulation in order to mount effective responses to specific infectious challenges and respond to vaccination. examples of the impact of this immunopolarization include decreased ifn-γ production by cd + and cd + t cells as compared with production in children and adults. , the likely significance of decreased ifn-γ production is a reduction in activation of other immune cells, such as macrophages. reports of lymphocyte function in infected newborns are very limited. expansion of lymphocytes after antigenic stimulation is important for development of sustained immunity. decreased lymphocyte proliferative responses have been shown during the first weeks of life in vlbw neonates, and may predispose the premature neonate to development of late-onset sepsis. for example, t-lymphocyte function was depressed in infected newborn infants, and especially in those with multiorgan failure, versus healthy term or growing preterm infants. similarly, production of lymphocyte-associated cytokines after stimulation of umbilical cord blood peripheral blood mono nuclear cells with gbs was significantly deficient in preterm and term infants compared with adults. cytomegalovirus infection in utero leads to the expansion and differentiation of mature cytomegalovirusspecific cd + t cells, which have characteristics similar to adult cd + t cells. these cells showed potent perforin-dependent cytolytic activity and produce antiviral cytokines, highlighting the potential for adult-like immunocompetence of neonatal t cells under specific circumstances. an important location for effective lymphocytic function during systemic bacterial infection is the spleen. the marginal zone of the spleen facilitates the clearance of bacteria, particularly encapsulated organisms, from the bloodstream. these functions are accomplished via the interaction of multiple leukocytes, including macrophages, dendritic cells, b cells, and t cells, within follicles of the spleen. the neonatal splenic marginal zone is immature, owing to a lack of antecedent antigen exposure and is virtually devoid of cd + b cells. as a result of this functional asplenia, there is decreased clearance of pathogens from the blood and potential for a more fulminant course with bacteremia. , b cells are critically important in the adult host response to sepsis. data suggest antibody-independent and antibodydependent roles for b cells in the outcome of sepsis. studies deciphering the role of b cells in neonatal sepsis are very limited, and thus the role b cells play in the neonatal host response is unclear. after gbs meningitis, the level of igm was increased, suggesting b cells from neonates can respond to pathogenic challenge. premature neonates with perinatal infection or nosocomial infection may show signs of humoral immunoparalysis, manifested by decreased igm/igg production ex vivo as compared with production in their healthy age-matched counterparts. sepsis in early life did not reduce serum antibody titers in preterm infants after heptavalent pneumococcal conjugate vaccine exposure but was associated with a reduced opsonization titer to a single serotype, suggesting the capacity to respond to vaccination or other immune challenge may be altered. in the setting of reduced classic adaptive immune function seen in early life as compared with the function in adults, innate lymphoid populations (which lack b cell receptor and t cell receptor) may play a significant role in protecting the neonate from infectious challenge. [ ] [ ] [ ] [ ] [ ] [ ] [ ] examples of innate lymphoid cells include γδ t cells, intestinal lymphoid cells, invariant nk t cells, mucosa-associated invariant t cells, and b cells. to the lymphoid and reticuloendothelial system roles of the spleen in the healthy adult, the spleen is normally a major site of erythropoiesis during fetal and neonatal life, to support rapid fetal and postnatal growth in the setting of significantly reduced erythroid reservoirs as compared with the adult reservoirs. , , a lack of effect on neonatal murine survival to polymicrobial sepsis after adoptive transfer or diminution of cd + erythroid splenocytes suggests that the impact of these cells on neonatal infection risk and progression may be limited. the contribution of the adaptive immune system in the neonatal host response to sepsis is uncertain. the -to -day interval required for development of an adaptive immune responsenamely, the selection and amplification of specific clones of lymphocytes (b cells and t cells) that results in immunologic memory-argues against a central role for adaptive immunity in the protective response to early neonatal bacterial sepsis. as a result, the neonate is thought to largely depend on innate immunity for protection from infection during the first days of life. in adults, absence or dysfunction of the adaptive immune system has a profound impact on survival in preclinical models. b cells (and in particular b-cell cytokine production) and not t cells were shown to be important in the early host response to experimental sepsis. studies using neonatal mice lacking an adaptive immune system showed no difference in polymicrobial sepsis survival as compared with survival of wild-type mice with an intact adaptive immune system. furthermore, there are many quantitative and qualitative differences in lymphocytes from neonates compared with lymphocytes from adults, each with a respective proposed clinical impact. as these findings illustrate, the contribution of adaptive immunity for protection and response against sepsis, and in particular which components are protective, is unclear in the most immature and requires further investigation. peripheral blood examination has yielded inconsistent changes in the percentage, number, and type of circulating lymphocytes during sepsis. , [ ] [ ] [ ] [ ] [ ] [ ] moreover, changes related to the timing of sepsis onset (early-onset or late-onset sepsis) and prematurity have been incompletely characterized. t regulatory cells are abundant and potent at birth, facilitating inhibition of t h cell immunity, and perhaps mediating a state of immunologic tolerance. although the numbers of splenic t regulatory cells are increased in murine neonates and adults with sepsis, depletion of t regulatory cells had no effect on survival of murine adults. , alterations in the number or function of t regulatory cells in human neonatal sepsis have not been reported. examination of peripheral blood to identify markers of sepsis has yielded a number of lymphocyte cell-surface molecules whose levels increase during sepsis. activation of neonatal t cells is evidenced by increased cd ro expression (present on t cells after antigenic stimulation) at the time of sepsis diagnosis, , , and with congenital infection, although changes in number may take several days to occur after stimulation. other markers of lymphocyte activation may be found at different time points during the course of infection. for example, expression of the activation marker cd is increased on t cells (cd + ) early in the infectious process, whereas cd and cd ro expression persists for several days. increased expression of cd + t-cell carcinoembryonic antigen-related cell adhesion molecule (cd a) in preterm infants with lateonset sepsis may contribute to sepsis-associated immune suppression. hla-dr expression is increased on multiple cell types during neonatal sepsis. in contrast to adults, a large portion of neonatal t cells produce cxcl , which activates of genes for innate immune and metabolic pathways with decreased levels of adaptive immune transcripts. using a proteomics approach, ng and colleagues identified proapolipoprotein cii and a desarginine variant of serum amyloid a as promising biomarkers for late-onset sepsis and nec in preterm infants. it is very likely that implementation of unbiased "omic" approaches will reveal critical age-appropriate pathways and opportunities for therapeutic interventions aimed at improving neonatal sepsis outcomes. sepsis that leads to shock and organ failure carries the worst prognosis. sirs contributes to the development of organ failure in neonates (see figure - ). , , , persistent decreases in capillary perfusion are associated with mods and death in adults. lethargy, shock, and birth weight less than g were independent predictors of sepsis-related death. in neonates, impairment of the cardiovascular system, manifested by poor perfusion or hypotension, is invariably associated with septic shock. sustained poor organ perfusion in neonatal sepsis and septic shock due to cardiovascular dysfunction is associated with mods affecting the kidney, , liver, gut, and central nervous system (see figure - ) . the mechanism of organ failure may be decreased oxygen utilization associated with mitochondrial dysfunction rather than poor oxygen delivery to tissue. , on the basis of available evidence, it has been speculated that the prolonged sirs associated with severe sepsis and shock leads to organ failure via a cessation of energy-consuming processes. , development of severe nec is also associated with severe sepsis, shock, mods, and death. , the need for intubation or initiation of vasoactive medications, and hypoglycemia, thrombocytopenia, increased prothrombin time, or excessive bleeding as presenting laboratory signs of sepsis are risk factors for sepsis-related death. , , independent predictors of in-hospital late-onset sepsis death during the birth hospitalization were the presence of congenital anomalies (or, . ; % ci, . to . ), neuromuscular comorbidities (or, . ; % ci, . to . ), and secondary pulmonary hypertension with/without cor pulmonale (or, . ; % ci, . to . ), underscoring the impact of organ-level comorbidities that increase neonatal sepsis mortality. the most common organ dysfunction associated with sepsis is cardiovascular dysfunction. cardiovascular dysfunction associated with sepsis may lead to shock that is a composite of hypovolemic, cardiogenic, and distributive shock. distributive shock is related to endothelial no production that leads to excessive vasodilation. cardiogenic shock may be related to mitochondrial death (induced by reactive nitrogen and reactive oxygen intermediates) with subsequent myocardial dysfunction. abnormalities in peripheral vasoregulation and myocardial dysfunction may play a larger role in hemodynamic derangements in pediatric patients, especially infants and neonates. in children, a non-hyperdynamic state with reduced cardiac output and increased systemic vascular resistance is most commonly observed in the setting of sepsis. [ ] [ ] [ ] [ ] [ ] the hemodynamic presentation in neonates is much more variable and is complicated by an unclear association between a normal blood pressure and adequate systemic blood flow. , microcirculatory flow is impaired in term neonates even with mild to moderate severity of infection. preterm neonates with sepsis have relatively high left and right cardiac outputs and low systemic vascular resistances. however, a decrease in right or left ventricular output of more than % has been associated with increased mortality in neonatal sepsis. an elevated left ventricular output mechanistic investigations that fully explore the role of these newly discovered populations in the neonatal host response to sepsis are likely to uncover novel therapeutic opportunities. systems biology and the use of "omic" approaches have the potential to produce significant insights into the pathogenesis of sepsis. genomic and proteomic approaches have yielded important data associated with septic shock in older populations. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the use of these modern techniques in the study of neonatal inflammation and response to pathogen challenge has only just begun. , , , the ability to profile genome-wide expression has significantly enhanced our understanding of the complexity of the host immune response to sepsis in children. , , , , for example, genome-wide expression profiling revealed zinc homeostasis as an important feature of pediatric sepsis. , , prophylactic zinc supplementation reduced bacterial load and mortality in a murine model of peritoneal sepsis. however, oral zinc supplementation did not alter mortality in neonates with probable sepsis. in a study of pediatric patients who met the criteria for septic shock, a unique whole-blood transcriptomic response was found in neonates as compared with infants, toddlers, and school-age children. neonates manifested the largest number of uniquely regulated genes, representing both innate and adaptive immune system pathways, and showed a predominance of down-regulated transcripts representing the adaptive immune system. the number of up-regulated genes increased in proportion with developmental age. investigation of the murine circulating leukocyte transcriptome revealed significant differences in the host immune response to sepsis across the age spectrum (neonate, young adult, elderly), despite similar increases in mortality among the neonates and elderly mice as compared with young adult mice. these data underscore the impact of developmental age on the host immune response and suggest that therapeutics, which may have efficacy in older populations, may be ineffective in or possibly detrimental to neonates. because the transition to extrauterine life is associated with dramatic changes in physiology, the whole-blood transcriptome is likely to be quite different between both uninfected infants and in the host response to sepsis by timing after birth. indeed, an unsupervised analysis of the whole-blood genome-wide transcriptome on prospectively collected peripheral blood samples from infants evaluated for sepsis revealed the major node of separation between groups (infected or uninfected) was the timing of evaluation relative to birth (early, lass than days or late, more than days). principal component analyses revealed significant differences between patients with early or late sepsis despite the presence of similar key immunologic pathway aberrations in both groups. this study highlights both the uninfected state and the host responses to sepsis are significantly affected by timing relative to birth. a study of vlbw infants with blood culture-proven late-onset sepsis ( % cons) identified a -gene signature associated with sepsis, with increased expression of the tnf-α network, including matrix metalloproteinase and cd among the most commonly up-regulated genes. elevated matrix metalloproteinase mrna expression and activity in septic shock correlated with decreased survival and increased organ failure in pediatric patients. matrix metalloproteinase is a direct activator of nf-κb. inhibition (genetic or pharmacologic) of matrix metalloproteinase leads to improved survival and a blunted inflammatory profile in a murine model of sepsis. most recently, a -gene network was uncovered and validated that accurately identified infected infants, who exhibited increased expression phosphorylation, ubiquitination, sumoylation) may occur after sepsis. , these dna alterations may modify transcription factor access of gene-specific promoter regions, ultimately leading to short-term and long-term changes in gene expression and immune function. the dna methylation pattern in the promoter region of the calca gene varied in different types of bacterial sepsis in preterm infants, suggesting its potential use as an epigenetic biomarker. trained immunity, the term coined to describe an adaptive innate immune response, may also be a positive or negative consequence of sepsis in early life. mechanisms that underlie trained immunity are beginning to emerge, and include dna methylation and modification of energy utilization pathways. , nonspecific vaccine benefits and resistance to subsequent pathogen challenge after innate immune priming or previous infection are likely manifestations of trained immunity in neonates. , , the cell types, extent, and duration of trained immunity-based modifications in neonates with sepsis have not been studied. myeloid suppressor cells manifest immunosuppressive activity with sepsis and were recently described in neonates. myeloid suppressor cells are present at high frequency at birth and decline in number with postnatal age. they inhibit t-cell proliferative responses and ifn-γ production. reactivation of viral infection that may contribute to morbidity and mortality has been demonstrated in infected adults. the impact of this phenomenon in neonates is unknown. acute hypoxic respiratory failure, ards, and acute lung injury are common pulmonary complications associated with severe sepsis. destruction of the alveolar capillary membrane leads to refractory hypoxemia. after direct or indirect insults to the lung, alveolar macrophages produce chemokines that mitigate pmn influx to lung parenchyma. activated pmns release reactive oxygen and reactive nitrogen intermediates that damage endothelial and epithelial barriers, leading to leakage of protein-rich edema fluid into the air spaces. other pulmonary complications with severe sepsis may include secondary surfactant deficiency, primary or secondary pneumonia, and reactive pulmonary hypertension. , infants with sepsis and persistent pulmonary hypertension of the newborn may require inhaled no in addition to optimized ventilation strategies such as highfrequency oscillatory ventilation. if oxygenation or tissue perfusion remains severely compromised despite optimal medical management, extracorporeal membrane oxygenation should be considered in neonates weighing more than kg without contraindications. , the detrimental neurodevelopmental long-term impact of sepsis has been demonstrated in multiple studies and has been reviewed in detail. [ ] [ ] [ ] [ ] [ ] central nervous system injury is predominantly white-matter injury (loss of pre-oligodendrocytes), manifested by focal cystic periventricular leukomalacia, diffuse necrosis, or a combination of these entities. , central nervous system injury is, in part, mediated by inflammation with or without direct pathogen invasion. , , the impact of sepsis on central nervous system injury is intensified with lower gestational ages, highlighting the detrimental effects of sepsis on the developing brain. the importance of evaluating the preterm infant for disseminated infection that may include meningitis cannot be overemphasized. a complete evaluation, including cultures of blood, urine and cerebrospinal fluid, is uncommon, although one third of the cases of culture-positive meningitis in vlbw infants are associated with negative concurrent blood cultures. clinically apparent seizures may occur in % of vlbw preterm infants with meningitis. low-voltage background pattern, sleep-wake cycling, and seizure activity on but normal ejection fraction in preterm neonates with septic shock suggests that septic shock in preterm neonates is predominantly due to vasoregulatory failure. neonatal sepsis may or may not be associated with left ventricular diastolic dysfunction; however, cardiac injury as manifested by elevated levels of cardiac troponin t may complicate the clinical picture. , abnormal peripheral vasoregulation with or without myocardial dysfunction is the primary mechanism for the hypotension accompanying septic shock in the neonate. therefore, infected neonates may present with hypotension and adequate perfusion (warm shock) or inadequate perfusion (cold shock). myocardial dysfunction can lead to ventricular wall stretch that in turn elevates b-type natriuretic peptide levels. b-type natriuretic peptide levels are elevated in children with septic shock, and increased levels have utility as prognostic indicators of death. plasma no level is elevated in neonates with sepsis and shock compared wit those with shock alone. elevated serum lactate level (> mmol/l) distinguished nonsurvivors from survivors in a pediatric study that included neonates. after severe sepsis or septic shock, there is an increased risk for subsequent infection and death in children and adults. this phenomenon is termed immunoparalysis and is associated with reduced mhc class expression and tnf-α production by mononuclear cells after endotoxin stimulation. in addition to altered monocytic responses, there is significant loss of lymphoid cd + t and b cells via caspase-dependent apoptotic pathways. , whether by clonal selection, apoptosis, or elevated endogenous glucocorticoid levels, [ ] [ ] [ ] lymphocyte loss may lead to a state of immune compromise after the acute phase of sepsis. , , [ ] [ ] [ ] [ ] [ ] new data suggests that il- may play an important role in promoting t-cell activation and prevention of apoptosis. the importance of immunoparalysis has been convincingly demonstrated in infected adults - and children. however, the clinical impact in the preterm neonate in whom adaptive immune function is less well developed is uncertain. , in examinations of peripheral blood and postmortem spleens from infected adults, there is significant loss of b and cd + t lymphocytes and dendritic cells, , resulting in decreased antigen presentation, antibody production, and macrophage activation. circulating peripheral absolute lymphocyte counts can drop significantly in adults with sepsis but this phenomena is also seen in critically ill adults who are not infected. sustained lymphopenia significantly increases the risk for secondary infection, mods, and death in children. extensive loss of lymphocytes (both b and t lymphocytes) has been described in postmortem specimens from the thymus and spleen in infected preterm and term infants. , , [ ] [ ] [ ] [ ] the number and the size of the follicles in the spleen decreased significantly and the total number of cells decreased by more than three times; similar changes were found in lymph nodes. however, these histopathologic splenic findings are in contradiction to earlier reports where no differences were described in infected and uninfected infants. splenomegaly may occur in infants with late-onset sepsis and may be due to splenic congestion in the absence of hyperplasia of white pulp. the mechanisms responsible for immune alterations after sepsis are beginning to emerge. the intensity of the inflammatory response may be modified by neural-based mechanisms. t cell-secreted acetylcholine acts on macrophages to reduce production of tnf, il- , il- , hmgb- , and other cytokines. the role of vagal tone in the neonatal host response to sepsis is unclear. discovery and characterization of the impact of epigeneticmediated immune system functional alterations after sepsis is an area of intense research. dna methylation and posttranslational modification of histone proteins (methylation, acetylation, prevented hmgb- level elevation, and was associated with longer survival times. increased plasma nitrite and nitrate concentrations are associated with the development of multiple organ failure in pediatric patients with sepsis , but have not been investigated in neonates. the incidence of neonatal sepsis remains high and outcomes remain poor despite considerable technologic advances in the field of neonatology. much remains to be learned about the impact of developmental age on the host response to sepsis and what facets are critically important. important considerations for future investigations include the development and implementation of a generally accepted definition for neonatal sepsis, the use of homogeneous systems (only neonatal components) for human ex vivo studies, and transgenic approaches in preclinical models, alongside observational studies in humans to ensure meaningful findings. complete reference list is available at www.expertconsult.com. the amplitude-integrated electroencephalogram may be helpful to predict neurologic outcome in infants with sepsis or meningitis. significantly lower resistance, vasodilatation, and higher blood flow were noted in all the cerebral arteries of infants with sepsis. increase in cerebral blood flow velocity was correlated with elevated il- concentrations. alterations in blood flow in preterm infants, in addition to factors associated with sepsis, such as respiratory distress, hypercarbia, hypotension, and patent ductus arteriosus, contribute to the risk for intracerebral hemorrhage. endocrine abnormalities may include altered thyroid function and adrenal insufficiency associated with refractory hypotension. inadequate adrenocortical responses are associated with increased mortality. , cortisol production in the neonate is significantly increased early in septic shock. however, very preterm neonates can have relative adrenal insufficiency that may contribute to hemodynamic instability and hypotension. hydrocortisone has not been evaluated in large prospective randomized clinical trials for the treatment of septic shock in the neonate but it has been shown to increase blood pressure, decrease heart rate, and decrease vasoactive medication requirements in preterm and term neonates in addition to its cytokine-suppressing effects. [ ] [ ] [ ] if hydrocortisone treatment is considered, the obtaining of a pretreatment serum cortisol level is prudent in order to differentiate contributing causes of hypotension. sepsis was the most common cause ( %) of acute kidney injury in term neonates and was associated with high mortality ( %) (n = ). the frequency of acute renal failure (defined as a blood urea nitrogen level greater than mg/dl) in infected neonates was % and oliguria occurred in % of acute kidney failure cases. acute kidney injury in preterm neonates is associated with high mortality. hepatic injury and dysfunction are frequent associations with severe sepsis. the mechanisms include reduced hepatic perfusion associated with septic shock and mitochondrial energy failure. reductions in coagulation and complement factors, acute-phase reactant proteins, and increases in the levels of transaminases and bilirubin are commonly seen, especially in association with decreased perfusion states. energy expenditure and oxygen consumption are increased during sepsis, and decreased mitochondrial oxidative function precipitated by hypoxia and the presence of reactive oxygen intermediates may lead to impaired growth, caloric deficiency, and energy failure. , sepsis that leads to mods carries a dismal prognosis. inadequate cardiac output and microcirculatory failure, which may be combined with formation of microthrombi and dic, can lead to poor perfusion to the kidney, , liver, gut, and central nervous system. , , , , recent studies suggest that the mechanism of organ failure in sepsis may relate to decreased oxygen utilization associated with mitochondrial dysfunction rather than poor oxygen delivery to tissues. , mitochondrial dysfunction can initiate activation of cell death pathways, including apoptosis, pyroptosis, necrosis, and netosis (i.e. cell death mediated by nets). damage-associated molecular patterns (including nucleosomes and microparticles) created by activation of these cell death programs 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concentrations and multiple organ failure in pediatric sepsis key: cord- -g u xg authors: wang, disen; fang, hui title: an adaptive response matching network for ranking multi-turn chatbot responses date: - - journal: natural language processing and information systems doi: . / - - - - _ sha: doc_id: cord_uid: g u xg with the increasing popularity of personal assistant systems, it is crucial to build a chatbot that can communicate with humans and assist them to complete different tasks. a fundamental problem that any chatbots need to address is how to rank candidate responses based on previous utterances in a multi-turn conversation. a previous utterance could be either a past input from the user or a past response from the chatbot. intuitively, a correct response needs to match well with both past responses and past inputs, but in a different way. moreover, the matching process should depend on not only the content of the utterances but also domain knowledge. although various models have been proposed for response matching, few of them studied how to adapt the matching mechanism to utterance types and domain knowledge. to address this limitation, this paper proposes an adaptive response matching network (arm) to better model the matching relationship in multi-turn conversations. specifically, the arm model has separate response matching encoders to adapt to different matching patterns required by different utterance types. it also has a knowledge embedding component to inject domain-specific knowledge in the matching process. experiments over two public data sets show that the proposed arm model can significantly outperform the state of the art methods with much fewer parameters. with the prevalence of intelligent personal assistant systems, it becomes increasingly important to build an effective chatbot that can communicate with humans fluently and help them fulfill different tasks. moreover, chatbots also play an important role in many other applications such as customer support and tutoring systems [ , , ] . one of the key components of any chatbots is the underlying response matching model, whose goal is to identify the most appropriate response from a pool of candidates given past conversations. the past conversations include both user inputs and the system's previous responses. as illustrated in fig. , a user is having a multi-turn conversations with the system (e.g., chatbot). given the current input "something is slowing me down bad" as well as the past utterances, the system is expected to identify the most appropriate candidate response, i.e., "what is the process name with the highest cpu usage?", among all the candidates. an optimal response matching model needs to address the following three challenges. ( ) it needs to capture a matching relationship that goes beyond simple lexical or semantic similarities. as shown in the fig. , the current input and its correct response do not share any similar words, so the models that are only based on word similarity would not work well for this problem [ , , , ] . ( ) it should be able to capture different matching relationships for different utterance types. past utterances include two types of information: past inputs from the user, and past responses from the system. intuitively, given a candidate response, its desirable matching relationship with past inputs should be different from that with past responses. for example, the correct response needs to address the questions or concerns described in the current input, while it needs to avoid repeating the same information as those mentioned in the past responses. ( ) it can utilize domain knowledge to understand the specific meanings of some utterances. for example, "htop" is a process monitoring application for linux. without understanding the meaning of this command, it would be difficult for the system to figure out that htop is related to the cpu usage. most existing studies on multi-turn response selection [ , [ ] [ ] [ ] ] mainly focused on the first challenge (i.e., modeling the matching relationships that go beyond semantic similarity), but little attention has been paid to address the last two. in this paper, we propose an adaptive response matching network (arm) to address all three challenges. specifically, a novel response matching encoder is proposed to model the response matching relationship between each utterance and response pair. for each utterance-response pair, instead of computing the matching score based on word or segment level, the encoder computes a response matching matrix through transfer matrices and the multi-attention mechanism. to adapt to the different utterance types, separate encoders are trained: one encoder is used to capture the relationship between current response and the past responses, while the other is used to model the relationship between the current response and the past input. finally, a knowledge embedding layer is added to the model, and such a layer enables the model to leverage domain knowledge during the response matching process. to validate the proposed arm model, we conduct experiments over two public data sets. experimental results and further analysis demonstrate the proposed arm model is able to achieve superior performance in terms of both effectiveness and efficiency when compared with the state of the art methods. given the past conversations, a chatbot can either automatically generate responses [ , , ] , or retrieve the most appropriate response from a pool of candidates [ , , , ] . this paper focuses on the retrieval-based models for multi-turn response selection, and we now briefly review the related work in this area. early studies concatenated all of the past utterances together and computed the matching score between the merged utterance and each candidate response [ , , ] . specifically, the dual-encoder model [ ] used two lstms to generate the embeddings for the utterances and candidate response respectively to compute the matching score. the deep relevance matching model (drmm) [ ] and the arc-ii model [ ] were proposed for ad-hoc retrieval tasks, but they were also applied to the response selection problem when past conversations were used as queries and candidate responses were used as documents. one limitation of these models is that they concatenated all the previous utterances before embedding. sequential matching network (smn) [ ] was proposed to address this limitation. it treated each utterance separately, computed the word-level and segment-level similarity for each response-utterance pair, and used a cnn to distill matching features. deep matching network (dmn) [ ] also treated each past utterance separately. it extracted relevant question-answer posts from the collection and utilize the pseudo relevance feedback methods to expand candidate responses with the extracted q/a pairs. these methods are mainly based on the word semantic similarity scores [ , ] . however, semantic similarity is not sufficient to capture the relationship between a past utterance and a candidate response as shown in fig. . more recently, inspired by the transformer structure [ ] , the attention mechanism has been applied to find better semantic representations of the utterances. deep attention model (dam) [ ] proposed self-attention and cross-attention to construct semantic representations at different granularity, and the multirepresentation fusion network (mrfn) [ ] has further applied multiple representation strategies for utterances and fused them in the final step to compute the matching scores. all these previous studies mainly tackled the first challenge discussed in sect. , and focused on developing models to capture the matching relationship between the past utterances and candidate responses. few of them studied how to adapt the matching model to different types of utterances and how to incorporate the domain knowledge in a more general way, which is the focus of our paper. the problem of multi-turn response selection can be described as follows. assume we have a training data set denotes a conversation consisting of multiple utterances. each utterance could be either a previous input (p i,j ) or a previous response (q i,j ). r i denotes a candidate response for u i , and l i ∈ { , } indicates whether r i is the correct response for u i . the task is to learn a model f based on d, which can compute the response matching score between any response r and utterance u. given a new utterance, the learned model f will rank a list of candidate responses based on their matching scores, and the one with the highest score will be selected as the final response. in this paper, we will also study how to incorporate the domain knowledge in the training process. specifically, we assume that the knowledge base can be denoted as follows: where c i is a domain-specific keyword, and g i is the corresponding description of that keyword. take the conversations shown in fig. as an example, c i could represent the command htop and g i would be the description of that command. both d and k will be used to learn the model f . the key challenge in multi-turn response selection model lies in how to model the matching relationship between a candidate response and the past utterances. almost all of the recent studies followed a three-step procedure: representation, matching and accumulation [ , ] . the first step is to represent candidate responses and utterances in various ways that can capture their semantic meanings. the second step is to compute the matching scores between the utterances and the candidate response based on these representations. and the last step is to accumulate all the scores into the final one. our proposed adaptive response matching network model (arm) also follows the above three-step procedure, with major differences in the first two steps. figure shows the overall architecture of the arm model. compared with the existing studies, arm aims to develop a matching mechanism that can adapt to the domain knowledge (in the representation step) and different types of utterances (in the matching step). in the arm model, the representation module first converts each utterance into a segment-level vector representation using word embedding and gru, and then enhances the vector representation with domain knowledge using knowledge embedding layer. after that, the matching module utilizes the representations of the utterance vectors and the response vectors to calculate the response matching score, and extracts features to feed into the accumulation module. more specifically, multiple transfer matrices are trained to transfer hidden states vectors into different representations spaces, and the combination of response matching matrix from each representation space is used to compute the matching score in the response matching encoder. the segments in an utterance that are important for recognizing appropriate response will have higher matching scores. the areas with higher scores in the final relevance matrix will be extracted by cnn network. finally, the accumulation module generates the final matching score based on the matching vectors provided by the matching module. a past utterance could be either an input from the user or a response from the system. both utterance types are useful to select the matching candidate response, but in a different way. an input often describes a request or a problem encountered by a user, so the correct candidate response is expected to be the solution to the inputs. on the contrary, a candidate response is expected to be a follow-up or clarification of previous responses. let us take a look at the example illustrated in fig. again. the correct response, i.e., "the process name with the highest cpu usage", is expected to be the solution to the request related to "something is slowing me down". on the other hand, it is a follow-up question to the previous response, i.e,. "do you have any task manager installed?" clearly, it is necessary to ensure the matching model be adaptive to different utterance types. in other words, when selecting a response for the given utterances, different matching mechanisms need to be used for past inputs and past responses. although a few models have been proposed to solve the problem of multi-turn response selection [ , , , ] , none of them studied how to directly adapt the matching mechanisms to different utterance types. instead, they mainly focused on exploring various complicated representations of the utterances with the hope that these representations can better capture the semantic meanings of the utterances. although the more complex representations can lead to better effectiveness, they often require more computational resources and longer time to train and test. in the arm model, we propose adaptive response matching encoders to learn different matching patterns according to the utterance types. the basic idea of arm encoders is to start with some basic semantic representations of the utterances/responses, and then learn new matching representations for each matching type. the new matching representations are expected to better capture the response matching relationship for each utterance type. we now describe the encoders in detail, and the important notations are summarized in table . following the previous study [ ] , we represent both responses (r) and utterances (either p for a previous user input or q for a previous response) using segment-level representation. specifically, we first apply word vec [ ] algorithm to generate the word embedding e ∈ r d for each word, where d is the number of dimensions in the word embedding. the model looks up a pre-trained word embedding table to convert p = [w p, , w p, , ..., w p,np ] into p = [e p, , e p, , ..., e p,np ], where w p,i is the i-th word and e p,i is the corresponding word embedding vector, and n p is the length of p. similarly, we can represent q as q = [e q, , e q, , ..., e q,nq ] and r as r = [e r, , e r, , ..., e r,nr ] where e q,i , e r,i ∈ r d , are the embeddings of the i-th word of q and r, and n q and n r are the length of q and r. to extract the contextual information in each utterance, we feed p , q and r into gru network [ ] and use the generated hidden states h p , h q and h r as basic representations of the responses and utterances. learning new matching representations: given the basic representations of a previous utterance and a response (e.g., h p and h r ), two transfer matrices (i.e., w p and w r ) are learned to transfer the basic representations to the new ones (i.e., rm p and rm r ) that can better capture the response matching relationship. formally, the new representations (i.e,. relevance matrix) of the utterance and the response can be computed using where w p , w r ∈ r h×m , h is the number of dimensions in hidden states, and m is the number of dimensions in the response matching encoder. w p and w r are transfer matrices and will be learned from the training data. the initial weights of these matrices are randomly initialized with different values, and the training data contain the labels indicating whether a utterance is a input or a response. with the newly learned matching representations of a utterance and a candidate response, we can compute a response matching matrix as follows. in order to capture different representations that could be useful for response matching, we apply the multihead attention mechanism [ ] to the response matching encoder. the structure of multi-head encoder is shown in fig. . we learn different transfer matrices to transfer the hidden states vectors into different representation spaces, and combine the matrices from each representation space to get the final matrix m . specifically, we stack matrices m , m , ..., m x into one matrix and multiply it with a transfer matrix w t to get the final response matching matrix m , where the elements of w t are learning parameters. w t learns how to combine information from multiple channels into the final response matching matrix. in the final response matching matrix m , elements with higher values mean the corresponding word pairs have higher semantic matching scores. this multi-head mechanism can expand the model's ability to focus on different positions, and it is able to capture more diverse response matching patterns through the multiple representation spaces (fig. ). an optimal response matching model also needs to adapt to the domain-specific knowledge. as explained in fig. , if we have domain-specific knowledge about "htop", we might be able to better match the candidate responses. assume the available knowledge base can be represented as pairs of domain-specific concepts and their descriptions (i.e., (c i , g i )), we now discuss how our model adapt to domain knowledge. a straightforward way to incorporate knowledge base is to replace the domain-specific words with their definitions or descriptions in the corresponding hidden states. however, words are ambiguous, and not every occurrence of a domain-specific word refers to the same meaning. for example, the word "install" is a command in the ubuntu system. but if a user asks "how to install the task manager", the word "install" in this utterance should not be replaced by the description of the command because it did not refer to the command. to tackle this problem, we propose to apply gating mechanisms to decide when and how to fuse knowledge into utterance representations in the knowledge embedding layer [ ] . for each word w i that occurs in either the utterances or the response, if it is one of the domain-specific word (c i ), we would extract its corresponding description from the knowledge base (i.e., g i ), feed g i into a gru unit, and the generated hidden state is used as knowledge embedding, which is denoted as h k i . after that, we can apply gating mechanism to fuse knowledge embedding h k i into word representation h i to generate the new representation h new where w s and w g are learning parameters, and [v , v , v ] means to stack the three vectors into a matrix. with the gating mechanism, the new word representation h new i has selectively adapt to the domain knowledge based on the context information. for each dimension in the vector h i , the gating mechanism decides whether to keep the original value or replace it with the value from knowledge embedding. without the gating mechanism, the model would replace values in all dimensions with those from knowledge embedding, which might not be always the best solution. the new representation h new i is then used to replace h i in the hidden state matrix h p , h q and h r , i.e., the input of the adaptive response matching encoders. the arm model can be regarded as an extension of the smn model [ ] with a couple of notable differences. first, the model uses adaptive response matching encoders to learn different matching patterns according to the utterance type, and the captured matching relationship is able to go beyond the simple semantic similarity. second, the model adds a knowledge embedding layer (in the representation module) to provide a general way to incorporate domain knowledge. these two differences enable the arm model better capture the response matching relationship, and explain why the arm model is more effective than the state of the art models. dmn [ ] is the only existing study that tried to utilize knowledge base. it extracted question-answer pairs from the collection as knowledge, and then utilized the pseudo relevance feedback methods to expand candidate responses with the extracted knowledge. on the contrary, the arm model presents a more general and robust way of incorporating domain-specific knowledge. in particular, the representation of the knowledge base is more general. the knowledge can come from either collection itself or external domain-specific resources. moreover, the gating mechanism in the embedding layer makes it possible to selectively apply the domain knowledge based on the context information, which can improve the robustness of the model. another major advantage of arm model lies in its efficiency. when designing the model, we intentionally use the basic semantic representations (i.e., word/knowledge embedding and gru outputs) and let the transfer matrices to learn useful matching relationships for each utterance type. such a learning process is more targeted than learning a general yet more complicated representation that could be useful to match all kinds of utterance types [ , ] . as shown in sect. . , our model uses much fewer parameters than the state of the art models, yet it is able to achieve better performance. data sets: we evaluate the performance of the proposed model on the two publicly available data sets from the dstc response selection challenge . ( ) the first data set is the ubuntu dialogue corpus, which contains conversations about solving an ubuntu user's posted problem. the domain knowledge includes the commands and their corresponding function descriptions. the training set contains million conversations, the development set and the test set each contains . million conversations. ( ) the second data set is the student-advisor data set. in each conversation, the advisor will guide the student to pick courses. the external knowledge is about the courses such as their descriptions and areas. the training set contains . million conversations, development set and test set each contains , conversations. following the previous studies [ , ] , the primary evaluation measures are r @k, which is the recall at position k in candidates. k is set to , , . since there is only one correct response for each conversation, the precision at position k always equal to the recall at position k divided by k. thus, only the values of recall are reported. parameter settings: in our model, dropout layer was added to the cnn network, and the dropout rate is set to . . zero padding was applied to make the size of interaction matrix same for all utterance-response pairs. the size of interaction matrix after padding is × . we also set the maximum length of utterance and response as words. the learning rate was set as . , and experiments show that larger learning rate would lead to a significant decrease in performance. we applied word vec algorithm to train the word embedding matrix, and the number of word embedding dimensions is . all these hyperparameters are chosen to be consistent with the previous study [ ] . we compare the performance of the proposed arm model with several state of the art baseline methods over both data sets. in particular, the baseline models include the following seven state of the art response matching models: dualencoder [ ] , drmm [ ] , arc-ii [ ] , smn [ ] , dmn [ ] , dam [ ] and mrfn [ ] , which were briefly reviewed in sect. . the implementations of the baseline models were obtained either through the code published by the authors or matchzoo on the github. the results of performance comparison are summarized in table . it is clear that our proposed arm model outperforms all the strong baseline models significantly on both data sets, which indicates the effectiveness of the arm model in addressing the limitations we discussed before. we conduct additional experiments to better understand the proposed model. the proposed arm model is an extension of the smn model [ ] with the addition of two important components: adaptive response encoders and the knowledge embedding layer. to better understand the effectiveness of these additions, we conduct additional experiments by disabling each of these new additions, and the results are shown in table . arm-s is the variation of arm model, where we disable the separate encoders and use the same response encoders for different utterance types. arm-k is the variation of arm, where we disable the knowledge embedding layer. when we compare the two variations with the arm model, it is clear that both type-adapted encoders and the knowledge embedding layer are useful to improve the performance. another key difference between arm and smn model is how to model the response matching relationships. smn mainly relies on the semantic similarity, while the proposed response matching encoder can capture other semantic matching relationship. the difference can be easily seen through an example shown in fig. . the darker color means a higher score. it is clear that the arm model can correctly capture the matching relationship between {slowing, me, down} from the input and {process, name, with, the, cpu, usage} from the correct response, while the smn cannot. furthermore, arm is shown to be more effective than the recently proposed dam and mrfn models according to table . in fact, it is also more efficient. similar to dam and mrfn, arm aims to capture the matching relationship that goes beyond simple semantic similarity. instead of learning general yet more complicated representations for all utterances, arm is more focused and it explicitly adapts to different matching patterns caused by different utterance types. as a result, arm is more efficient and requires much fewer parameters than dam and mrfn. with the same hyper-parameter values (e.g., the number of dimensions in word vec vectors), the numbers of parameters used in the arm, dam and mrfn models are millions, millions, and millions respectively. fewer parameters means significant improvement in terms of efficiency. in this paper, we propose an adaptive response matching network for multiturn chatbot response selection. existing models focused on modeling different relationships using multiple representation strategies, while the arm utilizes adaptive response matching encoders in the matching module to directly model different matching relationships for different types of utterances. moreover, arm has a knowledge embedding layer, which can adapt to external domain knowledge in a general way. empirical results over two data sets show that the proposed model outperforms various state-of-the-art models in terms of both effectiveness and efficiency. in the future, we plan to study how to incorporate unstructured domain knowledge to further improve performance. an experimental evaluation of apple siri and google speech recognition enhanced lstm for natural language inference learning phrase representations using rnn encoder-decoder for statistical machine translation learning to forget: continual prediction with lstm a deep relevance matching model for ad-hoc retrieval convolutional neural network architectures for matching natural language sentences a personabased neural conversation model training end-toend dialogue systems with the ubuntu dialogue corpus efficient estimation of word representations in vector space alime chat: a sequence to sequence and rerank based chatbot engine from eliza to xiaoice: challenges and opportunities with social chatbots a neural network approach to context-sensitive generation of conversational responses multi-reprentation fusion network for multi-turn response selection in retrieval-based chatbots attention is all you need a network-based end-to-end trainable task-oriented dialogue system ranking responses oriented to conversational relevance in chat-bots a sequential matching framework for multi-turn response selection in retrieval-based chatbots learning to respond with deep neural networks for retrieval-based human-computer conversation system response ranking with deep matching networks and external knowledge in information-seeking conversation systems multi-turn response selection for chatbots with deep attention matching network acknowledgement. the first author is grateful to the jp morgan chase scholarship he received from the ph.d. program in financial services analytics to support this research.an arm for ranking multi-turn chatbot responses key: cord- - p nr authors: schroyen, martine; tuggle, christopher k. title: current transcriptomics in pig immunity research date: - - journal: mamm genome doi: . /s - - - sha: doc_id: cord_uid: p nr swine performance in the face of disease challenge is becoming progressively more important. to improve the pig’s robustness and resilience against pathogens through selection, a better understanding of the genetic and epigenetic factors in the immune response is required. this review highlights results from the most recent transcriptome research, and the meta-analyses performed, in the context of pig immunity. a technological overview is given including wholegenome microarrays, immune-specific arrays, small-scale high-throughput expression methods, high-density tiling arrays, and next generation sequencing (ngs). although whole genome microarray techniques will remain complementary to ngs for some time in domestic species, research will transition to sequencing-based methods due to cost-effectiveness and the extra information that such methods provide. furthermore, upcoming high-throughput epigenomic studies, which will add greatly to our knowledge concerning the impact of epigenetic modifications on pig immune response, are listed in this review. with emphasis on the insights obtained from transcriptomic analyses for porcine immunity, we also discuss the experimental design in pig immunity research and the value of the newly published porcine genome assembly in using the pig as a model for human immune response. we conclude by discussing the importance of establishing community standards to maximize the possibility of integrative computational analyses, such as was clearly beneficial for the human encode project. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. anticipating the need to efficiently feed an estimated billion people, pig farms have significantly increased in size, which also unfortunately increases the risk of disease incidence. a better understanding of the immune system in swine is essential, because susceptibility to infectious diseases has great influence on pig performance (mellencamp et al. ; boddicker et al. ) . selection for improvement in economical traits such as feed conversion and prolificacy is already routine practice, and improvement of immune capacity through selection is gradually catching up (edfors-lilja et al. ; uddin et al. ; lu et al. ). in addition, pigs are an important biomedical model for humans as they share great similarity in anatomy, physiology, genetics, and genomics, including many genes in the immune system (dawson ; dawson et al. ) , which is very helpful when modeling human immune responses and diseases (lunney ; fairbairn et al. ; kapetanovic et al. ; meurens et al. ) . moreover, pigs have recently been genetically modified to serve as an improved model for human disease (suzuki et al. ) . for more genetically modified pig models for human diseases, we refer the reader to the reviews of ross and prather ( ) and walters et al. ( ) . at the genomics and transcriptomics level, the pig is a very appealing model since one can extrapolate more easily to human due to the high homology in gene sequence and chromosomal structure (groenen et al. ). the immune system components of the pig genome (immunome) have become the best-annotated genes in the current genome (dawson et al. ) . this improved pig genome annotation will greatly aid creation of biomedical models. when the cause of a medical condition in humans is found, the corresponding mutation(s) can be genetically engineered in pigs to mimic the disease, and (novel) drug therapies can be tested (walters et al. ) . differences in genotype associated with a different immune response phenotype in pig can easily be examined with the use of the porcine k high-density snp chip (boddicker et al. ; lu et al. ; wang et al. a ). the last decade witnessed a steep increase in the amount of transcriptomic pig immune response data using whole porcine genome microarrays. meanwhile, custom-made arrays enriched in annotated genes of the pig immune system were used to address specific research questions (gao et al. ). in addition, novel techniques are surfacing in studies focusing on the porcine immune response transcriptome. high-density oligonucleotide tiling arrays and next generation sequencing (ngs) have been further broadening the knowledge of the pig's immunome (mockler et al. ; morozova and marra ) . these techniques are able to identify and quantify known and unknown transcripts and can be used to extend recent efforts made on the pig genome annotation (gao et al. ) . the understanding of the function of diverse mirnas will profit immensely from ngs, and papers on the porcine mirnaome are being published sharbati et al. ) , as surely soon will follow on epigenetic changes such as dna methylations or histone modifications. several research groups have graciously provided information to our review on their recently published and unpublished studies, including whole genome microarrays to rna-seq, mir-na-seq, rip-chip, and some of those methylation and histone modification studies (table ) . the aim of this review is to summarize the use and results of recent transcriptomic research in pig immunity, identify current needs in the field, and anticipate future areas of progress. an overview will be given of studies using whole genome microarrays and immune-specific arrays, small-scale high-throughput expression methods, high-density tiling array, and ngs (table ; fig. ). experimental design concerns and studies using the newest sequencing techniques and epigenetic tools together with a systems biology approach to interpret the data will also be discussed. for this review, we searched the entrez pubmed database, using keyword search terms such as pig transcriptomics, pig immunity, microarray, immune-specific array, high-throughput qpcr, rna-seq and mirna-seq, and systems biology. in addition, references from the articles obtained by this method were checked for additional relevant material. the arrayexpress database from ebi was consulted to create supplementary table , using sus scrofa as organism and filtered on 'array assay' as technology. whole genome microarray studies, the start of global high-throughput transcriptomics the range of genes assayed with whole genome microarray depends on the array used (table ; fig. ). the first porcine whole genome array available, the qiagen-nrsp array, was a result of collaboration between qiagen-operon and the usda-nrsp- swine genome community (zhao et al. ) . until then, human arrays were used to tackle porcine transcriptomic issues, or swine arrays were developed for a specific research question, as described in the next section. the pigoligoarray was a second-generation porcine -mer oligonucleotide array (steibel et al. ). currently, the most well known are commercially available microarrays such as the porcine gene expression microarray of agilent and the genechip porcine genome array of affymetrix (tuggle et al. ; huang et al. ; zhou et al. ; bao et al. ) . freeman and colleagues recently introduced the affymetrix snowball microarray, which is more comprehensive than the first affymetrix chip in coverage both for transcript structure and for the transcriptome as a whole, containing more than double as many probes (freeman et al. ) . details and examples of these whole genome arrays are shown in table . an overview of pig immunity experiments submitted to the arrayexpress database is given in supplementary table . since global porcine microarray studies have been extensively described earlier , ), we will limit this section by highlighting the importance of using microarrays in a systems biology approach; we do provide a comprehensive table on all porcine immunerelated microarray experiments submitted to arrayexpress (supplementary table ) . several microarray studies have been conducted to study host response to porcine reproductive and respiratory syndrome (prrs) using commercial or more specific pig annotated microarrays (bates et al. ; genini et al. a; ait-ali et al. ; wysocki et al. ) . badaoui et al. ( ) recently illustrated how the information of multiple prrs studies could be used simultaneously to gain insight on host response to prrsv challenges. they collected all publicly available microarray data covering multiple porcine immunology studies and including many different breeds, tissues, pathogens, and array platforms. the data of general immune response arrays were assembled, and separate meta-analyses for differential expression were performed using these arrays as well as a subset of arrays specifically from prrs experiments (badaoui et al. ) . to find prrs-specific expression responses, they eliminated differentially expressed genes common to these two metaanalyses. other meta-analysis studies, examining the immune response to salmonella typhimurium, combine microarray information with data such as serum cytokine measurements or microbiota differences. the results of these meta-analyses performed on prrs or s. typhimurium will be discussed in the section ''overall value of transcriptomics in important infectious swine diseases.'' in addition, whole genome microarrays were used to study pig response to haemophilus parasuis infection by zhao et al. ( ) . in one of their earlier studies, a genome-wide affymetrix array experiment revealed differentially expressed genes between non-infected and h. parasuisinfected pigs in spleen at dpi ). among them were retn, s a , s a , and s a , all important innate immunity genes marking inflammation. chen et al. ( b) reexamined the raw data of this first experiment and used a more robust genechip rma (gcrma) normalization (wu and irizarry ) , and an improved annotation using anexdb (couture et al. ) . a network analysis revealed that the ps a /ps a -casp -slc a pathway played an important role in h. parasuis infection, and cebpb may act as a transcription factor of the two s family members (chen et al. b ). later, zhao and co-workers describe a systems biology approach starting from the same affymetrix data. with the use of the kegg and reactome databases, , transcripts from the affymetrix chip were flagged as immunogenes. with this reduced dataset, exploratory analyses such as a principal component analysis (pca) and a geneset enrichment analysis (gsea) were conducted. with this pca/ gsea method, they came to a core set of differentially expressed genes, indicative of an h. parasuis infection (zhao et al. ) . to construct the immunologically important topology involved in an h. parasuis infection, the c net algorithm was used (altay and emmert-streib ). several networks were created, and the largest network had the complement gene c r as hub; although c r was not differentially expressed on the microarray, it was predicted to play a crucial role in the immune defense. with this immunome-focused method, more subtle differences could be pulled out, and much new information was obtained as compared to the earlier whole genome array analysis (zhao et al. ). the immune-specific array, a focused microarray besides using commercially available or custom-made transcriptome-wide microarrays, or examining only immunogenes on those arrays, arrays focusing only on the genes involved in the porcine immune response are also used (table ). in the past, several of these arrays were constructed to specifically focus on key immune genes (ledger et al. ) , to explore gene expression in a specific immune tissue (dvorak et al. ; machado et al. ; niewold et al. ) , or to examine host interactions with a specific pathogen (zhang et al. ; skovgaard et al. ). more information on these studies is given in table . next to these small-scale arrays, other immunespecific arrays were made using an existing whole genome array and adding probes examining immune-related genes. in a study by flori and co-workers, the qiagen-nrsp array was extended by adding probes for pseudorabies virus rnas as well as probes for transcripts from the porcine major histocompatibility complex, also called the swine leukocyte antigen (sla) complex, referred to as the qiagen ? sla/prv in fig. . this array was the first to simultaneously examine viral transcripts and porcine immune transcripts (flori et al. ). gao and colleagues used the nrsp - k chip enriched with sla genes and immunity genes outside the sla complex and called it the sla-ri/nrsp - k chip (gao et al. ). in the future, when using microarrays to examine gene expression patterns, whole genome arrays or the extended versions of them are clearly preferred. when the objective of the study is to examine only a relative small number of genes, other techniques such as fluidigm digital array or nanostring (described in more detail below) can be performed. these low cost, user-friendly, and rapid approaches make the small-scale in-house printed arrays redundant. upcoming sub-genomic-scale, high-throughput methods: fluidigm and nanostring even though qrt-pcr is often used as the routine method to confirm microarray and rna-seq findings, it has been used in many pig immune studies as a primary means to measure expression levels of a dozen or more immune genes (borca et al. ; lastra et al. ; islam et al. a, b; uddin et al. ; martins et al. a, b; uddin et al. ), together with suitable housekeeping genes for normalization . although it is a very accurate and sensitive method, standard qrt-pcr has the major drawback that only a few genes in a few samples can be examined at once as compared to high-throughput methods (fig. ) . further, significant amounts of money and time are spent controlling sample and assay variation by using appropriate-and preferably multiple-housekeeping genes and validating pcr efficiencies or correcting for them by using standard curves. the use of highthroughput qrt-pcr methods, such as the fluidigm digital array, can help overcome these issues. the fluidigm digital array integrated fluidic circuit (spurgeon et al. ) is an example of a nanofluidic chip through which up to , qrt-pcr reactions can be performed at once, which represents genes tested on samples (ramakrishnan et al. ) (fig. ) . beyond sheer speed of analysis, an important advantage of nanotechnology is that a much smaller amount of rna per sample is necessary to perform assays. in livestock, the first fluidigm experiments have been published only recently, as a stand-alone expression study or as a validation of a microarray/rna-seq experiment (robic et al. ; skovgaard et al. ; sorg et al. ; pilcher et al. ) . in a h n influenza virus study, skovgaard and coworkers used fluidigm to examine the expression of several pattern recognition receptors, ifn and ifn-induced genes, cytokines, and acute response protein genes after hpi, hpi, and dpi. they expanded their study by including data on differentially expressed mirnas and mrnas in the lungs and saw several mirnas potentially targeting the differentially expressed mrnas (skovgaard et al. ) . another high-throughput method uses the nanostring ncounter system by which color-coded probes are counted to digitally measure gene expression (fig. ) . this nonamplification method can be seen as a small-scale, hybridization-based platform. per gene of interest, two sequence-specific probes are needed, a capture probe that has an affinity tag such as biotin to capture the gene on a surface, and a reporter probe with a multiple-fluorescent tag code that acts as a unique detector (geiss et al. ). its strength lies in the non-enzymatic approach, with no reverse transcriptase and no pcr amplification. this approach essentially eliminates primer-primer interactions, and up to transcripts can be measured at a time. the technique is not described in livestock species thus far. however, with the possibility to use degraded rna samples such as rna from formalin-fixed paraffin-embedded tissue samples, this method could become a very useful expression measuring tool. high-density oligonucleotide tiling array for gene expression and genome annotation high-density oligonucleotide tiling arrays have diverse genomic applications, such as transcriptome mapping and quantification. in general, tiling arrays are similar to microarrays as they both utilize oligonucleotide probes. however, through utilization of large numbers of probes, tiling arrays can span large genomic sequences without being restricted to annotated sequences (gao et al. ) . thus, tiling arrays may contain probes covering the entire genome in an unbiased manner (liu ) , and such partially overlapping probes can be used to find expressed genes, previously annotated or not. gao and co-workers used a nimblegen tiling array ( , probes) for the analysis of the transcription map of the sla complex (gao et al. ) . ninety-seven genes were found to be differentially expressed between un-stimulated peripheral blood mononuclear cells (pbmcs) and pbmcs stimulated with phorbol myristate acetate (pma)/ionomycin. these results are in nearly complete agreement with their previous experiment using the sla-ri/nrsp - k chip. furthermore, with the tiling array, they were able to confirm and refine the previous annotation of the sla genes (gao et al. ). transcriptome sequencing rna-seq is a recently developed method enabling the examination of the complete set of transcripts in a cell using a sequencing by synthesis approach ). with this technique, mrna is converted into a library of cdna fragments, and at one or both ends of these fragments, adaptors are ligated. each fragment is read in a highly parallel manner, and typical read lengths are between and bp, and go up to an average of , bp and more with the pacbio rs sequencer (ferrarini et al. ) . the resulting reads are aligned to a reference genome of the organism examined, and a genomewide transcription map can be made describing the structure and enumerating the expression level of each gene. de novo assembly of a transcriptome is also possible if the reference genome is not available or poorly annotated (martin and wang ) ; integrated approaches are also possible. rna-seq has already proven to be a cost-effective way to investigate the sequence of all mrna transcripts in a specific tissue and/or for a specific physiological condition. not only is the sequence obtained together with the quantitative level of transcripts, but also splice variants can be detected, which is a major advantage in contrast with techniques such as microarrays. moreover, with rna-seq, there is very low, if any, background noise, and no transcript specific primers or probes are required, so no prior knowledge is needed ). this latter attribute is important when working with livestock species for which genomic sequence assemblies are incomplete (bauersachs and wolf ). as such, esteve-codina and colleagues reported hundreds of un-annotated protein coding genes in the porcine genome while studying the gonad transcriptome (esteve-codina et al. ). however, projects costs are influenced by the research question asked. if the goal is to examine general expression pattern differences in well-annotated, moderately expressed genes, sequencing does not have to be very deep, and samples can be multiplexed in one lane. for novel transcripts or genes with a low expression level, higher coverage is needed, and costs will increase (the encode consortium project a). one may also consider depleting highly abundant genes of little interest, such as alpha and beta globin in a blood sample, prior to sequencing in order to improve detection sensitivity (choi et al. submitted) . in , xiao and collaborators performed a ' tag digital gene expression (dge) analysis of the porcine lung transcriptome on pigs infected with the prrs virus (xiao et al. a, b) . this dge technique can be seen as a predecessor of rna-seq. whereas rna-seq will sequence all transcripts containing poly-a ? tails, dge detects those transcripts containing catg recognition sites because the transcripts are 'tagged' through digestion of cdnas at a nlaiii restriction site (hong et al. ) . with dge, only a portion of the transcript is analyzed instead of the nearly full transcripts as seen with rna-seq ). both techniques offer similar estimates of gene expression levels but rna-seq has the advantage to be able to provide information about transcript structure and consequently can detect splice variants (hong et al. ). xiao and colleagues sequenced lung rna libraries of control noninfected pigs (n = ), prrs-affected pigs necropsied at dpi (n = ,) and prrs-affected pigs necropsied at dpi (n = ) after infection with the classical north american prrsv type (nprrsv) (xiao et al. a) or the highly virulent prrsv (hprrsv), typically found in asia (xiao et al. b ) and found , (hprrsv) and , (nprrsv) differentially expressed genes. for both types of prrsv, a higher expression of anti-apoptotic genes and a lower expression of pro-apoptotic genes could be seen as a viral strategy for replication and spread (xiao et al. a, b) . as such, suppressed expression of short type i interferon (spi ifn) and ifna, both important innate anti-viral genes, was detected. additionally, there was an upregulation of cd noted, which could indicate an increase in internalization of prrsv since a positive correlation is described between the expression of this prrsv receptor gene and prrsv infectivity (patton et al. ). patton and colleagues described that treatment with modulators such as lipopolysaccharide (lps) or il , affecting cells to express cd , as a consequence also affected the susceptibility of the host for prrsv, thereby increasing or decreasing viral infectivity (patton et al. ). miller and colleagues examined gene expression profile data obtained by sage tag analysis of trachea-bronchial lymph nodes of sham-or prrsv-infected pigs at dpi. infection occurred with both highly pathogenic hprrsv (n = ) as well as north american type nprrsv (n = ) (miller et al. ). the hprrsv strain showed a more altered gene expression profile with higher fold change differences relative to the controls than the nprrsv, indicating the increased pathogenicity of the hprrsv. among the top genes that were upregulated were three serum amyloid a acute-phase isoforms, resistin (retn), and three s calcium binding proteins, s a , s a , and s a (miller et al. ) . jiang et al. ( ) used the go, kegg, and reactome databases to analyze the data on nprrsv infection further and identified six biological system categories affected by prrsv, including cellular processes, genetic information processing, environment information processing, metabolism, organismal systems, and human diseases (jiang et al. ) . in a largescale prrsv infection study aimed at finding genes controlling variation in immune response outcomes, boddicker and colleagues identified a region on ssc , containing snp marker wur , was strongly associated with both weight gain and prrs viral load for days postinfection (boddicker et al. (boddicker et al. , . eisley and coworkers performed an rna-seq analysis of all genes in this wur region through comparing blood rna from pairs of littermates with one of two different wur genotypes. they identified a strong candidate gene differentially expressed between favorable and unfavorable genotypes (eisley et al. ) . several other immune-related rna-seq studies are forthcoming (table ) . their objective is to examine viral or bacterial immune responses in pig macrophages, dendritic cells, lymph nodes, globin depleted whole blood, and tissue samples from the gastrointestinal tract. undoubtedly, the knowledge of pig immune responses at the transcriptome level will greatly benefit from these recently published and to-be-published studies. next to exploring the ''traditional'' transcriptome, mir-naome analyses are also progressing in the pig (mcdaneld ; liu et al. ) . micrornas (mirnas) are small non-coding rnas that can regulate gene expression through degrading or interfering with the respective mrna sequence, often at the ' un-translated region ( 'utr) of the gene. during the last few years, high-throughput sequencing has lead to a steep rise in discovering novel porcine mirnas (xie et al. ) . moreover, ngs can facilitate distinguishing between mirnas and other small rna fragments, and thus, ngs is the most promising technique for exploring the mirnaome. criteria to distinguish true mirna from other rna fragments are listed in kozomara and griffiths-jones ( ) . mirna sequences and predicted targets for all important livestock animals can be found in mirbase, a comprehensive mirna information database (griffiths-jones et al. ) . with regard to immunological responses in pigs, recent studies have reported that porcine mirna can intricately engage itself in host-virus interaction networks (he et al. ; loveday et al. ; guo et al. ) . conversely, mir-nas expressed by the viral pathogen can promote a favorable host cell environment for enhanced viral replication by targeting porcine mrnas (skalsky and cullen ) . an example of this involving response to pseudorabies virus was published by anselmo and co-workers. using ngs, they analyzed both viral and host mirna expressions in infected dendritic cells, and identified viral mirnas, known porcine and new porcine mirnas (anselmo et al. ). another group used ngs techniques to analyze mirna expression profiles in pseudorabies virus infected porcine epithelial cell lines . eleven mirnas were detected in the viral genome, and known and novel mirnas assigned to the porcine genome were also found. wu and colleagues mainly focused on the viral mirnas, which were associated with regulation of viral gene transcription but also proposed to control gene expression in the host of genes annotated for immune processes, viral replication, cell death, as well as other processes. podolska and colleagues compared the mirnaome in necrotic and visually unaffected pieces of lungs from piglets infected with a. pleuropneumoniae and found conserved and candidate novel mirnas. twenty-nine were significantly up-or down-regulated between necrotic and unaffected tissue (podolska et al. ). timoneda and colleagues noted differences in mirna expression between aujeszky's disease or suid herpesvirus type (suhv- ) virus-infected and mockinfected animals, as well as differences when looking at a virulent strain of suhv- compared to an attenuated one (timoneda et al. ). mrna-seq and mirna-seq: a powerful combination toward the elucidation of mirna-rna interactions, endale ahanda and colleagues analyzed the 'utr variants of all genes of the sla region by analyzing rna-seq data. in this way, snps in mirna target sequences, potentially impacting gene expression, could be revealed. to investigate the co-expression between mrna and mirna, mrna-seq and mirna-seq data from an earlier study looking at liver, longissimus dorsi, and abdominal fat were used (chen et al. a; endale ahanda et al. ) . negative correlation between expression levels of mirnas and their predicted target genes could be found, which suggested that the prediction algorithms used were reliable (endale ahanda et al. ) . since mirnas can play an important role in hostpathogen interactions (scaria et al. ), gao and colleagues looked by means of mirna-seq at host mirnas that could target prrsv transcripts. deep sequencing was performed on pams inoculated with a mock dose or with prrsv. the resulting data were mapped against all known mirnas listed in the current mirbase. mirna target prediction revealed that one mirna family, the mir- members, seemed to suppress prrsv replication in vivo at the early stage. one mir- c target is the 'utr of cd mrna, which encodes an important prrsv receptor. mir- c is able to downregulate cd expression and thus interferes with viral attachment and penetration . similar results were seen for other mir- members. there is also good evidence that expression profile differences with regard to an s. typhimurium infection may be partially controlled by mirnas. huang and co-workers found that mir- was decreased in persistently shedding (ps) pigs in comparison with low shedding (ls) pigs ) after an s. typhimurium infection. mir- targets transcription factors cebpb and spi , which in turn control the expression of important immunogenes (adamik et al. ; wei et al. ) . bao and co-workers specifically investigated potential mirna-mrna regulatory interactions occurring during challenge with s. typhimurium. mrna-seq and mirna-seq data were collected on whole blood samples of ls and ps animals (bao et al. unpublished) . they found and mirnas up-and down-regulated at dpi when looking at ls and ps pigs together. several of them were thought to be involved in innate and adaptive immune responses. they also discovered mirnas to be higher expressed in ls than in ps pigs at day , which could be interesting candidates for biomarkers for selection toward low shedders. bao and colleagues subsequently used the sequence-based mirna target prediction software miranda to propose mirnas-mrna regulatory relationships associated with the s. typhimurium infection (bao et al. unpublished) . ye and colleagues searched for factors controlling susceptibility for enterotoxigenic e. coli with fimbria (etec-f ) in intestinal tissue in the sutai pig and found differentially expressed mirnas, and after examining regulatory networks with differentially expressed mrnas that are target of one of those mirnas, of them were shown as hubs for an enriched list of differentially expressed immune-related genes (ye et al. ) . several other porcine mirnaome studies are to be published soon focusing on the impact of mirnas after bacterial or viral infections (table ) . in addition to different expression measurement techniques to examine pig immune responses, various experimental designs to study immunity have been used in these studies. first, there are in vitro versus in vivo studies. whereas in vitro studies are performed outside the living organism, and thus can be more controlled, in vivo studies usually better reflect the underlying biology. choices can be made to compare challenged and non-challenged or vaccinated versus non-vaccinated pigs, and whenever possible preferably littermates are chosen for such comparisons. to reduce genetic background variation even more, treated and untreated tissue within animal can be compared, as seen with the small intestinal segment perfusion (sisp) technique (hulst et al. ) . one can also choose to challenge all animals with a specific pathogen, and not using separate uninfected animals as controls, but contrast high and low responders, as was done in the salmonella experiments (uthe et al. ; huang et al. ; knetter et al. ) . less controlled, but perhaps more realistic, are studies performed after an on-farm outbreak, comparing healthy and diseased pigs or low and high responders (serao et al. ) . another key variable concerns the type of samples to be collected. most pig immune studies conducted to identify host response to common porcine pathogens or to immune response stimulators such as lps or pma/ionomycin described in this review provide gene expression data from a single tissue or isolated cell type, and this at a limited number of times post-infection/stimulation. to study specific components of immune response, it is clear that dissection and analysis of primary or secondary immune tissue are required. in human and mouse studies, significant effort has been taken a step further-the analysis of the transcriptome of highly specific cell types. such samples are isolated on the basis of cell surface marker expression. the parameters for cell selection and isolation are often complex, utilizing a multifactorial list of cell surface markers to identify a highly refined cell population (novershtern et al. ; shay and kang ) . reports on, and options for, specific cell subsets are limited in swine (genini et al. a; kapetanovic et al. ) and are mostly due to the relative lack of immune-targeted reagents critical for such detailed cell phenotyping. examining the whole blood transcriptome has several advantages, including ease of collection and repeated sampling of the same individual during response to a stimulus, which is especially useful in controlling for baseline variation in the study of immune responses. blood rna profiling is advantageous in screening for biomarkers as well; it can be used to study variation in immune response and develop gene signatures predictive of inflammatory and/or disease status. an example is given by the prrs host genome consortium (phgc) project, where blood of infected animals per trial is collected at day and different times post-infection ). however, since whole blood comprises a number of cell types, gene expression differences should be handled with great caution. with the aid of complete blood counts, the transcriptional response data can be deconvoluted to help identify the unique regulatory control of specific cellular responses to pathogens (shen-orr et al. ) . at the end of the day, the ultimate goal is to see how the results of all these individual transcriptomic studies fit into an improved understanding of porcine immune response. recently, such a meta-analysis was performed by combining results of several microarray-based pig immune studies to find prrs-specific responses (badaoui et al. ) . this meta-analysis successfully summarized the general pathway(s) believed to be induced by prrsv. several interferon regulatory factors (irfs) were highlighted in this analysis, and interferons clearly play an important role in viral infections. in agreement with the digital gene expression experiment (xiao et al. a) , in several microarray and qpcr experiments, a dampened expression type i ifn response can be seen, which indicates an inadequate stimulation of the innate anti-viral immune response (genini et al. a; xiao et al. a; ait-ali et al. ; garcia-nicolas et al. ). genini and colleagues observed a strong elevation of ifnb at hpi, but only a slightly elevated expression of ifna (genini et al. a ). ait-ali and colleagues noted a similar low ifna expression, but an, albeit late, accumulation of ifnb expression. they stated that prrsv could delay type i interferon transcriptional response in an attempt to counteract the host's early immune response (ait-ali et al. ) . van reeth and colleagues measured the ifna levels in bronchoalveolar fluids during prrsv infection and saw that its presence was low, a thousand-fold lower than with an infection with swine influenza virus or porcine respiratory coronavirus (van reeth et al. ). however, different prrsv isolates were shown to invoke different (and sometimes significantly higher) ifna expression levels, but no detectable ifna protein levels were found by elisa (lee et al. ). zhang and colleagues found that prrsv does not fail to induce ifna or ifnb mrna expression in monocyte-derived dendritic cells, but the protein seems to be blocked post-transcriptionally . although all described data point out to a weakened ifn response, greatly responsible for a persistent viral infection, the data by these last two studies demonstrate the incomplete information achieved from looking solely at transcriptomic data. another overall prrs finding is the induction of proinflammatory chemokines and cytokines. the differential expression of a cell surface receptor involved in cytokine regulation, trem , was found through the meta-analyses by badaoui et al. ( ) and was also present in the list of top ten upregulated transcripts in the rna-seq experiment of miller et al. ( ) . in the meta-analysis study, trem changed, among others, the expression of chemokines such as ccl and ccl , interleukins il , il , and il b, and toll-like receptors tlr and tlr . xiao et al. ( a) showed an upregulation in the inflammatory response toll-like receptor genes tlr , tlr , cytokines (among which il b), and chemokines. the acute-phase protein saa and inflammasome genes retn, s a , s a , and s a were upregulated in the rnaseq experiment by miller and colleagues. for s. typhimurium, as mentioned earlier, transcriptomic studies in blood have examined differences between ls and ps pigs. in the in vivo study by knetter and colleagues, when looking at cytokine presence in serum at dpi, the pro-inflammatory cytokines il b, tnfa, and ifnc levels were higher in ps pigs compared to ls and control pigs, and the anti-inflammatory il was upregulated in both ls and ps pigs, while only cxcl was elevated in the ls animals (knetter et al. ) . also, uthe and colleagues saw a correlation between ifnc levels and shedding status. it seems that the ps animals have a much more extreme inflammatory response, as if the ps animals respond less quickly and thus extend their inflammatory response (uthe et al. ). additionally, looking at gene expression differences at dpi compared to dpi, ps animals showed a more extensive transcriptomic response, both in number of differentially expressed genes, as well as in level of expression compared to the ls animals. the most overrepresented regulation networks in ps animals at dpi involve the stat , ifnb , and ifnc networks, showing a complex pro-inflammatory profile (knetter et al. ) . the genes casp , tnfa, and il were also found upregulated in these networks, and hence, a nice correlation between serum cytokines and gene expression could be noted. other important regulators cebpb, spi , and tlr in the ps upregulated expression pathways as well as the tnfa and ifnc pathways were earlier reported by huang and colleagues in similar challenge studies . not surprisingly, the microbiota that differ between ls and ps on dpi point to microbiota that play a role in gastrointestinal inflammation (bearson et al. ) . there are, however, regional differences in the inflammatory response to s. typhimurium expression pattern in the gut on dpi (collado-romero et al. ) . while cytokine genes such as tnfa, il , il b, and ifnc are upregulated in the jejunum and colon, they are not induced in the ileum. collado-romero and co-workers proposed that the ileum mucosa reacts slowly against the pathogen. martins and colleagues and wang and colleagues both examined transcriptomic differences due to s. typhimurium in mesenteric lymph nodes. martins and colleagues describe an elevation of ifnc, il b, cxcl , cxcl , casp , slc a, defb , tlr , and nod at dpi. nfjb was significantly down-regulated at and dpi (martins et al. a ). wang and colleagues also note an early repression of the nfjb pathway from to hpi in mesenteric lymph nodes . when looking at gene expression at only , , or hpi in jejunal scrapings, elevated gene expression was observed of inflammatory genes such as il , il b, pap, and s a (hulst et al. ) . they also noted an upregulation of nfkbia, an nfjb inhibitor at this early time point. a study using in vitro stimulation with endotoxin of blood of animals prior to infection was also able to find cytokine differences between ls and ps pigs, showing an attenuated response in ls animals, in contrast to a clear pro-inflammatory response in ps pigs (knetter ) . interestingly, while gene expression on day showed a similar magnitude of response in ls and ps pigs, response differences to lps in blood at dpi between ls and ps pigs were dramatic. only probesets were differentially expressed in ls animals after endotoxin stimulation, while probesets in ps animals changed significantly, showing an apparent tolerization mechanism in ls animals. since differences in gene expression patterns between ls and ps animals on day were not significant enough to create predictor sets of genes in this and earlier studies, kommadath and co-workers used the more sensitive weighted gene co-expression network analysis (wgcna) technique after rna-seq profiling of ls and ps animals. wgcna creates modules of co-expressed genes that are significantly correlated with shedding levels. they include interesting genes such as cytokine genes, genes involved in tlr, nfjb, and nod-like receptor pathways and genes linked to bacterial infections (kommadath et al. ) . for over a decade, microarrays have provided an enormous amount of information concerning different immune-related questions in pig transcriptomics. they have become increasingly inexpensive tools to search for gene expression differences between distinct immune phenotypes. recently, rna-seq, and other sequence-based methods such as mirna-seq, bs-seq, chip-seq, and medip-seq, experiments have become more cost-effective. the advantage of gaining information about all expressed and modified genes, different regulation of splice variants, as well as information about sequence-specific and histone code regulation, is a big plus for rna-seq over microarrays. however, in the near future, the biggest challenge lies in comparing all existing data from many different kinds of platforms, so as to integrate such orthogonal data and better understand the physiology behind the disease phenotype and to find regulatory networks or biomarkers for disease resistance. such meta-analyses use a set of statistical techniques to combine results from different studies (badaoui et al. ) , requiring only that the platform elements can be matched. it is not necessary that the exact same questions were addressed; e.g., an experiment looking at high and low responders to an infection can be compared to a study with infected versus control animals. or an acute response study can be matched to a chronic response study. adding to this, the possibility to integrate new (and broader) information obtained from upcoming next generation sequencing studies would really improve our transcriptomic and epigenomic insights into pig immunity. an example of combining microarray studies for deeper insight is given by pérez-montarelo and co-workers. a meta-analysis was performed on independent gene expression studies, using data from of the same affymetrix array. by doing so, the expression of , genes could be checked in tissues and they could identify tissue-specific genes and tissue-specific regulatory networks and transcription factors (perez-montarelo et al. ) . the meta-analysis study by badaoui and colleagues described above illustrates how a meta-analysis experiment could be achieved for a prrs-specific research question even across different platforms. disparate microarray elements that mapped to the same ipa cdna assembly (couture et al. ) were considered to be comparable. , such elements could be compared across all datasets used, of which more than a third was investigating prrs. to facilitate these kinds of meta-analyses, lessons can be learned from the human encode project by conducting experiments in a similar way and processing and archiving data using standard procedures (the encode consortium project b; birney ; landt et al. ) . in this way, data quality is assured, data utility can be extended, and it becomes easier to compare datasets, combine computational analyses, and consequently perform meta-analysis. besides cross-platform meta-analyses, also cross-species meta-analyses are very promising, and r packages are freely available to conduct them (kuhn et al. ; kristiansson et al. ). however, until now, the main goal of these cross-species expression comparisons is often to employ model organisms for human diagnostic or therapeutic research (yu et al. ; grigoryev et al. ). kapetanovic and colleagues show how the clustering software biolayout express d (be d) can be used to visualize inter-species expression comparisons (kapetanovic et al. ). when comparing differential gene expression in mouse, human, and pig macrophages after lps stimulation, they noted that pig macrophages act more human-like than do mouse macrophages. in a first paper, this group used be d to identify and visualize sets of genes responding similarly over time to lps. they found that a subset of these genes had similar patterns of induction with human macrophage response, but not with mouse, where much lower stimulation or even repression was observed (kapetanovic et al. ) . they extended this work in a later paper by using the larger snowball array. taking only the differential genes between mouse and human, and removing those that did not have pig orthologues on the snowball array, be d showed a large upregulated cluster in human compared to mouse macrophages which contained ido as the hub gene. this cluster was highly upregulated in pig macrophages as well. other genes such as those clustering around nos a also behaved in a murine-specific manner, with an upregulated expression in mouse macrophages while no differential expression in human or pig (kapetanovic et al. ) . in addition to cross-platform and cross-species comparisons, it is important to investigate simultaneously different biological levels influencing the pig's immune status. in the past, disease research very often focused only on one part of the host-pathogen interaction (smits and schokker ) . one examined a portion of the host response, and looked at its ability to fight off an infection and examining its degree of disease susceptibility. or one focused solely on the pathogen, describing level of virulence among different pathogen variants. as well, very often, only a small part of the host immune response was measured, such as a specific cell type or tissue, or only one particular timeframe was targeted. narrow time windows or even single stages can be quite limiting, as immune response varies dramatically over time, and thus, time is a particularly crucial variable in an expression study of immune responses. with a systems biology approach, many levels of knowledge are gathered on both host and pathogen in a challenge study (genomic, transcriptomic, epigenomic, and metabolomic) and at different time points (smits and schokker ; tuggle et al. ) . the ultimate goal is to combine that data to fully explain host-pathogen interactions and discover emergent properties of the system that are difficult to reveal with current approaches. to disseminate public data and improve transcriptomic and epigenomic data mining, a livestock expression and epigenetic database (epidb) is under development. epidb includes data from chickens, cattle, pigs, sheep, and horses and provides a useful repository source, as well as tools to process and visualize expression and epigenetic data ). examples of systems biology approaches integrating transcriptomic and epigenomic data can already be found in many mirna-seq experiments, where one mirna can regulate a network of several mrnas (giles et al. ; valdmanis et al. ; szeto et al. ; yang et al. ) . a porcine mrna-seq study often precedes a mirna-seq experiment on the same sample set to test for correlations between mrna and mirna in order to predict influences of specific mirnas on components of the whole transcriptome (bao et al. unpublished; endale ahanda et al. ) . however, the accuracy and depth of understanding stand or fall with the quality of the pig draft genome assembly and its annotation. recently, the immunome response annotation group (irag) was able to improve the characterization of the pig immunome by manual annotation of almost , transcripts mapping to over , genes (dawson et al. ). this was accomplished using the latest swine genome assembly version . . for the genes without porcine rna sequence evidence, rna sequences of other species (often of human) were used to annotate more than , transcripts using the alignment tools in otterlace (searle et al. ; loveland et al. ) . furthermore, gene expression clustering after infection or stimulation for many independent challenge experiments provided evidence for the involvement of over genes not previously annotated for function in immune response processes (dawson et al. ). ongoing improvements of the draft assembly and additional annotation of the immunome will greatly improve the value of pig disease transcriptomic studies as well as further support the pig as model for human immune response. since immune networks are very complex (gardy et al. ), a deeper understanding of such complexity is needed for advancements in unraveling porcine disease response mechanisms and in developing the pig as a viable model for human immunity. it is encouraging that substantial new high-throughput data have been reported in this area, and that analysis of such data is moving toward a systems biology approach by integrating different methods and combining multiple datasets. with the even higher throughput whole genome techniques coming to the forefront and performed at a relatively low cost, these comprehensive experiments will become more commonplace in the near future. the making of 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pleuropneumoniae key: cord- -yfaji cv authors: kim, yong-kyun; sohn, hong-gyoo title: disaster theory date: - - journal: disaster risk management in the republic of korea doi: . / - - - - _ sha: doc_id: cord_uid: yfaji cv to find a conclusive definition for contemporary purposes and uses, we look at many of the various definitions of disasters through cataclysmic events, historical records, public policies, laws, and organizational usage. our natural progression leads us to modern theories of disaster and disaster risk management (drm) that have had to tackle new types of disasters that are being brought about by the interconnectivity of societies, people, diseases, technology, etc., increasing in magnitude and complexity like what was seen in fukushima, japan, in and on-setting disasters like climate change. after looking at all the historical evidence, we come to a definition for the term disaster for modern usage and what it means for policy implications. risk as "the probability of harmful consequences, or expected loss (of lives, people injured, property, livelihoods, economic activity disrupted or environmental damaged) resulting from interactions between natural or human induced hazards and vulnerable conditions" (undp ) . in the national infrastructure protection plan (nipp ), department of homeland security (dhs) considered risk elements as "threat nature and magnitude, vulnerability to a threat, and consequence that could result." haddow et al. ( ) noted that risk is composed of ( ) the probability and frequency of a hazard occurring, ( ) the level of exposure of people and property to the hazard, and ( ) the effects or costs, both direct and indirect, of this exposure. the korean word for risk is "wiheom" or "wiheomdo." finally, the origin of the word "crisis" is the greek word "krinein (separate; critical moment);" and it has been used as the medical term of "decisive moment of life and death." quarantelli ( ) viewed that a crisis of a certain organization appears in the three following interrelated conditions: ( ) a type of threat including organizational value, ( ) sudden occurrence of an unexpected event, and ( ) need to respond collectively as the outcome may seem more negative otherwise. the term "crisis" is defined as "a difficult or dangerous situation that needs serious attention" in the merriam-webster's learner's dictionary (webster dictionary ) . the korean word for crisis is "wigi." the term indicates a dangerous crucial moment or time and the state to make the final decision on a critical matter. the term "wigi" is a combination of the two characters: "wi" meaning danger and "gi" meaning chance. in the same context, lee et al. ( ) defined crisis as an "incomplete state that desperately needs a significant change or the turning point to determine whether to proceed with, modify, or end an event or a behavioral process." disaster means a condition where the damage cannot be overcome without national or external assistance due to lack of capacity or resources of the damaged community or local/state governments uniqueness korea a presidential decree regulates the magnitude of disaster, which can be financially supported by the national government usa disaster is classified into emergency and major disaster declaration depending on the magnitude of the damage japan disaster caused by natural hazard was historically emphasized in act unisdr a comprehensive approach considering human, physical, economic, or environmental losses and impacts is emphasized fritz considers disaster as the disruption of the essential functions of the social system quarantelli considers disaster as what is caused by natural or technological hazard that is beyond the knowledge of modern society jeong ( ) saw the word "crisis" as natural disasters such as typhoons, heavy snowfalls, and floods; human and technical disasters such as explosion, traffic accidents, and collapse; comprehensive risk situations in various contexts such as terrorist attacks, status of diplomatic relations with north korea, failure of various policies, financial difficulties of firms, stores, and state, crimes, spread of diseases, and various scandals. the terms related to disaster can be summarized as table . . summarizing the discussions above, "disaster" can be conceptualized as follows: • a state that cannot be recovered to the original state with the ability of the community due to great damages in human life and property • inclusive of disasters triggered by natural, technological, or social hazard the term "disaster" will be used embracing natural, technological, and social disasters that the whole community need to cope together reflecting the characteristics of contemporary societies. therefore, in this book, we will define "disaster" as "a status of community or nation's being seriously damaged by natural, technological or social cause and difficult to recover from the damage with its own resources, requiring the whole community to cope together." this definition is similar to the definition of disaster by unisdr and consistent with the definition by laws in korea, japan, and the usa. originates from the old french word "sauf" meaning "not damaged or not harmed," or the latin word "salvus" meaning "not damaged or healthy" the condition of being safe from undergoing or causing hurt, injury anbo security originates from se (¼without, liberation) + cura (¼care, concern, or distress) the state of being protected or safe from harm wiheom (do) originates from the spanish word "risque," known to be used from the mid-seventeenth century a probability of harm that encompasses threat/hazard, vulnerability, and consequence wigi crisis originates from the greek word "krinein" (separate) dangerous crucial moment or time in this context, referring to "jaehae" as natural disaster and "jaenan" as human-caused disaster is not appropriate, which had been commonly used in korea from to ; it is still partially used. both "jaehae" and "jaenan" should be used as terms pointing out natural, technological, and social disasters. to be more precise, "jaehae" means the damage caused by hazards, and "jaenan" means the situation that creates or is likely to create damage. like the concept of disaster, its categorization is also very diverse. the typical one is a categorization by disaster cause. the other ways include categorization by the characteristics of the event, phase-related categorization, and categorization by surgical and medical disaster. in korea, the current framework act on the management of disasters and safety classifies disasters into natural and social disaster. its original version enacted in classified disasters into natural, human-caused, and social disasters, but the same act when it was amended in integrated "human-caused disaster" and "social disaster" into "social disaster." thus, disaster in the current korean law is divided into "natural disaster" and "social disaster." the categorization by disaster cause is a typical way about disaster type. most of renowned organizations dealing with disaster data, such as center for research on the epidemiology of disasters (cred) and munich re, and many scholars have used disaster cause as the principle of disaster categorization. the official categorization of disasters in korea is "natural disaster" and "social disaster" regulated by disaster-related laws, which are differentiated by causing phenomenon. the countermeasures against natural disasters act, formerly the disaster control act and the disasters and safety act, defined disaster-related terms as below: • countermeasures against natural disasters act (act no. , amended on march , ) the purpose of this act is to prescribe necessary matters concerning natural disaster prevention or recovery and other countermeasures against natural disasters, in an effort to preserve national land and to protect lives, bodies, and properties of nationals as well as key infrastructures from disasters caused by natural phenomena, such as typhoons, floods, etc. this act defined "jaehae" as any damage caused by "jaenan" and encapsulated "jayeon-jaehae," meaning natural disaster and "pungsu-hae," meaning disaster triggered by hydrological hazard as specific examples of "jaehae." article (definitions) the definitions of the terms used in this act shall be as follows: . the term "jaehae" means any damage caused by "jaenan" under subparagraph of article of the disasters and safety act (hereinafter referred to as the "framework act"); . the term "jayeon-jaehae" means any disaster caused by typhoon, flood, heavy rain, strong wind, wind wave, sea wave, tidal water, heavy snowfall, lightning, drought, earthquake (including any earthquake-caused tsunami), yellow dust or other natural phenomena corresponding thereto, from among disasters falling under subparagraph ; . the term "pungsu-hae" means any disaster caused by typhoon, flood, heavy rainfall, strong wind, wind wave, sea wave, tidal water, heavy snowfall or other natural phenomena corresponding thereto. • disaster control act (enacted on july , , act no. and abolished on june , ) this act, abolished when the disasters and safety act was established on march , , states that the purpose of the act is to establish a disaster and safety control system of the state and local governments, establish a system for prevention of and countermeasures against disasters as well as emergency rescue, declare a special disaster area, and define matters such as emergency measures necessary for disaster management in order to protect the lives and property of citizens from disasters due to artificial causes. this act was enacted to prepare for human-caused disasters, such as arson and building collapse, rather than natural disasters, such as drought and floods. thus, it targets technological disasters, such as explosions, collapse of bridges, traffic accidents, and chemical, biological, and radioactive accidents, like the leakage of hazardous material and environmental pollution incidents. • framework act on the management of disasters and safety (enacted on march , , act no. ) the framework act on the management of disasters and safety that took effect in categorized disaster into natural, human, and social disaster and defined them as follows: article (definitions) the terms used in this act shall be defined as follows: . the term "disaster" means any of the followings, which actually causes or is likely to cause any harm to the lives, physical safety and property of citizens and the state: (a) disasters caused by a typhoon, flood, downpour, strong wind, wind and waves, tidal wave, heavy snowfall, lightning, drought, earthquake, sandy dust, red tide, ebb and flow and other natural phenomena equivalent thereto; (b) damage beyond the scale prescribed by presidential decree, such as a fire, collapse, explosion, traffic accidents, chemical, biological and radioactive accidents, and environmental pollution incidents and other accidents similar thereto; (c) damage caused by the paralyzation of the state's backbone systems, such as energy, communications, transportation, finance, medical treatment and water supply, and by a spread, etc. of infectious diseases. . the term "overseas disaster" means those which actually cause or are likely to cause any harm to the lives, physical safety and property of citizens of the republic of korea outside the territory of the republic of korea, and which require the government to take measures thereon. • framework act on the management of disasters and safety (partially amended on august , , act no. ) types of disasters were categorized into natural and social disasters in the disasters and safety act partially amended on august , , defining them as follows. the korean government classifies standardized disaster types on the standard crisis management manual as shown in table . according to the statutory classification of disasters summarized above. article (definitions) the terms used in this act shall be defined as follows. . the term "disaster" means any of the followings which actually causes or is likely to cause any harm to the lives, physical safety and property of citizens and the state. (a) natural disasters: disasters caused by a typhoon, flood, downpour (the korean word "howoo"), strong wind, wind and waves, tidal wave (the korean word "hae-il"), heavy snowfall, lightning, drought, earthquake, sandy dust (the korean word "hwangsa"), red tide (the korean word "jeokjo") outbreak, ebb and flow (the korean word "josu"), and other natural phenomena equivalent thereto (b) social disasters: damage beyond the scale prescribed by presidential decree, such as a fire, collapse, explosion, traffic accidents, chemical, biological and radioactive accidents, and environmental pollution incidents and other accidents similar thereto, and damage caused by the paralyzation of the state's critical systems, such as energy, communications, transportation, finance, medical treatment and water supply, and by a spread, etc. of infectious diseases under the infectious disease control and prevention act, and contagious animal diseases under the act on the prevention of contagious animal diseases enforcement decree article (scope of disasters) "damage beyond the scale prescribed by presidential decree" in subparagraph (b) of article of the disasters and safety act (hereinafter referred to as the "act") means the following harm: . harm to human life or property for which measures at the level of state or local government are required; . other harm deemed by the administrator of the national emergency management agency to be necessary for disaster control, and which is equivalent to the harm referred to in subparagraph damage on cultured marine products and fishery production facilities due to a discoloration of seawater to red or light brown caused by an explosive growth in phytoplankton density ※ article of the act on the prevention of and countermeasures against agricultural and fishery disasters 「methods regarding the monitoring, prediction of red tide, and damage prevention」(directive of ministry of maritime affairs and fisheries) drought a prolonged shortage of water supply in a certain region due to a period of below-average precipitation or insufficient available water resources: it can be categorized as a meteorological drought, a hydrological drought, an agricultural drought, or a socioeconomic drought depending on its usage tidal wave damage by level rise due to the effects of tides, weather tidal waves, swells, and abnormal waves in costal or estuarine waters social disaster forest fire trees, weeds, fallen leaves, etc. in a forest or in an area adjoining to a forest are burned by a fire artificially or naturally ignited ※ article of the forest protection act chemical accident all situations that occur because a chemical flows out or leaks out to humans or the environment due to the fault of a worker as at the time he/she works, such as replacement of facilities, defects in facilities or deterioration of facilities, a natural disaster, a transport accident, etc. ※ article of the chemicals control act (continued) disaster type definition water pollution accident that oil, toxic substance, or sewage or wastewater is discharged to river, causing continuous large-scale suspension of water intake or perish of fishes and significant effects on the people's living and natural ecosystem large-scale marine pollution accident accident caused by the crack of oil tank due to sinking, stranding, or collision of a ship resulting in a significant and extensive damage to the people's living and natural ecosystem and requiring comprehensive measures at the government level utility-pipe conduit accident occurred on the facility installed underground for smooth maintenance, aesthetic improvement, preservation of road structure, and smooth traffic flow by accommodating underground facilities (electricity, gas, waterworks supply facilities, communication facilities, drainage system, etc.) commonly collapse of dam accident of leak or overflow occurred due to natural disaster such as large-scale flood or earthquake or structural defect of dam, concerning the collapse of dam large-scale subway accident accident occurred due to the collision, derailment, fire, explosion or flooding of electric train in operation, causing casualty or expecting the suspension of train operation for a long period of time large-scale high-speed railroad accident accident occurred due to the collision, derailment, fire, or explosion of high-speed train in operation, causing casualty or expecting the suspension of train operation for a long period of time large-scale fire at multiuse facility accident at a facility used by the many and unspecified general public concerning which has a high concern of large casualty and property damage in case of large-scale fire radiation exposure from a neighboring country situation requiring national protection measures due to a large-scale radiation exposure or radiation contamination exposure accident from a nuclear facility of neighboring country marine vessel accident accident occurred due to the collision, fire, explosion, stranding, sinking of vessel, loss of loaded cargo, or other hull damage large-scale casualty accident at workplace accident of large-scale casualty such as physical explosion related to tasks at the workplace, largescale collapse accident at construction site, choking accidents due to oxygen deficiency, acute poisoning accident due to exposure to chemicals, or other equivalent accidents multiuse facility large building collapse accident accident at a facility used by the many and unspecified general public concerning large casualty and property damage in case of collapse of building (continued) paralysis of essential national financial information function due to natural disaster, strike, terrorism, and electronic infringement on the financial information system which is the key national infrastructure safety of nuclear facility disaster caused by radiation leak or radioactive contamination from a nuclear power plant and research reactor facility electric utility serious impediment to national life and paralysis of national functions due to anxiety about the electricity demand and supply caused by increased demand, defected facilities, and social conflicts demand and supply of oil serious hazard to the national safety and the stability of national economy due to imbalance between demand and supply of oil caused by foreign oil producing countries health and medical services serious hazard to national health due to the paralysis of healthcare system drinking water national crisis situation such as suspension of drinking and industrial water supply due to water pollution, destruction of facility due to natural and artificial disasters, and strike and cyber terror cargo transport situation which may cause or lead a significant crisis in the national logistics system due to refusal of (continued) by cred and munich re the cred is a research institute founded in by lechat, a professor in belgium and has been studying international disasters and disputes for over years. it has been a world health organization (who) collaborating center since and is operating an international disaster database called emergency events database (em-dat). the disaster classification system of em-dat is in four levels: generic group, subgroup, main type, and subtype. generic group, the highest level of the system, classifies disaster into natural and technological disasters. the natural disaster category is divided into six subgroups: biological, geophysical, climatological, hydrological, meteorological, and extraterrestrial disasters. each subgroup in turn covers disaster types and more than subtypes. more detailed information can be found in the em-dat report (cred ) . natcatservice provided by munich re group in germany is another database related to disasters that wins global recognition, using a natural disaster classification system similar to em-dat. natcatservice enters all natural disasters that cause human and physical damages regardless of scale, but it does not mention artificial/technological disasters (munich re ) . in , cred and munich re developed and presented a joint standard classification system for natural disasters (cred and munich re ). this can be considered the international standard classification system for natural disasters. this system classifies disasters into five levels, dividing disaster into two generic disaster groups of natural disasters and technological disasters. natural disasters are then divided into six disaster groups, geophysical, meteorological, hydrological, climatological, biological, and extraterrestrial, each of which is then classified into main type, subtype, and sub-subtype. however, this system is limited in that it cannot present a detailed classification system for artificial/technological disasters, as munich re does not have the classification for such disasters. threat and hazard identification and risk assessment the threat and hazard identification and risk assessment (thira) is a four-step common risk assessment process that has been designed by the dhs in the usa. this process helps the whole community identify risks, and enables the community to build local resilience to the risks. according to thira, threats and hazards are divided into three disaster type definition people in cargo truck transport business to cargo transport as a group without justifiable grounds resulting in suspension or setbacks of cargo transport gps signal interference service interruption of national infrastructure due to intentional or unintentional interruption of gps signal reception for national core infrastructure and service using gps cosmic radio wave disaster related to radio wave occurred due to change in electromagnetic energy existing outside of the earth's atmosphere types as listed in table . . the natural hazards are the types that result from acts of nature, such as flood, earthquakes, drought, pandemics, epidemics, or more. the technological hazards are the types that result from the accidents or the failures of systems and structures, such as transportation accidents, collapse, hazardous materials spills, or more. the human-caused incidents are the types that result from the intentional actions of an adversary, such as terrorism, sabotage, cyber incident, chemical attack, biological attack, or more. classification of disasters by their cause has been useful for deciding governmental actions toward disaster recovery because it clearly indicates the responsibility of who will pay for what, e.g., supporting disaster victims and/or providing disaster relief. however, the categorization is not useful for disaster response because the response way is not different depending on its cause. therefore, a new prism is needed. kim et al. ( ) have analyzed the occurrence of large-scale disasters in korea since and the government's response to them and have proposed new optics for classifying disaster types for advancing disaster responses. to this end, two important elements were considered: the evolutionary or devolutionary pattern of the magnitude of disaster damage and the roles and responsibilities of responding agencies. regarding the first element of the pattern of disaster damage, three types can be derived: ( ) events with measurable prediction of large-scale damage, ( ) events with unpredicted high social shocks with quick evolution and devolution independent of its magnitude, and ( ) events slowly evolving to large scale. large-scale damage caused by typhoons, torrential rain, and heavy snowfall can be predicted according to weather forecasts and precedence. train crashes and building collapses are types of disasters that suddenly evolve and devolve, leaving not only physical damage but also a shock to the social fabric. finally, foot-andmouth disease, infectious disease, and red tide are disasters that commence on a small scale, or with little notice, but over time become considerable disasters that require large-scale disaster response tactics. the reason why this classification is important is that the response system, such as the timing of the operation of the central disaster and safety countermeasure headquarters (cdschq) and the central disaster management headquarters (cdmhq), is different depending on the progress or evolution of the damage. secondly, the scope of the overall coordination that cdschq should undertake varies depending on whether there are one or more disaster management authorities. the role of the coordinating authority becomes important for typhoons, torrential rain, and heavy snowfall, since they are types of disasters that simultaneously cause damage to various facilities, which will result in various agencies, such as the ministry of construction, the ministry of agriculture, the ministry of environment, etc., engaging the disaster together. for effective response to these types of disasters, it is important for the coordinating authority to support the human and material resources necessary for the various disaster responses by the disaster management agencies. on the other hand, for the disasters such as aircraft accidents, infectious diseases, and dust storms, expertise and experience are concentrated in specialized disaster management authorities, meaning it is efficient for the agencies responsible for those disasters to respond. the category by kim et al. ( ) is very useful in developing an optimum disaster response system fitting the disaster cause and evolutionary path. in this book, we will propose the disaster category for effective disaster response based on the category by kim et al. ( ) as outlined in table . . dombrowsky ( ) suggested event-related concept and phase-related concept as disaster categorization. the event-related concept is composed of time, space, and severity, while the phase-related concept is composed of pre-emergency phase, emergency phase, warning, and post-emergency phase. methods of classifying disasters include one in terms of emergency and medicine. in the field of emergency and medicine, disaster is classified into surgical and medical disasters. the purpose of this categorization is to treat efficiently patients during disasters by securing effectiveness of first aid on-site and hospital treatment. surgical disasters are mostly those in which victims are injured, and they refer to disasters in which damage from physical disasters appears in the form of injury. medical disasters or disease disasters refer to chemical disasters that cause hindrance to respiratory organs and metabolic systems due to the leakage of chemicals, radioactive matters, or toxic agents. this classification method has significance in that it is possible to predict the conditions of the patients in disasters and effectively manage medical resources accordingly (kim and lim ) . in case of instant disaster with single primary response agency, such as dam failures, major rail accidents, and maritime accidents, the cdschq and the cdmhq are established simultaneously; the cdmhq takes charge of response, and the cdschq coordinates the pan-governmental support primary response agency takes charge of its own responsibility in the early stage of the event. as the damage evolves, the charge is transferred to the cdschq. this type of event includes infection, animal disease, red tide, and green tide contemporary society is becoming vulnerable to new types of disasters, such as new epidemics including ebola virus and middle east respiratory syndrome coronavirus (mers-cov), new animal diseases including bovine spongiform encephalopathy (bse) and hpai, and complex disasters such as the tohoku earthquake and tsunami in along with typical types of disasters like typhoon, fire, and building collapse. in addition, the characteristics of complexity in modern times require new and innovative approaches that are different from current response ways. there has recently been much research in various fields, such as sociology and science of public administration, in order to understand what increases disaster complexity in modern times. this section will review representative disaster theories and find how these theories explain major disasters that occurred in korea and around the world. based on this understanding, we will recommend the desirable policy change for effective disaster response in korea. representative theories, such as heinrich's law, normal accident, risk society, and complexity theory, will be reviewed in the following section. herbert william heinrich, who was an assistant superintendent at an american insurance company, presented an important study in based on his analyses of a wide range of accidents. he coined the notion of heinrich's law, which states that, for every major accident, there have been preceding minor accidents and signs of anomaly (heinrich ). heinrich's law is therefore also known as the law of : : . in other words, an accident that is large enough for people to notice is only a small tip of an iceberg and is always antedated by countless other accidents and happenings that warn of the upcoming disaster. through a scientific statistical approach on industrial disasters that were once believed to take place unexpectedly, heinrich found that massive disasters occurred due to negligence on minor defections. originally, the heinrich's law was applied to industrial disasters, but it is now extended to rules related to accidents, disasters, and failures throughout all kinds of areas in contemporary society. the : : rule is meaningful in that it sets up a rule to explain quantitatively the process of an evolutionary incident, starting from a small accident and resulting in a catastrophic event. he also applied the domino theory to disaster areas and indicated that in order for disasters to take place, an inappropriate "direction of flow" occurs sequentially: many causes interact with complexity, and those causes generate interwoven influences. as a result, phenomenon called disaster or accident takes place, and ultimately it causes human and physical disasters, which is a major argument of the domino theory. the theory emphasizes fundamental elements before accidents to take place and explains there are three potential elements as follows: the first condition -human genetic component or socially undesirable phenomena the second condition -flaws caused by the first condition the third condition -unsafe actions, mechanical and physical, according to the second condition among the three conditions that cause a disaster, the first condition of inherited elements or social environment and the second condition of inherited or acquired human defects are hard to be rectified; however, the risk by the third condition can be significantly reduced through safety education and strengthened safety devices. in other words, heinrich insisted that if the third condition is effectively eliminated, disaster can be prevented before it occurs (heinrich ) . the sampoong department store collapse accident in korea is a typical example of the heinrich's law. the accident happened because no countermeasures had been taken even though there were many signals before the accident. the department store opened its business with inborn structural problems during the construction process: about tons of installation equipment was installed on the rooftop, which was four times more than its original design load, and reinforcing bars were out of place. along with problematic construction, poor management was another cause of the accident: minor symptoms such as cracks in the ceilings and damage to the floor of the rooftop (potential elements of ) were overlooked by staff and maintenance crews. in addition, although customers and employees raised several concerns about the general health of the building, such as vibrating sound from an air conditioner and many cracks in the wall, no specific actions were taken, even after receiving evaluations from experts ( small accidents). neglecting potential elements eventually led to one huge accident with casualties (one massive accident) (lee et al. ) . we found that most of the major social disasters, such as the seongsu bridge collapse, the sampoong department store collapse, the daegu subway fire, and the sewol ferry sinking accident, have common things to cause the disasters: improper alteration of use, insufficient safety culture, insufficient safety inspection, and insufficient safety infrastructure. heinrich's law indicates the direction of disaster response that korea should follow: institutional reforms for strengthening disaster and safety management, improvement of safety inspection, expansion of safety education, and enhancement of disaster and safety infrastructure. in the book normal accidents, charles perrow, a professor of sociology at yale university, insisted that complex systems, such as nuclear power plants, chemical factories, aircrafts, ships, dams, and gene manipulation, hold a risk of tragedy. thus, there are some unavoidable accidents called normal accidents that have a high probability of occurring regardless of safety measures and devices (perrow ) . charles perrow proposed the normal accident theory based on the empirical evidence of the three mile island (tmi) nuclear power plant accident that took place in pennsylvania in , the first nuclear power plant crisis to have occurred. perrow ( ) defines a normal accident, which he also refers to as a system accident, as "an event that involves the unanticipated interaction of multiple failures, reflecting the characteristics of high-risk system in which multiple and unexpected interactions of failures are inevitable." perrow's theory insists that accidents in today's cutting-edge technological societies are closely related with complex technological and mechanical structures with built-in risks and those risks are therefore a normal part of our lives. an extremely complex system, in which individual technologies interact with one another closely and inseparably through an endless loop, is prone to catastrophe caused by a series of breakdowns whenever one of the interwoven elements begins to dysfunction. it is nearly impossible for humans to predict such technical failures (park ) . perrow noted that conflicts of interests might exist between a given organization and its members and that organizations are also subject to external political and social environments. technical solutions are therefore not enough, particularly as it is impossible to ensure the total control and containment of risks in such highly complex private facilities as nuclear power plants and petrochemical factories, at which minor errors can lead to complete failures (perrow ). the three mile island accident is considered as the typical example of normal accident. this brought widespread panic to the usa as large portions of one of the reactor's cores partially melted, releasing radioactive gases and hazardous iodine into the surrounding environment. the direct cause of the accident was officially recorded as a mistake by operational staff; however, the complexity of the disaster is a hidden root cause of the catastrophic event. therefore, as perrow indicated, complex systems with built-in intensive risk, such as nuclear power plants, require that centralized and decentralized management should be considered simultaneously. there have been a lot of industrial accidents around the world. accidents, such as the india bhopal chemical factory accident, the chernobyl nuclear power plant radiation leak, and the bp gulf oil spill, indicate that industrial safety measures need constant improvement through comprehensive understanding about high-risk technology and systems. some scholars criticized that the normal accident theory overemphasizes the vulnerability of high-risk facilities; the high reliability theory is a typical example of the critics. professors from uc berkeley and weick, an organizational theorist, proposed the high reliability theory based on research of organizations fraught with disaster risk, such as national aeronautics and space administration (nasa), the nuclear industry, aviation, nuclear carriers, swat, and massive petrochemical facilities in the usa. the high reliability theorists insist that sophisticated quality controls, a settled safety culture, built-in safety by cross-check, and continuous education and training can prevent disasters at high-risk facilities. irrespective of these critics, perrow's normal accident theory provides a beneficial lens through which we may view and analyze risks in contemporary society. because the theory explains risks as a matter of organizational characteristics of the ecosystem, it overcomes the shortcomings of the science and technology-centered approach. it provides sociological explanations for organizational risks that are likely to cause massive collateral damage, such as in petrochemical factories and nuclear power plants (jeong ). after the earthquake that occurred on september , in korea, social interest in the safety of high-risk facilities such as nuclear power plants and old industrial complexes has increased. in accordance with etkin ( ) , who noted that careful thought should be given to the construction of complex tightly coupled systems, the korean government should entirely overhaul the safety of risky facilities such as nuclear power plants, industrial complexes, and chemical plants. moreover, there is a need for society-wide attention and efforts to provide a more sophisticated safeguard system. around the s, an in-depth exploration on new risks that occurred in modern society was conducted by european scholars, such as luhmann, giddens, and beck (lee ) . ulrich beck, the german sociologist, suggested the concept of risk society as a solution to structural and deep-rooted problems of industrial societies, including science and technical safety issues, which started in the mid- s (lee et al. ) . in his book world risk society ( ), ulrich beck argued that the modern society is a "risk society" replete with risks all throughout and emphasized the multiplication of cross border risks and international dangers that single nationstates cannot tackle on their own (beck ) . during his lecture at seoul national university in , beck pointed out the similarities between the asian financial crisis in the s and the chernobyl nuclear plant accident in , stressing the importance that all nations must work together to reduce the global risk on the basis of a common understanding that they are facing the same global risk. beck also considers this emerging risk as a result of the de-bounding of traditional national boundaries in the spatial, temporal, and social dimensions. beck defined risk society as a society where socially produced risk is inherently accompanied by socially produced wealth (oh ) . he also insisted that industrial society should be addressed through a comprehensive perspective, which includes social, historical, and technological views (beck ). as the world entered into a contemporary society, new types of risks, combined with secondary, supernatural, and artificial uncertainty, had arisen, and those risks were beyond a dimension of traditional response methods (lee ) . the background with such phenomena includes a complex structural change that is understood as the term of postmodernity. generally, risk, unlike danger that indicates direct and physical loss, is based on the probability of prediction or control: a new concept of "uncontrollable risk" needs urgent attention because this type of risk denies the applicability of currently available risk theories and risk control mechanisms, arising new threats (beck ). the concept of risk society by beck has a critical meaning to korea's drm. the increased risk can be also found in urbanization in korea. lee et al. ( ) emphasized the increased risk due to urbanization by showing the increased disaster risk following urban development with the change of green space to paved road. korea has also suffered from newly emerging disaster risks, such as severe acute respiratory syndrome (sars), foot-and-mouth disease, and mers-cov. therefore, an innovative and cooperative approach to disaster risk suggested by beck needs to be reflected in designing resilient future. complexity theory began from researching complex natural phenomena such as meteorology. just as lorenz proved with the butterfly effect, the contemporary society embedded with complex network needs to take different countermeasures against disaster. the fukushima nuclear power plant accident of japan, a great flood in thailand, and new types of epidemics such as mers-cov are representative examples of disasters that the contemporary society is newly facing. the complexity theory is useful for understanding the characteristics of contemporary disasters and finding appropriate countermeasures. on march , , the fourth strongest earthquake struck japan, since japan started to observe earthquakes, with a mega tsunami and aftershock that caused more than , deaths. also, the earthquake destroyed the nearby fukushima nuclear power plants and caused radiation leakage. this disaster showed signs of complex disaster. due to the fukushima accident, air, soil, ocean, and underground water were exposed to radioactivity, and damages from contamination influenced largely not only japan but also the whole world continuously. the severe flood in thailand is a typical example that a disaster that happened in one country affected the regional economy. due to a heavy rain, combined with low topographic characteristics of thailand and high tide of seawater, two thirds of the land in thailand, including parts of bangkok, was flooded, causing significant damage to the entire manufacturing industry. the flood did not affect just thailand. for example, many japanese companies' production facilities located in the central region, having developed the area for the manufacturing of automotive and electronic goods, were inundated during the flood. the intensity of the flooding caused all of the facilities to be shut down, which caused tremendous economic loss for both countries. additionally, the shutdown of the automotive plants caused a reduction of japanese auto and parts deliveries to other major markets like japan, the usa, and europe. in case of the electric and electronic industry, hard disks became in short supply due to flooding of hard disk manufacturing factories. due to this, the production of semiconductors by intel became inadequate, resulting in a short supply of semiconductors to the world's semiconductor market. moreover, it caused a price increase of computers worldwide. also, since the severe flood occurred right before the tourist season, the number of tourists in the fourth quarter decreased by approximately %, and additionally induced damages occurred in other industries, such as transportation and food services. due to the influence of the heavy flood, the estimated economic growth rate of thailand in was lowered from . % to . % (korea institute for international economic policy ). table . shows the summary of damage that occurred to the japanese economy due to the heavy flood in thailand. this table shows that the flood was not just problematic for thailand but indicates that a disaster occurring in one country does influence other countries. the contemporary society is facing new types of epidemics and animal diseases, such as sars ( ), h n virus ( ), foot-and-mouth disease ( ), and mers-cov ( ), which did not exist in the past, and such symptoms have a high possibility of becoming more frequent due to increase in trade and traveling with foreign countries. pelling ( ) argued that complexity theory possesses a very important lesson to understanding ways to cope with disasters. for example, one of the characteristics of complexity theory is "emergence," which shows a trait of disasters that take place with unexpected causes at an unexpected place. according to drabek and mcentire ( ) , "emergence" appears while people change organizations in the process of making temporary organizations and responding to disaster situations. beck's risk society and perrow's normal accident theory share the similar understanding with complexity theory in that the emerging risk in modern society is closely related with its increased complexity. therefore, the core elements of complexity theory, such as nonlinearity, self-similarity, fractal, self-organization, and emergence, are essential in understanding disaster characteristic and innovating disaster response methods in modern society. as disasters started to become complexed and intensified, scholars and stakeholders began to look for new theories and methods to improve disaster risk knowledge and organizational capabilities. starting in the s, several theories, such as petak's four-phase model, mcloughlin's comprehensive disaster management procedures, and the new public administration theory, were developed to deal with these new complex and intensified disasters and to help modernize and reform institutions that were becoming too antiquated to deal with modern disasters. organizations such as the usa's fema, created in the s, used the progressing knowledge and concepts to help it mature in the s, and the un and world stakeholders, who came together in the early s to produce the hyogo framework for action (hfa), used the foundations' insights to increase disaster resilience around the world. investigating the historical progress of such knowledge and the evolution of response organization is important in figuring out what logical steps should be made for a resilient future as well as in strengthening organizational capabilities to better deal with complex and intensified disasters. in , petak proposed a four-phase model to identify the role of governments and stakeholders in each disaster management phase. he divided pre-disaster management and post-disaster management according to the progress of disasters and countermeasures and explained disaster management procedures in a timesequential manner: . disaster mitigation . disaster preparedness . disaster response . disaster recovery petak emphasized that the clear delineation of roles and responsibilities of all levels of governments and stakeholders is essential for effective disaster management four main areas in japan reduced production by % nippon steel corporation adjusted release of the crude steel in japan due to effect of reduced automobile production honda suspended the operation of four-wheeled vehicle plant in malaysia toshiba had trouble in hard disk drive (hdd) production pioneer had trouble in the production of car navigation system, relocated from thailand to malaysia deteriorated business results expects billion yen decrease in sales automobile possibility of sales decrease of billion yen for five automobile companies suspended distribution suspended the transport based on flooded areas paid insurance tokio marine and fire insurance co., ltd. began investigation to provide insurance to japanese client companies (petak ) . petak's model influenced the basic structure of the framework act on the management of disasters and safety (hereafter "disasters and safety act") in korea, which is shown in fig. . . the new public administration theory emphasizes that a government can provide better public service to citizens by adopting business management principles to public management (rosenbloom and goldman ) . the theory aims to overcome the problems of a typical bureaucracy and to improve the efficiency of the public sector by adopting business management skills and by emphasizing output and outcome of public policy rather than the input to implement the policy (rosenbloom and goldman ) . the theory was accepted through the national performance review project during the clinton administration, and laid the foundation for fema's reformation (waugh and streib ) . the demand for the reformation of fema started from hurricane hugo, which hit north carolina and the virgin islands in , causing $ billion in damages. in the aftermath of hurricane hugo, the loma prieta earthquake struck california, a fire broke out in oakland, and hurricane andrew struck florida and louisiana, dealing a massive blow to the two states. disappointed by fema's ineffective response to these massive natural disasters, political groups and citizens in the usa raised the need to improve fema's performance, which was endorsed by the clinton administration in (anna et al. ). james lee witt, appointed as director of fema by president clinton, emphasized disaster mitigation and shifted from recovery-oriented policy to preventionoriented policy. additionally, he insisted that disaster mitigation investment should be based on the assessment of the effectiveness. consequently, government policy has continued to embrace this line of thinking. to quantify the future savings of hazard mitigation activities, the multihazard mitigation council (mmc) of the national institute of building sciences (nibs) analyzed three major hazard mitigation grant programs: the hazard mitigation grant program, project impact, and the flood mitigation assistance program. the independent study proposed the following significant findings: ( ) for every dollar spent on mitigation, four dollars was saved from future spending; and ( ) fema mitigation grants beget nonfederally funded mitigation activities (nibs ) . the new public administration theory affected the development of disaster management in korea: the increased investment to structural and nonstructural measures for disaster mitigation and the evaluation of the effectiveness of disaster mitigation and recovery projects in the s are typical examples of the effect of the theory. although the theory receives criticism in that it does not consider the characteristics that disaster management has as a public service, it can provide a useful prism to improve the effectiveness of disaster management policy by focusing on the outcome or performance of the policy, rather than the input. public governance has been acknowledged as a way to improve public-private partnership in disaster management. in the public governance theory, rules govern the behavior of actors not as the result of official authority or market equilibriums, but according to the consensual process among participants, based upon networks and cooperation. the concept of collaborative networks in disaster management entails the assumption that parties involved in disaster management have diverse cultural backgrounds and are bound to experience conflicts. effective cooperation in this setting requires cultural sensitivity and mutual understanding from all participants. collaborative networks, moreover, are becoming important because, in modern society with emerging and complex risks, it is not possible to ensure perfect preparations and resources for all possible disasters and because a specific organization or one single agency cannot perfectly control all response agencies involved in managing disasters (waugh and streib ) . the advantage of public governance theory is that it strengthens shared responsibility by enabling diverse social groups to participate in the decisionmaking process so as to tackle uncertainty with social intellect and make policy decisions based on the social consensus. the theory emphasizes cooperation, public participation, problem-solving, and openness as key principles of disaster management. it aims to form a collaborative risk governance system consisting of diverse actors, including governments, businesses, and civil society organizations, in which national government serves as facilitator in promoting the development of a decentralized collaborative network among local governments, nonprofit organizations, and various public services. however, there is an opinion that the public governance theory is hard to be applied to emergency response. in other words, in an urgent disaster response process where there is not enough time to make a consultation, the process of negotiation through mutual discussion and consultation is not appropriate (waugh and streib ) . the argument seems reasonable, but recent researches have shown that interagency cooperation is becoming more important in an emergency situation. moynihan pointed out that establishing network governance in advance are an essential element in achieving the two objectives of "interagency cooperation" and "coherent response" in a crisis situation for effective disaster response (moynihan ). public governance theory is also important in the development of the disaster response system in korea. the recent major disasters in korea, such as the mauna ocean resort gymnasium collapse accident, the sewol ferry sinking accident, and the middle east respiratory syndrome, taught korea important lessons that cooperation among all relevant organizations, such as on-site response agencies, local disaster and safety countermeasure headquarters (ldschq), various line ministries involved in the cdmhq, and the cdschq, is essential for effective disaster response and relief. therefore, the korean government should develop an interagency cooperation plan and execute a joint field training program; demanding the participation of all relevant organizations specified above. in late , more than federal organizations related to civil engineering and defense had fragmented responsibilities for hazard mitigation and disaster response, resulting in no coordinating organization taking the full responsibility for the entire phase of disaster management. president jimmy carter created fema in for this reason. the establishment of fema made it possible to unify the fragmented responsibilities of emergency preparedness and response resources (anna et al. ) . mcloughlin ( ) proposed an integrated emergency management model. considering disaster as an incident or condition that threatens the survival of organizations, he was concerned with the fact that troubles in cooperation among related agencies repeatedly occurred during disaster response since various public and private groups had been engaged in disaster response without comprehensive coordination. he argued that a comprehensive and integrated emergency management system could sustain administrative capabilities during emergencies and protect property and life through a series of circulation processes under the cooperation of the federal, state, and local governments. this model emphasizes that each local government and the federal government should cooperate to protect life, property, and government functions through a program of mitigation, preparedness, response, and recovery (cho ) . quarantelli ( ) also emphasized that comprehensive disaster management is essential in modern times in order to build a comprehensive and unified organization that manages all types of disasters. he argued that a dispersed or separated approach, by which each line ministry is responsible for its own disaster; devoid of a coordinating agency, cannot deal with complex and intensified disasters in modern times. additionally, he asserted that a comprehensive and integrated method be used when managing disasters because: first, disasters have become more complex and capable of destroying the functionality of typical community operations, meaning that dividing disasters into natural or human-caused ones is inadequate for effective response; second, there is an underlying commonality among governmental departments to respond to disasters; therefore, comprehensive measures are required for a unified effort; third, the planning process and its contents for disaster response by each department has commonality, thus, it is ineffective that each department develops its own response plan; finally, the shared response resources among the governmental departments reveals that a move to a comprehensive management format is feasible since vital resources are similar in each department (quarantelli ). global cooperation for effective disaster relief had been a pivotal issue among the international society. to improve the international cooperation for disaster relief to affected nations, the united nations established a responsible agency, labelled as "the united nations disaster relief organization (undro)," in . since then, the international community has been working for developing a systematic disaster management framework; disaster response plan, disaster prevention measures, and scientific and technical solutions to disaster risk have been discussed and adopted. in particular, technical approaches, such as vulnerability analysis for disasters and early warning measures, have been tried. additionally, the scientific approach played a key role in the evolution of disaster management through a variety of research on how to identify hazard and assess vulnerability (unisdr ). in , the international society began to prepare the international decade for natural disaster reduction framework to promote more systematic disaster management (unisdr ). the international community established the un humanitarian emergency assistance and the international search and rescue advisory group (insarag) mechanism in . also in , systematic mechanisms for disaster response were introduced by building the united nations disaster assessment and coordination (undac) mechanism. the international conference held in in japan adopted the yokohama strategy that emphasized the paradigm shift from recovery-oriented policies to prevention-oriented policies. in , the name of the international strategy for disaster reduction was adopted with the title "safer world in the st century: disaster and risk reduction." since then, a comprehensive policy framework to cope with increasing global risk has been developed in earnest (unisdr ). in december , a tsunami hit countries in the eastern-western-southern asian regions, such as indonesia, sri lanka, and india. in the wake of the catastrophic event, national governments, international organizations, un agencies, and other stakeholders agreed to adopt "the hyogo framework for action (hfa): building the resilience of nations and communities to disasters," to mitigate global disaster risk for - . with the hfa, disaster management moved toward building resilience based on social consensus and strengthening shared responsibilities among all stakeholders. in march , all national governments also agreed to the sendai framework for disaster risk reduction - (sfdrr), which is the first major agreement on disaster management in line with post- development agenda. the sfdrr aims for the significant reduction of disaster risk and losses with the following four priority actions: understanding disaster risk, strengthening disaster risk governance to manage disaster risk, increasing investment in disaster risk reduction for resilience, and enhancing disaster preparedness for effective response and to "build back better" in recovery, rehabilitation, and reconstruction. the drr-related global agenda in various fields will be further reviewed and analyzed in chap. in order to link them with disaster resilience in the future. in the s, massive disasters continued to occur throughout the world. national governments have reformed their disaster response system to deal with massive disasters. the usa reformed its drm system after / in , shifting from a typical natural and human-caused disaster management paradigm to comprehensive security (park and cho ) . within this context, since the s, civil defense and civil protection became important concepts in disaster management. civil defense was originally designed to protect citizens from calamities, such as war, but it was gradually expanded to encompass drm, reducing civilian casualties during disasters. alexander ( ) suggested that the role of the state and the civilian in disaster preparedness be shifted from civil defense to civil protection. this is because civil protection is an appropriate concept to reinforce the protection of people against external risks, meeting the need to protect people from typical disasters, such as natural and technological disasters, as well as emerging risks, such as new infectious diseases, terrorism, and cyber attacks, while civil defense was useful to cope with the invasion of an outside force which was a severe threat during the cold war era. one of the most important features of civil protection suggested by alexander is a shift from providing public safety service by the government to encouraging public participation. when a national government provides public safety services, it usually prefers to use a top-down approach: command and control, a hierarchical decision-making system excluding citizen's participation, strengthening law and order, and rules through the principle of confidentiality. however, the civil protection concept by alexander put stress on a bottom-up approach: emergency preparedness and response ensuring public participation and cooperation, problemsolving approach, and openness principles. as a result, risk governance is the most critical issue in civil protection, ensuring the cooperative interaction among the government, the private sector, and civil organizations to replace the existing hierarchical bureaucratic system. alexander ( ) addressed hurricane katrina as a typical failure case of civil defense with a top-down approach, requesting a shift from a top-down approach to a bottom-up approach, along with improved risk governance. this section will describe disaster response institutions in korea, the usa, japan, and other nations. the analysis of one nation's disaster response institution requires huge efforts and a large amount of work. due to limited time and space, this section will focus on the basic structure of disaster management organizations for normal times and emergency situations at national and local levels. the disaster response institutions in korea, the usa and japan will be analyzed first, and then those in australia, germany, switzerland, and the united kingdom (uk) will be analyzed. in korea, the ministry of public safety and security (mpss) is responsible for the overall coordination of the nationwide disaster response based on the disasters and safety act. after the presidential election on may , , the ministry of interior (moi) has been preparing for the revision of the government organization act, which includes the establishment of ministry of public administration and safety (mopas) by integrating mpss with moi and the establishment of korea fire service and korea coast guard as ind ependent agencies. the act is expected to be reviewed and decided by the national assembly in late . in addition, the crisis management center under the national security council in the blue house (presidnet's office) is expected to work as a control tower for national crisis, such as the sewol tragedy. the korean government has developed its disaster response system suitable for normal times and emergency situations at the national and the local levels. organizations for normal times are composed of disaster management agencies, disaster-management supervision agencies, emergency rescue agencies, emergency rescue, and relief support organizations. in addition, central, city/do, si/gun/gu committees are being operated to deliberate matters on disaster and safety management under their responsibilities. disaster management agencies take charge of all phases of disaster management activities that are related to their responsibilities. the agencies include national administrative agencies, local governments, local administrative agencies, public institutions and organizations, and other organizations prescribed by presidential decree. disaster-management supervision agencies are responsible for disaster response when a disaster or an accident occurs in accordance with the responsibilities prescribed by presidential decree. for example, the ministry of education is responsible for disasters in schools and school facilities, the ministry of environment is responsible for environmental pollution accidents, and the ministry of employment and labor is responsible for large-scale human accidents occurring in places of work. table . shows the disaster-management supervision agencies by disaster or accident type in korea. emergency rescue agencies are responsible for carrying out life rescue, first aid, and other necessary measures to protect the lives and property of citizens when a disaster is likely to occur or when a disaster occurs. the agencies in charge include fire hqs and coast guard hqs, under the mpss, city/do fire headquarters and si/gun/gu fire stations, and regional headquarters of the korea coast guard and coast guard stations. to support rescue activities, presidential decree prescribed emergency rescue and relief support agencies, which are equipped with human resources, installations, equipment, operation systems, etc., that are necessary for emergency rescue and relief service. to deliberate and decide crucial matters related to disaster and safety management at the national level, the central safety management committee, chaired by the prime minister, is operated. in addition, the safety policy coordination committee, chaired by the minister of public safety and security; the central disaster broadcasting consultative committee, chaired by a person appointed by the minister of the ministry of science, ict and future planning; the central private-public cooperative committee, chaired by the vice minister of public safety and security; and a civilian representative are being operated to advise, consult, deliberate, or sometimes decide disaster and safety management issues under their responsibilities. at regional and local levels, a city/do safety management committee, a si/gun/gu safety management committee, a city/do disaster broadcasting consultative committee, and a si/gun/gu disaster broadcasting consultative committee are operated. when disaster occurs or is likely to occur, emergency response organizations are established and operated to take timely and proper measures at national, regional, and local levels. the emergency response organizations at the national level include the cdschq, chaired by the mpss; the cdmhq, chaired by the head of the relevant disaster-management supervision agency; and the central emergency rescue control group (cercg), chaired by the head of the central fire headquarters for disaster occurring on land and the chief of the central rescue center for disaster at sea, respectively. the local emergency response organizations include the city/do disaster and safety countermeasure headquarters (city/do dschq), chaired by mayor or governor, and the si/gun-gu disaster and safety countermeasure headquarters (si/gun/gu dschq), chaired by the head of si/gun/gu. for rescue activities, the local emergency rescue control group (lercg), chaired by the head of the fire headquarters and a chief of a fire station, is operated; when disaster occurs at sea, the head of a si/gun/gu emergency rescue control group and the head of a city/do emergency rescue control group shall be respectively construed as the chief of a regional rescue center and the chief of a metropolitan rescue center under article of the rescue and aid at sea and in the river act. when disaster occurs or is likely to occur, the mpss will immediately hold a situational meeting to supervise the initial response, rescue, and first aid operations. in particular, in the event of a major disaster, the ministry will operate the cdschq and coordinate the intergovernmental disaster response activities. in case of an oversea disasters, the minister of foreign affairs shall exercise the authority of the head of the central countermeasure headquarters, and in cases of radioactive disasters, the chairperson of the nuclear safety and security commission shall exercise the authority of the head of the central countermeasure headquarters, respectively. in case government-wide integrated response is necessary, the prime minister may exercise the authority of the central countermeasure headquarters. in such cases, the minister of public safety and security, the minister of foreign affairs (limited to cases of overseas disasters), or the chairperson of the nuclear safety and security commission (limited to cases of radioactive disasters) shall be the vice head. when a disaster occurs in a jurisdiction, the mayor or provincial governor and the head of a si/gun/gu shall set up their own dschq and coordinate the response and recovery operations. in order to efficiently operate the cdmhq under article - ( ) of the disasters and safety act, the head of the disaster-management supervision agency shall predetermine necessary matters for organizing, operating, etc. of the cdmhq and exercise the authority of the head of the headquarters. also, local disaster management headquarters shall be established to work as an action team of cdmhq in the disaster area. the disaster response organizations during emergency situations is shown in fig. . . the disaster response plan in korea consists of three parts: the standard risk management manual, the working-level manual for risk response, and the manual for actions-at-scene. as of may , kinds of standard risk management manuals, working-level manuals for risk response, and , kinds of manuals for actions-at-scenes have been prepared and utilized. the standard risk management manual, prepared by a disaster-management supervision agency, delineates roles and responsibilities of related agencies in disasters at the national level, which shall be the guidelines for preparing the working-level manual for risk response. the working-level manual for risk response is a document stipulating the measures and procedures necessary for responding to actual disasters in accordance with the functions and roles of the disaster-management supervision agency and support agencies, which are stipulated in the standard risk management manual. the manual for actions-at-scene, prepared by implementing agencies, such as local governments, stipulates in detail the procedures for actions to be taken by an agency that directly performs its duties at a disaster scene. the standard risk management manual shall be prepared by each disaster management supervision agency that is regulated in table . . however, the standard risk management manual for disasters involving many disaster management authorities, such as typhoon and drought, can be prepared by the minister of mpss. the working-level manual for risk response shall be prepared by related support agencies that are designated by the standard risk management manual. the manual for actions-at-scene shall be prepared by an agency designated by the working-level manual for risk response. the head of the si/gun/gu may develop several disaster types of manuals for actions-at-scenes in consolidation as needed. the manuals delineate roles and responsibilities of the disaster-management supervision agency, related support agencies, and implementing agencies. on the other hand, the operational functions of disaster response of each agency shall be designated by action plans for disaster response by function under article - of the enforcement decree of the disasters and safety act. the functions for disaster response, similar to the emergency support function (esf) of national response framework (nrf) in the usa, are described below: . managing disaster situation . supporting emergency livelihood stabilization . supporting emergency communications . emergency restoration of facilities damage . restoring damaged energy supply facilities . supporting disaster management resources . traffic countermeasures . supporting medical and disinfection services . environmental arrangement at disaster scenes . supporting and managing volunteer work . maintaining social order . searching, rescuing, and emergency support at disaster areas . publicity of disaster management in summary, the two axes of the disaster response plan in korea are three levels of manual and functional action plans. the manual describes the roles and responsibilities of primary response ministries and related agencies, and the functional action plans describe how each agency performs its key response functions in line with its roles and responsibilities. the disaster response organizations during emergency situation are: cdschq, city/do dschq and si/gun/gu dschq for overall coordination; cdmhq and ldmhq for the implementation of their own responsibilities; cercg and lercg for search and rescue; and support agencies. both the establishment and evolution of fema and dhs in the usa had influenced the disaster management system in korea; in particular, the establishment of the national emergency management agency (nema) in and the establishment of the mpss in . in addition, the integrated disaster management formed by the dhs, the nrf, and the national incident management system (nims) has also influenced the evolution of the disaster response system in korea. dhs, established in , is responsible for national security and disaster management. fema under dhs is in charge of all phases of disaster management, including national preparedness, public and private capacity assessment, mobilization of resources for emergency management and disaster relief, and long-term recovery plans. at the state level, the disaster management department focuses on strengthening the linkage between the federal government, the state, and the local governments and assisting the local government in disaster prevention, preparedness, response, recovery, and relief. when a disaster occurs, the emergency operations center (eoc) commences operation and responds to disasters in accordance with a preestablished disaster response plan (eop, emergency operation plan, or cemp, comprehensive emergency management plan). local governments have a primary responsibility for the whole process of the localized disaster, prevention, preparedness, response, and recovery of disaster, and for the activation of eop followed by executing the eoc to respond to disasters. when a disaster that exceeds the capacity of a local or a state government occurs, federal government's assistance and involvement is requested and is provided through the joint field office (jfo). the usa performs disaster management through nrf and the nims. be that as it may, the catastrophic events on september , ( / ), and august , (hurricane katrina), saw the federal government's failure to provide proper support to state and local governments for effective disaster response. after that, there is an increasing demand for the federal government to respond to disasters proactively by federal emergency declaration to large-scale disasters and pre-deployment of federal resources to states. however, the basic principle of disaster management in the usa is still that local governments are primarily responsible for all disasters, with the support from state and federal governments concerning disasters that exceed the capacity of local governments. the disaster management organizations of the usa are summarized in fig. . . the dhs was established in january to integrate the prevention of terrorism and the function of disaster management under one department's coordination in the wake of the september , terrorist attacks and the mailing of anthrax spores. dhs took charge after the commencement of the national strategy for homeland security and the homeland security act. the dhs carries out tasks that include the suppression of terrorists' attack, minimization of damage, prevention, preparation, response, and recovery in the emergency plans for all domestic and international dangers that threaten the usa. the dhs consists of the office of intelligence and analysis (oia), the information analysis and infrastructure protection (iaip), the chemical and biological (cb) weapon management service, the state affair safety service, secret service (ss), and fema. fema, which is the most well-known organization among the various organizations of the dhs, is responsible for various tasks that include the disaster risk reduction at all levels, the reduction of property loss through various risk-based emergency management programs for the preparation, response, and recovery, and the protection of people's lives and main facilities. fema was established in as an organization coordinating the response and recovery of disasters during the carter administration. however, the initial phaseout of fema was not that high. it had grown into a member of the administrative cabinet and a ministerial level organization during the clinton administration through the establishment of an integrated response system and the strengthening of preventative and mitigation programs after failing to respond effectively to several large-scale disasters such as hurricane hugo. through the establishment of fema, various agencies related to disaster response were integrated into one agent under the direct control of the president and capable of comprehensively responding to various human-caused disasters and natural disasters. after the dhs was established, fema was incorporated and operated as a bureau under the dhs. in the early days, under the dhs, fema's status was weakened since antiterrorism and security were the first priority of the nation. however, after hurricane katrina, the importance of disaster management has been highlighted, and fema has also been strengthened as the independent deputy-minister level agency. fema, headquartered in washington, dc, operates local offices and provides regular disaster support personnel who can respond immediately in case of a disaster. the central organization for when an emergency occurs includes the jfo. the jfo is established by fema for coordinating between state governments and federal agencies after the president declares a state of emergency, and jfo plays a pivotal role in providing coordination between federal, state, and local governments and non-governmental organizations (ngos) and private sector accident supports. the head of a jfo is called the federal coordinating officer (fco), who is designated by the president and carries out the comprehensive coordination and the management of support activities using the resources secured by the federal government. in general, the state and local governments have an organization dedicated to disaster management: a disaster management department at the state level and an emergency office at the local level. a local emergency office (leo) normally consists of divisions handling emergency operation, information and communication, administrative task related to disasters, prior identification of risk, and the modification and supplementation of a risk management plan. in the event of a disaster, the eoc is set up to coordinate or support the disaster response at the site and responds to the disaster in accordance with the incident command system (ics). disaster response plans in the usa are developed at the federal, state, and local levels. at the federal level, the nrf and the nims constitute overall responsibilities of the dhs, fema, line ministries, and other agencies. the purpose of the nrf was to connect government agencies with ngos and the private sector and transparently assign and coordinate key roles and responsibilities nimbly. the nrf consists of the base document, the esf annexes (esf annex), the support annexes, and the incident annexes. table . shows the organization of the nrf. the local disaster response plan is carried out through the preparation of an eop by each area. the plan incorporates all aspects of disaster management in any given area, and it guides the roles and responsibilities of all related agencies for disaster response, depending on the size and complexity of a disaster. eop consists of the basic plan enclosed with the annex including the esf, the administrative and financial support plan, and the incident annex. nims provides national response doctrine for the whole community to work together based on the principle of the nrf. the nims defines standardized command and control principles to enable various response agencies to coordinate in the event of a terror and disaster. it provides a standardized response principle that allows federal, state, local governments, and nongovernmental organizations to respond consistently to a disaster regardless of the cause, size, location, or complexity of the disaster. to this end, it defines concepts, principles, organizations, rules, procedures, and terminology that provide a structured framework that is flexible, applicable, comprehensive, and geographically balanced for all types of disasters. the on-scene commander of the nims directs and controls the response at the incident site, and the head of the eoc is responsible for supporting the incident site while coordinating and managing local resources from outside the incident site. the components of the nims include preparedness, communication and information management, resource management, technical support, continuous management, and maintenance. in , fema developed "state nims integration" to facilitate states to adopt the nims. the guidelines require states to develop both an "emergency operation plan" and a "procedural document." the eop is a response plan that adapts the disaster response principles and the emergency support functions specified in the nrf tailored to the situation of the state. the procedural document consists of general principles for disaster response, standard action procedures, on-site operation guides, and job aids. each standard operating procedure (sop), the instruction for carrying out esf tasks to enable the smooth support for disaster response in the field and how to carry out those disaster response tasks, contains the guiding principles listed in the esf. it can be drawn from fema's actions that the modified us disaster management system would emphasize comprehensive, integrated, and mutual cooperation among the relevant organizations and stakeholders. to solidify this new emphasis, fema announced in seven guiding principles to cope with national emergencies: comprehensive, progressive, risk-driven, integrated, collaborative, coordinated, flexible, and professional (emergency management ). describe key roles and responsibilities around the nation and structures for implementing nationwide response policy and operational coordination for all types of domestic emergency events emergency support function annexes specify the federal resources and capabilities to provide emergency support for functional areas and identify coordinator, primary agency, and support agency for each functional area support annexes describe common and basic supports to the majority of incidents: critical infrastructure and key resources support, financial management support, international coordination support, etc. describe the response methods for seven incident categories: biological, catastrophic, cyber, food and agriculture, mass evacuation, nuclear/radiological, and terrorism. the basic act for disaster management in the usa is the stafford disaster relief and emergency assistance act. it gives the state the right to request support from the federal government and the right of the federal government to supplement resources to requesting state. additionally, it gives the president the right to declare emergency or major disaster in order to provide federal assistance. the homeland security act and the post-katrina emergency management reform act are also important disaster-related laws. japan has been exposed to various natural and human-caused disasters, such as earthquake, typhoon, and hazardous material contamination. to cope with those threats, japan has developed a comprehensive disaster management system at the national and local levels. the "basic act on disaster control measures," enacted in , functions as the backbone of the disaster management system in japan. the japanese government enacted the law in the wake of typhoon vera (isewan typhoon), which caused deaths, , injures, and property damage of approximately . trillion yen. since then, the "basic act on disaster control measures" has become the foundation for carrying out all measures related to disasters including emergency countermeasures and recovery as well as the disaster prevention, and this law handles disaster prevention measures in each field comprehensively. after the great hanshin-awaji earthquake on january , , japan revised the master plan for disaster prevention completely, and the "basic act on disaster control measures" was partially revised to reflect the lessons learned from the earthquake. the great hanshin-awaji earthquake, which led to a full modification of the master plan for disaster prevention, resulted in deaths, , houses destroyed completely, and property damage of trillion yen, which accounted for . % of gross domestic product (gdp) at that time. with the occasion of several huge disasters, the disaster management system in japan has been appropriately modified so that a more systematic disaster response can be provided through central disaster prevention meetings. a central disaster prevention meeting (chairman, prime minister) consists of the prime minister, the minister of disaster prevention, related ministers, the representatives of designated public agencies, and people with knowledge and experience designated by the prime minister. this meeting carries out the preparation and implementation of a master plan for disaster prevention and emergency measure plans and the examination of important items regarding disaster prevention according to the advice of the prime minister. the fire and disaster management agency under the ministry of internal affairs and communications, which is equivalent to the central firefighting headquarters in korea, was established based on article , paragraph of the national government organization act and article of the firefighting organization act. the fire and disaster management agency under the ministry of internal affairs and communications takes charge of planning and drawing up firefighting administration procedures and policies of various laws and standards. this agency has no direct right to command firefighting at the local level but handles tasks in an adversarial, instructional, and coordination role. the general affairs and planning department (bureau) and disaster prevention sections in the general affairs division have been established to carry out cooperation and coordination tasks since the cooperation and coordination between relevant local divisions are important for disaster management. the local disaster prevention meeting is divided into prefectures and municipalities. the local crisis meeting of prefectures consists of heads of local administrative agencies, self-defense forces, and superintendents of education with the prefecture governor as the manager. the head of the municipality takes charge of the municipality local crisis meeting. these two agencies take charge of contact and coordination between relevant agencies in case of a disaster and play a role in establishing and implementing a disaster prevention plan to handle each step effectively including disaster prevention, emergency disaster measures, and disaster recovery. the local emergency organization is the local disaster relief center. the local disaster relief center is installed according to a local disaster prevention plan in case a disaster is expected or a disaster occurs. the local disaster relief center is responsible for carrying out disaster prevention and emergency disaster measures related to the relevant prefecture or municipality according to the local disaster prevention plan of the relevant prefecture or the local disaster prevention plan of the municipality. the central/local and normal times/emergency response institutions of japan are summarized in table . . the disaster management system in japan has been established into central and local disaster management systems and regular and emergency disaster management systems to enable a smooth communication among divisions. japan, where various disasters occur frequently, has established various systematic disaster response plans just as the disaster management system. the disaster response plan consists of a "master plan for disaster prevention" for the central government and a "local disaster prevention plan" for the local level. the "master plan for disaster prevention" is a comprehensive long-term plan for disaster prevention prepared by the central crisis meeting, and it is based on the basic act on disaster control measures. it is responsible for determining the comprehensive and long-term plan for disaster prevention, the key points for the disaster prevention task plan and the local disaster prevention plan, and the preparation standard for the disaster prevention task plan and the local disaster prevention plan. based on such disaster prevention plans, japan has prepared for disaster prevention in three steps including prevention, emergency measures, and recovery and redevelopment. each step contains the basic policy for disaster prevention, mutual linkage around the country between public agencies and local governments, and measures to share disaster prevention information between disaster prevention agencies and residents. the central disaster prevention plan and the local disaster prevention plan are executed identically. disaster management in australia utilizes a comprehensive and integrated approach (ema ) . each provincial and local government, in accordance with federal guidelines, establishes a disaster management act that puts forward how local disaster entities should actively and effectively prepare against regional risks. such a decentralized legal system gives each state or local government the responsibility and the flexibility to carry out disaster countermeasures tailored to the characteristics of disasters, which have occurred or are to occur in each area of australia. one of the most influential events that moved opinion for the improvement of disaster management in australia was a large-scale fire called the tasmanian bushfire that occurred on february , . this large-scale fire resulted in deaths, displaced from families, and , ha of land damaged. this fire made the australian government recognize the importance of a disaster management system at the federal level, which resulted in the establishment of the natural disaster organization (ndo) in . this organization was strengthened in due to the needs of more systematic disaster preparedness, and its name was also changed to emergency management australia (ema). currently, ema performs its duty as the standard central organization for disaster management and, based on the commonwealth government disaster response plan (comdisplan), takes charge of planning and the coordination for disaster management at the state, district, and local governmental levels in australia. disaster management in australia is divided into four levels: federal, state, district, and local. the disaster management system is handled by ema at the federal level, whereas disaster management groups (dmg) -state/district/local -manage the disaster management system at the state, district, and local levels. at the federal level, the australian government has established separate disaster management systems suitable normal times and emergency situations, respectively. the ema division in the attorney general's department carries out normal disaster management: national disaster management, planning, coordination, -h disaster status monitoring, international support, and cooperation tasks. in case of emergency situations, the federal government carries out disaster management through the australian government crisis coordination centre (ccc), an emergency center coordinating disaster responses for all line federal departments and state, district, and local governments. the state, district, and local governments in australia also have two types of disaster management systems suitable for normal times and emergency situations, respectively. during normal times, the dmg is the primary organization to deal with disaster management, particularly focusing on prevention and preparedness, which is managed by the department of the premier and cabinet (premier of state government), consisting of the premier of each state government, all state ministers, and the army commander of each state government. the dmg develops and operates disaster management plans, strategies, and policies and supports state, district, and local management groups. in case of an emergency, the manager of the dmg should appoint a disaster coordinator in advance for the cooperation and coordination between the federal government, other state governments, and relevant organizations within state governments. on the other hand, the disaster coordination centre-state/district/local (dcc) was established to handle and operate a disaster management system during emergencies more systematically. the dcc consists of divisions representing each state government, the bureau of meteorology under the federal government, the australian defense force, the australian red cross, and the australian government ) insurance parliament at the senior officer level, and is responsible for decisionmaking and coordination regarding support of resources to local, district, and state governments according to the level of the disaster situation occurrence. the disaster response plans in australia have also been separated into a federal level and a local-level disaster response plan. the disaster response plan of the federal government is the comdisplan, which specifies competent disaster organizations in the six states and seven territories of australia, and the contents and procedures for resource support and cooperation between state governments. the disaster response plan at the state, district, and local levels is called the disaster management plan (dmp) or guideline-state/district/local and consists of four parts: prevention, preparedness, response, and recovery for strategic policies to deal with disaster by delineating roles and responsibilities for each phase in disaster management. the roles and responsibilities of each department and agency are specifically described in esfs. for more specific response activities, the response phase is subdivided into four steps: alert, lean forward, stand up, and stand down. two record-breaking disasters have occurred in australia since . the black saturday bushfires that occurred on february , , resulted in large-scale forest fire damage in the southeastern region of australia. more than forest fires occurred simultaneously resulting in a more significant damage occurrence, and these forest fires resulted in deaths and forest loss of , ha (victorian bushfire reconstruction and recovery authority ). a series of floods called the queensland floods occurred in western brisbane, queensland, on december , . these floods resulted in more than dead, more than missing, and approximately , homes and businesses flooded (queensland floods commission of inquiry, ). these floods were recorded as the largest floods within years, and the continuous rain for weeks made the damage greater. the australian government is in the process of improving its disaster management system in the wake of these two catastrophic events, which is worthwhile to be monitored. according to germany's basic law, the primary responsibility for disaster management rests on local and state governments, and the federal government provides financial, human, and physical assistance to local and state governments when a large-scale disaster occurs that exceeds the capacity of the local government or the state government. in the event of a disaster beyond the capacity of the state, the state will be supported by police departments and military forces in the neighboring states. and if necessary, the state receives the support of the police department, military forces, and technical support from the federal government. the federal ministry of the interior (bmi, bundesministerium des innern) is in charge of coping with major disaster or nonmilitary crisis that require the intervention of the federal government. in particular, the federal office of civil protection and disaster assistance (bbk, bundesamt für bev€ olkerungsschutz und katastrophenhilfe), established in may under the ministry of the interior, is responsible for responding to disasters in an integrated manner through systematic cooperation with other federal, state, and local governments in the event of largescale disaster. bbk has been expanded from the federal office of administration (bundesverwaltungsamt) as the importance of civil protection has increased in germany, in the wake of the september terrorist attacks in the united states in and the floods of the elbe river in germany in august . the main responsibilities of the bbk include the development of a comprehensive civil protection plan, dissemination of emergency information to citizens in crisis situations, critical infrastructure protection, and education and training for civil defense. the joint situation and information center (gmlz, gemeinsames melde-und lagezentrum von bund und ländern) in the bbk is responsible for monitoring disaster situations, disseminating disaster information, and international requests for help. when a large-scale disaster occurs in germany, the federal ministry of home affairs calls up the crisis task force (krisenstab). the task force works jointly with relevant departments within the federal government, agencies under the ministry of interior, and state liaison officers. in the event of a major type of catastrophic event that can be a national crisis, the ministry of interior will form the joint task force with responsible ministries. for example, if a nuclear accident or an illegal use of radioactive materials happens, the bmi and the federal ministry for the environment, nature conservation, building and nuclear safety (bmub, bundesministerium für umwelt, naturschutz, bau und reaktorsicherheit) will form the joint crisis management task force. when pandemics or biochemical terrorism occurs, the bmi and the federal ministry of health (bmg, bundesministerium für gesundheit) will form the joint task force. the german local system consists of the state (länder) government, the city or county (kreis) government, and the municipal government (gemeinde). disaster management organizations in germany's local system are different depending on the characteristics of each region; however, departments dealing with internal affairs mostly take charge of disaster management. in recent years, there have also been a growing number of states establishing disaster risk protection departments (gefahrenabwehr) by experiencing increased terrorism and massive natural disasters due to climate change. the central/local and normal times/emergency institutions of germany are summarized in table . . each state government develops and operates its own disaster response plan. for example, hessen is the state government that has the disaster response plan under the title of katastrophenschutzpläne (emergency plans). katastrophenschutzpläne (emergency plans) of hessen specifies the contents regarding necessary information in the event of disaster and means to be used. also, in case of a very severe disaster such as nuclear risk that targets a specific object, it is required to establish and operate sonderschutzpläne (special protection plans) separately. figure . shows the katastrophenschutzpläne (emergency plans) in hessen, germany, and it shows the contents regarding the sonderschutzpläne (special protection plans). the english translation of fig. . is: § disaster protection plans the disaster protection plans shall contain, in particular, the necessary information on the emergency aid, the alarm, and the means of assistance available in a disaster. they must be coordinated with the neighboring disaster control authorities. special protection plans are to be drawn up for special dangers. the uk, based on the tradition of local autonomy and accountability of citizen and local governments, utilizes a bottom-up disaster management system. in , the cabinet office, a guide to emergency response and recovery was released with eight core principles: anticipation, preparedness, subsidiarity, direction, information, integration, cooperation, and continuity (cabinet office a). the basic act for disaster management in the uk is the civil contingencies act (cca), . the uk has developed its disaster management system suitable for normal times and emergency situations at the federal and at the local levels (korean association for local government studies ) . at the federal level, the civil contingencies secretariat (ccs) was established in to take charge of the overall coordination of disaster management during normal times, whereas the cabinet office briefing room (cobr) and the civil contingencies committee (ccc) take charge of disaster response in case of emergency situations. the ccs is headed by the permanent secretary, vice-minister level official in the cabinet office, and it takes overall responsibility for disaster management in the uk. if needed, the minister of the home office (or the cabinet office) reports to the national assembly for sharing emergency information or hearings after hit by disaster. the ccs carries out the emergency response tasks including anti-terrorism and disaster restoration tasks and is responsible for identifying and making preparations for a crisis during an emergency situation. the cobr and the ccc are activated for the federal government to act when catastrophic events occur. the activation process and procedures are decided depending on the level of emergency, specifically at level and level ; and at . disaster theories and progress of disaster management in modern times level , the responsible minister takes the leading role as chairman, and at level , the prime minister serves as chairman. table . shows the three levels for disaster response, in which the roles and responsibilities of each agency at the federal and the local levels are delineated differently. the cobr and the ccc focus on cooperation between the federal and local governments and support tasks rather than command and control. also, highranking officials can participate in the cobr, receiving and processing a report of situation, for prompt disaster management. figure . shows the block diagram of cobr. the cca categorizes agencies for disaster response into two categories according to their roles and duties with different obligations. the category responders are the agencies that have priority for response. the category responders consist of police services, fire and rescue services, health bodies, maritime and coastguard agency, local authorities, and environment agency. the police normally control and coordinate the activities at and around the scene. there are, however, exceptions, for example, the fire and rescue service takes the responsibility at the scene of a major fire. category responders consist of a wide range of private sector bodies that have an important role, but not routinely involved in the core of multi-agency emergency response and recovery work. utilities, telecommunications and transport providers, highways agency, strategic health authorities, and health and safety executives are included in the category responders. a local resilience forum (lrf) is a private and public joint organization of local governments, military officials, corporations, and civic groups that analyzes risk factors and establishes the risk management plan. the main aim of the lrf is to assist multi-agency and multi-sectoral cooperation. the lrf has regular meetings at least once every months to strengthen regional resilience, and category responders should participate in the meetings. the lrf is not a legal entity, nor does the lrf have powers to direct its members. nevertheless, the cca and the regulations provide that responders, through the forum, have a collective responsibility to plan, prepare, and communicate in a multi-agency environment. the police serve as chairman for the lrf, and a manager is assigned to each of the areas classified according to the police administrative district. a regional resilience forum (rrf) is a high-level organization of the lrf to coordinate plans for large-scale emergencies that are difficult to handle locally and to coordinate with the central government. the rrf divides areas into local units, and a table . disaster step as the standard of disaster management system in the uk ((cabinet office, b)) step disaster support system by step step (significant) the competent agency provides supports and cooperation at central government level step (serious) the national crisis committee is organized with the competent minister as the chairman step (catastrophic) the national crisis committee is organized with the prime minister as the chairman local government office is located in each local unit, and the regional director of government offices serves as chairman. the strategic coordinating group (scg) and the regional civil contingencies committee (rccc) are local emergency organizations for coordinating multiagency cooperation. the scg is a type of accident response and recovery center consisting of government branches, military, police, fire, hospitals, and private local committees in the relevant area at the local level for accident response, and all government officials dispatched to the jurisdiction of the central government are under the direction and control of the local director. the rccc is established when an emergency crisis exceeds the local level, and its composition and system are similar with the rrf. the disaster management in the uk is shown in table . . the disaster response plan in the uk is also operated separately into national and local disaster response plans. the national disaster response plan in the uk is carried out through the national contingency plan (ncp). the ncp describes initial response, accident response, responsibility and compensation, role and responsibility of central government, international support, and cooperation for each type of disaster, and it becomes the foundation for the disaster plan of the central government. the uk has also prepared a disaster response plan for flood under the ncp since flood occurs frequently, which is the guidance for accessing specialist flood rescue mutual aid. the guidance for accessing specialist flood rescue mutual aid specifies the strategic approach of the government for preparing for and responding to a flood, and it is responsible for providing comprehensive emergency response guidelines to all flood rescue service providers including public and private volunteer organizations including the utilization of existing and future flood relief assets. also, the guidance for accessing specialist flood rescue mutual aid presents the role and responsibility for each agency regarding floods as well as the action procedure (sop) to enable prompt response in case of a flood. the local disaster response plan in the uk is carried out through the strategic emergency plan (sep). the purpose of the sep is to present the direction of regional response activities and methods, through which the disaster response strategy and disaster response plan of each cooperation organization is prepared. the sep is managed through the lrf organized in each area, and the lrf is responsible for developing a more detailed implementation plan on the basis of what was given by rrf. in switzerland, cantons and communes have the authority and responsibility for disaster response, and the federal government intervenes only in the event of a national crisis. particularly, switzerland has a disaster management system based on a close cooperation among the federal, state, and local governments, a proactive role of the military in disaster response, and a strong civil defense system. the basic act of disaster management in switzerland is the federal civil protection and civil defence act (bzg, bundesgesetz über den bev€ olkerungsschutz und den zivilschutz). at the federal level, the federal office for civil protection (babs, bundesamt für bev€ olkerungsschutz) under the federal department for defense, civil protection and sport (vbs, eidgen€ ossisches departement für verteidigung, bev€ olkerungsschutz und sport) is responsible for disaster management. under babs, national alarm center (naz, nationale alarmzentrale) operates h all around the year in order to monitor situations, disseminate disaster information, and respond to disasters when necessary. since its establishment in , naz has designated radioactivity, chemical accidents, and the collapse of bridges due to a natural disaster as major disasters, making preparations for such disasters, and naz has also established the linkage system between major facilities and competent authorities to judge the situation in case of a disaster. the office for civil protection, sport, and military (bsm, amt für bev€ olkerungsschutz, sport und militär) takes charge of the normal-time disaster . rccc (regional civil contingencies committee) . lrf (local resilience forum) . scg (strategic coordinating group) management system at the local level. bsm is divided into four departments, and the department related to disaster management is included in the citizen and nation protection division. the manager of bsm changes according to the conditions of the state government. the five local-level emergency services utilize an "integrated system" (verbundsystem) which allows for an autonomous or a cooperative response, depending on the severity of the situation. in an extended or severe disaster situation, any of the police, fire brigades, health and ambulance services, technical agencies, or civil defense organizations can partner up to handle the crisis; this can be done at the communal or at the cantonal level. additionally, based on federal government guidelines, which includes the basic principle of disaster management and civil defense, the physical plan for disaster response, and the technical and structural countermeasures, state and local governments develop and manage their own disaster response plan. for planning and preventative measures for natural hazards, the five civil-security agencies follow the national platform for natural hazards (planat) guidelines. the swiss civil-security agencies, basing their planning activities on "risk-based planning" and "integral risk management," have developed common prioritization procedural guidelines (riko) and an online tool (econome). the guideline and the online tool, based on "protection objectives" (schutzziele), are also designed to keep natural hazard management projects cost-effective. when it comes to implementing a project, the babs have developed a tool (kataplan) to identify and classify various risks and the planning of response measures by cantonal agencies. the babs have also developed a learning program (lernrisk) and assessment software (riskplan) (babs ; bfu and babs ) . until now, we have described the disaster management system in six countries. table . shows the national and local disaster management organizations during normal time and emergency situations. social advancements in contemporary society beget new techniques and technologies. thus, new risks due to the application of the new technologies are continually springing up, and difficulties predicting uncertainties increase in parallel with new risks. furthermore, due to the development of information technology, the rise of interdependence increases propagation or chain properties to expand continuity in type, range, and scope (oh ) . the contemporary society can be featured with a change in natural environment such as climate change, socioeconomic environmental change such as aging, urbanization, and polarization, international environmental change such as each country being networked, and an entrance of complexity which indicates characters such as emergence, self-organization, and adaptation. due to these changes in environment, the contemporary society can be represented as increase of complex general set up the strategic objective for national safety, crisis management, and disaster relief and established the comprehensive drm system. in summary, most developed countries are in the process of shifting from a top-down, fragmented, and hazard-oriented disaster risk management approach into a comprehensive, integrated, and human-centered approach. while this is happening, special attention is being paid to "low probability and high impact" focusing events that are located in the long tail of the power-law distribution. symbolic and practical interpretations of the hurricane katrina disaster hazard mitigation and preparedness babs ( ) leitfaden kataplan: kantonale gefährdungsanalyse und vorsorge bfu and babs ( ) pragmatisches risikomanagement mit riskplan online risikogesellschaft: auf dem weg in eine andere moderne. frankfurt am main, suhrkamp beck u ( ) world risk society cabinet office ( b) responding to emergencies the uk central government response concept of operations disaster data: a balanced perspective national incident management system dhs ( ) threat and hazard identification and risk assessment guide: comprehensive preparedness guide again and again: is a disaster what we call a "disaster? emergent phenomena and the sociology of disaster: lessons, trends, and opportunities from the research literature principles of emergency management supplement emergency management australia (ema) ( ) emergency management in australia: concepts and principles disaster theory: an interdisciplinary approach to concepts and causes disaster relief and emergency assistance act, as amended, and related authorities fema ( ) national response framework: third edition fritz ce ( ) disaster and community theory introduction to emergency management, th edn hessisches gesetz über den brandschutz, die allgemeine hilfe und den katastrophenschutz national comprehensive crisis management: theory and reality research on effective disaster relief planning disaster classification for optimal disaster response in korea economic loss due to thailand flood in and policy implications to korea business korean association for local government studies ( ) a study on the comparison of disaster and safety management system in major advanced countries study on the change of risk structure in korea and its social and cultural effect a terminological and etymological study about safety damage situation in thailand big flood and implications for korean companies building an integrated emergency management system in korea introduction of disaster prevention. donghwa tech, hwaseong-si mcloughlin d ( ) a framework for integrated emergency management the network governance of crisis response: case studies of incident command system standard crisis management manual munich re ( ) natcatservice, global distribution of insurance premiums per capita crisis management system in korea: focusing on nuclear power plant and oil transportation the role of u. s. military forces and its implication for the korean military during disaster management natural disaster and development in a globalizing world. routledge, london perrow c ( ) normal accidents: living with high risk technologies what is a disaster? the need for clarification in definition and conceptualization in research technological and natural disasters and ecological problems: similarities and differences in planning for and managing them queensland disaster management arrangements public administration: understanding management, politics, and law in the public sector victorian bushfire reconstruction and recovery authority ( ) victorian bushfire reconstruction and recovery authority day report living with hazards, dealing with disasters: an introduction to emergency management collaboration and leadership for effective emergency management oecd studies in risk management: innovation in country risk management most developed countries have put stress on developing an effective disaster response system to cope with emerging risk interwoven with complexity and climate change. the policy implications through the comparative analysis can be summarized as follows:first, many countries aim for an integrated organization that considers both natural disasters and social disasters. the usa has endeavored to build an integrated disaster management system through the establishment of fema, and since the / terror attack, the dhs has been integrated to take charge of disaster management and terrorism. the dhs set the national preparedness goal to reflect the insights and lessons learned from hurricane katrina and to build a comprehensive organizational capacity for the entire nation that includes federal, state, and local governments and the private sector.japan and the uk have an organization that control and coordinate disaster and emergency response directly under the prime minister. the cca of uk and the cabinet office of japan are both small in size, but they are characterized by a very high level of authority for the overall coordination of disaster policies. according to the types in the reorganization of disaster management proposed by the organization for economic cooperation and development (oecd), the case of the usa corresponds to the first case, "super-ministry," and japan and the uk correspond to the second type, "a relatively small and highly influential body under direct authority of the head of government" (wyman ). disaster response plans are also being developed within national and local governments using an integrated disaster management style for all hazards approaches (waugh ) .second, all stakeholder engagement for disaster response is highly recommended. the usa successfully established the disaster risk management system by promoting the engagement and commitment of all levels of governments, citizens, and ngos. australia and japan also developed standard operating procedures indicating citizenry roles and activities in case of emergencies. in addition, australia developed checklists to enhance citizenry participation for disaster preparedness. japan has enhanced citizenry participation and preparedness by strengthening citizenry education and training. korea should improve the participation of local governments, ngos, and citizens to drm by providing guidelines and incentives and enhancing the governance-based approach.finally, many countries have stressed the clear accountability of each government and interlink between the national government and local governments. most countries, such as australia, germany, japan, and the usa, empowered local governments for disaster response and engaged in the national crisis situation. in addition, the interlink between national and local disaster response planning was also emphasized. the usa improved interlink between nrf and nims at the federal level and eop at the state and local levels. in australia, the attorney key: cord- -iz bo authors: shaw, dominick; portelli, michael; sayers, ian title: asthma date: - - journal: handbook of pharmacogenomics and stratified medicine doi: . /b - - - - . - sha: doc_id: cord_uid: iz bo asthma is a common respiratory disease with a complex etiology involving a combination of genetic and environmental components. current asthma management involves a step-up and step-down approach based on asthma control with a large degree of heterogeneity in responses to the main drug classes currently in use: β( )-adrenergic receptor agonists, corticosteroids, and leukotriene modifiers. importantly, asthma is heterogeneous with respect to clinical presentation and the inflammatory mechanisms that underlie it. this heterogeneity likely contributes to variable results in clinical trials, particularly when targeting specific inflammatory mediators. these factors have motivated a drive toward stratified medicine in asthma based on clinical/cellular outcomes or genetics (i.e., pharmacogenetics). significant progress has been made in identifying genetic polymorphisms that influence the efficacy and potential for adverse effects of all main classes of asthma drugs. importantly an emerging role for genetics in phase ii development of newer therapies has been demonstrated (e.g., anti-il ). similarly, the stratification of patients based on clinical characteristics (e.g., blood and sputum eosinophil levels) has been critical in evaluating newer therapies (e.g., anti-il ). as a proof of concept, anti-ige is the latest therapy to be introduced into clinical practice, although only for severe, allergic patients (i.e., in a stratified manner). as new asthma genes are identified using genome-wide association, among other technologies, new targets (e.g., il /il receptor (il rl )) will emerge and pharmacogenetics in these development programs will be essential. in this chapter we review the current understanding of asthma pathobiology and its clinical presentation, as well as the use of stratified medicine, which holds great promise for maximizing clinical outcomes and minimizing adverse effects in existing and new therapies. umbrella definition will require integrating bench and bedside approaches using data from ongoing genomic and proteomic profiling studies of large, well-characterized asthma populations-such as the current eu-wide ubiopred study (formally, "unbiased biomarkers in prediction of respiratory disease outcomes")-with feedback to improved in vitro and in vivo animal models and human studies. the international consensus report on the diagnosis and treatment of asthma defines asthma as "a chronic inflammatory disorder of the airways in which many cells play a role, including mast cells and eosinophils. in susceptible individuals this inflammation causes symptoms which are usually associated with widespread, but variable, airflow obstruction that is often reversible either spontaneously or with treatment, and causes an associated increase in airway responsiveness to a variety of stimuli." the interaction of these features determines the clinical manifestations and severity of the disease and the response to treatment [ ] . how these features relate to each other, how they are best measured, and how they contribute to clinical manifestations of the disease remains unclear. age of disease onset also complicates understanding, although there are many shared features in the diagnosis of asthma in children and in adults, there are also important differences. the differential diagnosis, the natural history of wheezing illnesses, and the ability to perform certain investigations are all influenced by age [ ] . according to the world health organization (who), between and million people around the globe (roughly equivalent to the population of the russian federation) suffer from asthma. worldwide, deaths from asthma have reached more than , annually. overall, asthma affects - % of the population in many developed countries. one study found that the annual estimated incidence of physician-diagnosed asthma ranged from . - . per persons. risk factors for incident asthma among children include male gender, atopic sensitization, parental history of asthma, early-life stressors and infections, obesity, and exposure to indoor allergens, tobacco smoke, and outdoor pollutants. risk factors for adult-onset asthma include female sex, airway hyper-responsiveness, lifestyle factors, and work-related exposures. while the exact cost of asthma care is difficult to determine, a systematic review [ ] found eight national studies reporting a national total cost. the total costs in in u.s. dollars were high and varied widely: singapore, $ . m; canada, $ m; switzerland, $ m; germany, $ m; united states, $ m. although the costs of asthma care vary by country, it is estimated that worldwide the number of disability-adjusted life years (dalys) lost from asthma is about million per year. worldwide asthma accounts for around % of all dalys lost, which reflects the high prevalence and severity of asthma. the number of asthmacaused lost dalys is similar to that for diabetes, cirrhosis of the liver, and schizophrenia. costs are related to other factors, including asthma control, demographics, medical history, and doses of inhaled corticosteroid (ics) prescribed [ ] . improving asthma control is associated with decreased cost. asthma classically causes wheeze, cough, chest tightness, and breathlessness (dyspnoea). the pattern of symptoms can be a clue to the diagnosis. episodic symptoms worse at night and in the early morning, or in response to exercise, allergen and cold air exposure, or after taking aspirin or beta blockers are very suggestive of asthma. moreover, symptoms that are relieved by bronchodilators point toward asthma as the underlying cause. symptoms that lower the probability of asthma include those of prominent dizziness, light-headedness, and peripheral tingling (all point toward dysfunctional breathing); a chronic productive cough in the absence of wheeze or breathlessness (more likely bronchiectasis); a repeatedly normal physical examination of chest when symptomatic; and voice disturbance, symptoms with colds only, or a significant smoking history. taking a detailed patient history and recording spirometry when the patient is symptomatic is crucial to the accurate classification of the disease. there are several differential diagnoses that must be considered when making a diagnosis of asthma. these are outside the scope of this chapter. a good guide to the clinical diagnosis and management of asthma are the british thoracic society asthma guidelines, which are updated regularly and are available at www.brit-thoracic.org.uk/clinical-information/ asthma/. triggers for asthma symptoms include allergens (proteins) derived from, among others, house dust mite (hdm), cat and dog dander, cockroach, and fungi, especially aspergillus fumigatus. there are numerous other nonallergic symptom triggers, including weather and atmospheric change, air pollution, exercise, menstruation, emotion and laughter, viral infection, gastroesophageal reflux, and rhinitis. various occupations can both cause and worsen asthma (e.g., welding, baking, paint spraying). asthma symptoms often overlap with other allergies, including oral allergy syndrome and food allergies. delineating between causes of symptoms can be difficult. if a specific aeroallergen is identified that triggers asthma (classically tree or grass pollen), a course of either sublingual or subcutaneous desensitization can be undertaken to reduce symptoms on exposure to that specific allergen. there are multiple other symptom triggers that can include both allergic and non-allergic mechanisms; examples include exposure to chemicals, such as perfumes, paint and bleach, plants (e.g., ligustrum vulgare-privet hedges), salicylates, and sulphites. multiple factors have been shown to either directly cause or worsen symptoms of asthma [ ] . these are listed in box . . other factors not directly related to the disease can worsen symptoms and are associated with more frequent exacerbations. these include dysfunctional breathing, vocal cord dysfunction, psychosocial problems, and poor adherence to treatment regimes [ ] . other forms of asthma are recognized, among them occupational, exercise-induced, and pregnancy-related asthma. these subdivisions are somewhat arbitrary, but may have different causes, prognoses, and complexities. for example, occupational asthma includes that triggered by ige-mediated mechanisms, that due to specific agents with unclear pathophysiology, and that secondary to irritants (also known as rads-reactive airways dysfunction syndrome). underlying asthma can also be made worse by occupational exposure (work-aggravated). most experts regard exercise-induced asthma as a different disease, albeit one resulting in similar symptoms. exercise-induced asthma is often diagnosed with different investigations, including eucapnic voluntary hyperventilation or exercise testing. it is associated with certain sports (swimming and cross country skiing in particular) and has a unique pathophysiology directly related to damage of the airway epithelium. asthma control can deteriorate in pregnancy, due to both hormonal effects and the physical effects of a gravid uterus on diaphragm and respiratory muscle function. pregnancy can also impact treatment decisions. leukotriene modifiers are generally contraindicated, and the health of the fetus as well as that of the mother needs to be considered. poor adherence is probably the biggest single issue affecting asthma control. studies have shown that people with asthma over-report use of ics; one study found that although % of responders said they used their inhalers, only % actually did. adherence to ics therapy is associated with a lower rate of death, whereas increasing use of reliever (salbutamol/bricanyl) medication, which improves symptoms but does not treat underlying airway inflammation, is associated with increased mortality [ , ] . a report entitled evaluation of the scale, causes and costs of waste medicines: report of dh funded national project (trueman et al. york health economics consortium and university of london school of pharmacy, ) put figures to the possible cost benefits from improving asthma treatment adherence in the united kingdom. it estimated the resulting net benefit associated with compliance to be £ per patient, with a reduction in expected annual treatment costs of approximately £ per patient per year. based on an asthma point prevalence of . %, the findings suggest that there are almost . million asthmatics in the united kingdom who are noncompliant. if interventions were available to change the behavior of all partially compliant medicine users to raise the percentage to % or more, the report estimated that over £ million in treatment cost savings could be realized in the united kingdom alone. a more modest target of doubling current compliance rates would result in savings of approximately £ million per year. the aim of asthma treatment is to improve symptoms, maintain lung function, and prevent exacerbations. from a patient's perspective, these are simple aims, but for the purposes of study design and treatment trials they do not address the complexity of the disease process. accordingly, guidelines now specifically address asthma control (symptoms and lung function) and severity (need for treatment) separately. the current global initiative for asthma (gina) guidelines assess levels of control based on daytime symptoms, limitation of activities, nocturnal symptoms/awakening, need for reliever/rescue treatment, lung function (pef or fev ), and exacerbations [ ] ( treatment steps are defined by the treatment level required to maintain asthma control (table . ), with patients at steps - having well-controlled asthma requiring little treatment, and patients at step having poorly controlled asthma despite four or five different drugs. the step approach is similar to that used by the british thoracic society (bts) guidelines [ ] . asthma is a variable disease both temporally and clinically. there is also overlap between symptoms and exacerbations. this overlap was the subject of a recent consensus document that defined asthma exacerbations as "events characterized by a change from the patient's previous status." severe exacerbations are "events that require urgent action on the part of the patient and physician to prevent a serious outcome, such as hospitalization or death from asthma," and moderate asthma exacerbations are "events that are troublesome to the patient, and that prompt a need for a change in treatment, but that are not severe…clinically identified by being outside the patient's usual range of day-to-day asthma variation" [ ] . the most practical definition of an asthma exacerbation is probably an episode of worsening symptoms not responding to increasing bronchodilator therapy. this definition was employed in an elegant study on the time course of peak flow changes [ ] . for the purposes of clinical studies, most authors define exacerbation as the need for rescue courses of systemic corticosteroids (prednisolone/prednisone). although the majority of patients with asthma are treated and investigated in primary care settings, the main burden of asthma is due to its severe form (i.e., step ) and exacerbations. using the united kingdom as an example (population million), asthma is responsible for more than deaths per year and for over , hospital admissions, with an annual expenditure of £ million on pharmaceutical costs alone. it was recently estimated that a patient controlled at the mildest end of the spectrum (british thoracic society (bts) guidelines: step with no exacerbations) would cost times less to provide his or her package of care than would a patient at the worst end of the spectrum (bts guidelines: step with exacerbations) [ ] . it is also calculated that % of asthma patients are responsible for at least % of the total healthcare burden [ ] ; the most expensive individual cost is an icu admission (bts level ) due to a life-threatening exacerbation. in the u.s. more than half ( %) of asthma sufferers in had an asthma attack, and of these half of the children and one-third of the adults missed school or work because of it; on average, children missed four days of school and adults missed five days of work. exacerbation pathogenesis is not fully understood. although research has focused on infective agents, especially viral, there may be other explanations. asthma exacerbations are associated with both inflammatory and immunological cell infiltration. the inflammatory cell infiltrate is composed of varying numbers of eosinophils, neutrophils, and lymphocytes. this airway inflammation, combined with smooth-muscle hypertrophy step (intermittent) fewer than once/week; asymptomatic, normal pef between attacks two or fewer/month % or more less than % step (mild persistent) more than once/week, fewer than once/day; attacks may affect activity > times a month >/= % - % step (moderate persistent) daily; attacks affect activity more than once/week %- % more than % step (severe) continuous; limited physical activity frequent % or less more than % and thickening of the lamina reticularis, is accentuated by mucus plugs, serum protein deposition, inflammatory cell and cellular debris, leading to blockage of the airway (airflow obstruction) and wheeze. approximately % of exacerbations are associated with respiratory tract viral infections, with rhinovirus responsible for about two-thirds of cases [ ] . asthmatic subjects also have much more severe lower respiratory tract illness with rhinovirus infection than do healthy control subjects. the mechanism for this is not fully understood. infection induces inflammation, increasing levels of neutrophils, eosinophils, cd + cells, cd + cells, and mast cells through increased mrna expression and translation of il- , il- , il- , eotaxin, ifn-γ-induced protein (ip- ), chemokine (c-c motif) ligand (ccl /rantes), and other proinflammatory cytokines. other viruses implicated in exacerbations include enterovirus, coronavirus, influenza, parainfluenza, respiratory syncytial virus, metapneumovirus, adenovirus and bocavirus [ ] . although influenza vaccination is recommended for all individuals with asthma, there is currently no hard evidence that this improves outcomes. bacterial infection has also been implicated in asthma exacerbation. individuals with asthma have an increased risk of invasive pneumococcal disease [ ] , and an increased frequency of detection of chlamydophila pneumoniae [ ] . one study found mycoplasma pneumoniae infection in % of patients hospitalized for severe asthma [ ] . several trials have evaluated the role of macrolide antibiotics in treating and preventing asthma exacerbations. one study randomized adults with exacerbations to the ketolide antibiotic telithromycin or placebo; the telithromycin group had a small but significant improvement in symptoms and lung function from exacerbation to the end of treatment [ ] . further studies of macrolide antibiotics (which have immunomodulatory as well as antibiotic properties) are under way, but as yet macrolide antibiotics are not recommended by international asthma guidelines. new techniques for assessing the airway bacterial microbiota have established that the airways are not normally sterile, and may permit the role of bacterial infection in airways disease to be further delineated. a recent study found that pathogenic proteobacteria, particularly haemophilus species, were much more frequent in the bronchi of patients with asthma or copd than in controls [ ] . prolonged exposure to aeroallergens can result in chronic airway inflammation via th -driven ige mechanisms. this immunological reaction may intensify airway inflammation, increase inflammatory cell activation, and stimulate mucus glands to hypersecrete, leading to airway obstruction. studies of bronchoalveolar fluid before and after allergen challenge show eosinophilic inflammation as the major airway response, associated with late-phase responses of airflow obstruction. in addition, il and il are significantly raised. exposure to seasonal allergens has been implicated in sudden asthma-related deaths; hdm, cat, and cockroach allergen sensitization are risk factors for emergency treatment [ ] . grass pollen sensitization, or ''thunderstorm asthma,'' has also been associated with epidemics of exacerbations [ ] . fungal allergens are found both outdoors and indoors, and sensitivity to them is a risk factor for the development, persistence, severity, and mortality associated with asthma. individuals with asthma are often sensitized to fungi such as cladosporium species, alternaria species, penicillium species, and candida species [ ] . alternaria species sensitization and exposure are associated with symptoms and a -fold increased risk of respiratory arrest in subjects with asthma [ ] . fungal exposures may worsen disease by different mechanisms; fungalassociated proteases may lead to the development of allergic airway inflammation along with ige-mediated responses; fungal allergen-induced asthmatic reactions evoke an il response with increased eosinophil recruitment and degranulation [ ] . the relationship between fungal exposure and asthma is complicated by the degree of sensitivity to fungal allergens and the resultant allergy. a specific disease entity called allergic bronchopulmonary aspergillosis (abpa) exists in which colonization of the respiratory tract with the ubiquitous aspergillus fumigatus is associated with an increased allergic response (both type and type ) and severe asthma exacerbations. trials in both abpa and a related disease, severe asthma with fungal sensitization (safs), have shown some benefit with the oral antifungal itraconazole [ ] , but good-quality trial data are lacking. unlike other chronic conditions, there is no gold standard for the diagnosis of asthma. although tests of airway function can be used to diagnose and study different aspects of airway disease (airway inflammation/airway hyper-responsiveness), diagnosis still depends on the presence of specific symptoms, which include wheeze, cough, chest tightness, and difficulty breathing. these symptoms are often variable, worse at night, or worse in response to triggers. they often respond to asthma therapy and may be associated with atopy/allergy and a family history of similar problems. the lack of a gold standard affects clinical provision and studies alike; most estimates suggest that asthma is wrongly diagnosed in approximately % of patients [ ] . the development of a cheap and reliable noninvasive test that can diagnose asthma is seen as the holy grail for diagnosisbased research. measurements of airflow limitation provide an assessment of the severity, reversibility, and variability of airflow obstruction, and can help confirm the diagnosis of asthma. spirometry (forced expiratory volume in second (fev ), forced vital capacity (fvc), and fev /fvc) is the preferred method of measuring airflow limitation, as it is more repeatable and less effort dependent than peak flow. a fev /fvc ratio of less than . is consistent with a diagnosis of airflow obstruction, and an increase in fev of > % (or > ml) after administration of a short-acting bronchodilator indicates reversible airflow limitation consistent with asthma [ ] . importantly, most asthma patients will not exhibit reversibility at each assessment, and repeated testing is advised to confirm a diagnosis. consequently, an absent response to bronchodilators does not exclude asthma. peak expiratory flow (pef) measurements can be used to monitor asthma. they are ideally compared to the patient's own previous best measurements using his or her own peak flow meter. an improvement of l/min (or > % of the prebronchodilator pef) after inhalation of a bronchodilator, or diurnal variation in pef of more than % (with twice-daily readings, more than %), suggests poorly controlled asthma. other approaches have been utilized to diagnose asthma, both to confirm a formal diagnosis and to identify corticosteroid response. these approaches include noninvasive measurements of airway inflammation or assessment of airway hyper-responsiveness. measuring airway inflammation is relatively easy, either using techniques to induce sputum and then counting the differential eosinophil and neutrophil cell numbers present, or by measuring the fraction of nitric oxide (no) present in the exhaled breath (f e no) using portable chemical analyzers. airway hyper-responsiveness can be assessed by several different methods. all are designed to provoke bronchoconstriction, which is measured using spirometry. the concentration (or dose) at which bronchoconstriction occurs is then used to categorize the degree of airway responsiveness and the presence or absence of asthma. although these tests are widely employed in research settings, their use in a clinical setting is hampered by their varying sensitivity and specificities for asthma diagnosis [ ] , as both airway inflammation and airway hyper-responsiveness can be present in healthy individuals without symptoms. the symptoms of asthma in children are recurrent wheezing, cough, difficulty breathing, and chest tightness. evaluation of these symptoms is critical to the diagnosis, treatment, and outcome measures used in clinical studies; however, asthma triggers and inflammatory cell type are increasingly used to define childhood asthma phenotypes. pediatric asthma is complicated by wide variations in symptom prevalence. the majority of children with asthma have mild or moderate disease; % of all asthmatic children have chronic symptoms and/or recurrent exacerbations, despite maximum treatment with conventional medications [ ] . asthma in childhood is heterogeneous in several ways, including etiology, clinical presentation, and response to treatment. several attempts have been made to stratify treatment on the basis of different asthma phenotypes in children, with varying degrees of success. stratifying treatments in pediatric asthma is more complicated because of patient age and the inability (dependent on age) to perform more complicated or invasive tests. in children, the onset and progression of asthma result from a complex interplay between genetic background and environmental exposures. the complexity is confounded by wheezing illnesses consisting of several distinct disease entities, with no general agreement on their number or underlying mechanisms [ ] . among the environmental factors that influence the risk of asthma are viral and bacterial respiratory infections. exposure to environmental tobacco smoke is also associated with increased rates of early viral illnesses. other factors associated with the risk of wheeze include physical factors associated with increased breathing (exercise, laughing, crying, and excitement) or allergens (aeroallergens and food allergens). there are considerable age-related changes in the relative importance of trigger factors for wheeze in children. human rhinovirus (hrv) has been implicated in both the etiology of asthma and exacerbations; infants who have hrv infections with wheezing are at a significantly increased risk for subsequent asthma, and over % of childhood asthma exacerbations are triggered by hrv. it is important to note that exposure to hrv does not lead to wheezing illness in all children, nor does wheezing illness result in asthma in all cases, suggesting that the host genotype also plays a role. a recent study of two cohorts of children with asthma found that variants at the q locus were associated with asthma in children who had hrv wheezing illnesses, implicating an interaction between hrv wheezing illness in early childhood and the q genotype (see section . . ). the presentation of asthma symptoms and exacerbations in childhood also varies. some children have frequent exacerbations with few daily symptoms, while others have recurrent symptoms without airway inflammation and exacerbations. other diseases coexist with childhood asthma, including gastroesophageal reflux disease, severe asthma with fungal sensitization, obesity, and vocal cord dysfunction. targeting these coexisting disorders has met with varying clinical success [ ] . an important component of the development of asthma is the inflammatory cascade. this involves the infiltration of a number of inflammatory cells, such as eosinophils, neutrophils, b-and t-lymphocytes, macrophages, mast cells, dendritic cells, and basophils, into the airway, and the release of inflammatory mediators (e.g., leukotrienes, histamine, cytokines, and chemokines) by airway, structural cells (including epithelial, smooth-muscle, endothelial, and fibroblast cells). however, since the degree of inflammation in asthma is not directly related to asthma severity, this suggests that other factors, such as structural changes in the airways, also play a role in disease modulation and progression. these structural changes have been termed airway remodeling and may exist in the presence or absence of inflammatory mechanisms in the airway [ ] . the inflammatory response in asthma is a result of excessive activation of mast cells in the airway, where in the early response to allergen challenge, degranulation of mast cells results in the release of a number of proinflammatory factors such as il , leukotrienes, and histamine. this causes an immediate hypersensitivity response that in turn leads to airway narrowing. the concomitant release of other inflammatory factors such as cytokines and chemokines from the same mast cells provides an optimal environment for recruitment to the airways of other inflammatory cells such as eosinophils, basophils, neutrophils, and t-lymphocytes. once in the airways, these cells act in tandem with allergen-activated macrophages, resulting in what is termed the late asthmatic response (occurring at - hours postchallenge). here, through the action of a number of released potent immunomodulators (e.g., tnfα, il , il , il , and il ), airway narrowing occurs. this may occur in periods that last more than hours. of the inflammatory cells involved in the asthmatic response, it is the infiltration of eosinophils that is considered characteristic of the asthmatic phenotype, particularly that of mild/moderate allergic asthma ( figure . ) . for a more extensive description of inflammatory mechanisms underlying allergic asthma see hodge & sayers [ ] . although inflammation in asthma has been extensively studied, the relationship between inflammation and clinical symptoms of asthma is still unclear. this may be partially explained by a possible link between airway inflammation and airway hyper-responsiveness [ ] . here, airways become more susceptible to sensitizing agents, which would otherwise be unable to trigger an airway response, because of (a) increased release of mediators such as histamine and leukotrienes, (b) abnormal smooth-muscle behavior, and (c) airway thickening. airway remodeling plays an important role in asthma pathogenesis. airway structural changes characteristic of airway remodeling occur because of prolonged airway inflammation. here, prolonged release of inflammatory factors results in: l thickening of the smooth-muscle bronchial walls, leading to airway narrowing l denudation of the bronchial epithelium l hyperplasia and hypertrophy of the airway smoothmuscle l hyperplasia and hypertrophy of the epithelial goblet cells, resulting in the formation of large mucus plugs liable to occlude the airway and increase airway hyper-responsiveness airway remodeling also involves changes occurring in the extracellular matrix and its constituent proteins (collagens i/iii/iv, fibronectin, and laminin) and structural changes such as angiogenesis, vasodilation, increased airway blood flow, and changes in autonomic neurological function [ ] . the development of asthma results in a degree of airway remodeling. the structural changes that define airway remodeling are an important feature in asthmatic airways, where even in newly diagnosed asthma patients, a degree of structural change can be identified in the bronchial wall [ ] . airway remodeling can be defined as a process of sustained disruption and modification of structural cells and tissues leading to the development of a new airway wall morphology [ ] . airway remodeling ( figure . ) is initiated either through various inflammatory pathways, highlighting the importance of the inflammatory pathway [ ] , or through bronchial hyper-responsiveness, where airway remodeling occurs independently of inflammation [ ] . our understanding of the extent and nature of genetic variation in the human genome has dramatically improved since the publication of the first draft genome sequence in , figure . asthma pathophysiology. exposure to allergens-for example, dermatophagoides pteronyssinus (der p ) and dactylis glomerata (dacg )-originating from various sources, including pollen, house dust mite, and mold, are taken up by dendritic cells, b-lymphocytes, epithelial cells, and macrophages, which present the antigens to t-lympocytes. this activates the t-lymphocytes to produce cytokines that regulate immunoglobulin e (ige) production by the b-lymphocytes. bound ige activates mast cells in the airways. activated mast cells initially release a number of factors, including il , leukotrienes, and histamine, that cause airway hyper-responsiveness and result in an early response to allergen stimulus via bronchoconstriction. the concurrent release of cytokines and chemokines from the mast cell recruits eosinophils, basophils, and t-lymphocytes to the airway. these cells, in association with t cell-activated neutrophils and with antigen stimulated macrophages, release chemokines and leukotrienes over an extended period of time, resulting in a late response to allergen stimulus via bronchoconstriction. prolonged stimulation of the late-response factors ultimately leads to the airway structural changes such as mucus hypersecretion, myofibroblast, and airway smooth-muscle proliferation, which are common to the asthmatic lung. and it continues to improve exponentially with initiatives such as the , genomes project, which aims to genome-sequence this number of uk subjects, and the encyclopedia of dna elements consortium (encode), an initiative to identify the genome's regulatory regions. recent figures suggest that more than . million singlenucleotide polymorphisms (snps) or single-base-pair changes exist in a genome consisting of billion base pairs. similarly there is a growing realization that deletions, insertions, and expansions of tandem repeats also represent significant variation. these genetic polymorphisms are found throughout the genome and have the potential to influence gene function in several ways leading to human disease: ( ) a coding-region nonsynonymous polymorphism can alter the amino acid sequence and structure/function of the protein; ( ) a polymorphism can introduce a stop codon leading to the production of a nonfunctional protein; and ( ) a polymorphism in a regulatory region can regulate the expression levels of a gene. it has long been known that atopic diseases such as asthma run in families. in , using atopic probands and nonatopic controls and their respective families, it was shown that . % of atopic probands had a family history of atopy, compared with . % in the control population [ ] . similarly, a study of families showed a very high concordance of asthma, hayfever, and eczema in parents and children [ ] . twin studies have been useful in identifying a significant concordance of asthma that is higher in monozygotic twins (identical genotype) versus dizygotic twins (on average sharing half of their genes). in the largest study to date, using , danish twin pairs, it was suggested that % of susceptibility to asthma was genetic, with a substantial environmental component [ ] . more recent studies suggest hereditability estimates of - % for asthma [ ] . therefore, asthma is considered a complex genetic disorder and, in contrast to single-gene disorders (e.g., cystic fibrosis), involves multiple genes, with expression influenced by both genetic and environmental factors. multiple environmental factors are important in asthma development, including tobacco smoke exposure, respiratory viral infections, antibiotic use, diet, and allergen exposure. gender and ethnic background also make a significant contribution. this complex mode of inheritance combined with the heterogeneity in the presentation of the disease has made gene discovery a challenge. early studies investigated inheritance through families containing multiple affected children, using linkage analyses and candidate gene approaches based on biology or location in the genome. however, the figure . asthmatic airway pathology. this schematic comparison of a normal airway with that observed in severe chronic asthma indicates histological changes that accompany recurring inflammation seen over time. unlike that from the unaffected individual, the bronchial mucosa from the severe asthmatic displays thickening of the basement membrane, airway smooth-muscle hypertrophy, leukocyte infiltration, epithelial cell desquamation, goblet cell hyperplasia in the epithelial lining accompanied by mucus hypersecretion, and plugging of the bronchial lumen, as well as edema and collagen deposition in the submucosal area. (not drawn to scale.) source: reproduced with permission from hodge and sayers [ ] . reproducibility of these findings was disappointing primarily because of inadequate power, subject heterogeneity (different phenotype definition), population stratification, and multiple testing without correction. more recently, hypothesis-free genome-wide association studies (gwas), which examine association with typically , + common (> % frequency) polymorphisms spanning the genome in cases and controls, using very stringent statistical thresholds ( figure . ), have been very successful. via these multiple approaches, over asthma genes have been described in more than publications. the major reproducible findings for asthma follow below (for more comprehensive reviews see [ , ] ). although this chapter focuses on the genetics of asthma diagnosis, subphenotypes in asthma (e.g., atopy, serum total ige levels, and lung function (e.g., fev )) have also been investigated for genetic influences. positional cloning involves linkage analyses (region or whole genome) followed by fine mapping using association. linkage analysis uses family data to follow the transmission of genetic information spanning the entire genome (commonly short tandem repeat markers) across generations. these data are used to determine if a genetic marker is close to, or linked with, a gene involved in a particular disease using families with multiple affected children. once a specific chromosomal region has been identified in this hypothesis-free approach, snps spanning the region are tested to see if they are more common in people with asthma compared to people without. the first gene to be identified with confidence using positional cloning was disintegrin and metalloprotease (adam ) [ ] . using a cohort of families, linkage was demonstrated to a locus on chromosome p for asthma, bronchial hyper-responsiveness (bhr), and total ige levels. subsequently, polymorphisms spanning adam were associated with asthma in the second stage. adam is thought to contribute to airway remodeling via its enzymatic functions. using positional cloning, multiple genes have been identified with functions varying from transcription factors to epithelial differentiation and tissue remodeling. these genes have provided a novel insight into potential mechanisms that are altered in asthma (table . ). polymorphisms spanning the urokinase plasminogen activator receptor (plaur or upar) were also associated with asthma susceptibility and were associated with rate of decline of lung function in asthma through linkage/association analyses on chromosome q [ ] . upar is a serine protease receptor involved in multiple mechanisms, including cell proliferation, migration, and extracellular matrix degradations via plasmin generation. these data suggest that genetic factors may be important in determining airway structural changes or "remodeling" in asthma [ ] . genotype for typically , + snps in large numbers of cases and controls using arrays compare differences to identify disease-specific snps using stringent correction for multiple testing candidate gene studies are hypothesis-driven and based on the suggested biology of a gene product or the location of the gene in a chromosomal region previously linked to asthma. such studies commonly employ asthma cases versus controls, although family-based association has also been used. for excellent reviews of candidate gene studies in asthma, see ober and hoffjan [ ] and undarmaa et al. [ ] . the primary highly reproducible genes identified using candidate gene approaches are multiple components of the interleukin / axis (see table . ), including the cytokine genes themselves (il /il ); related receptor (il ra) and downstream signaling effector signal transducers and activators of transcription (stat ) . the role of the il /il axis in the pathogenesis of asthma has been extensively documented [ ] . human studies have identified elevated numbers of cells expressing il mrna in the bronchial tissue of atopic and nonatopic asthmatic subjects [ ] ; administration of recombinant il in mouse lungs resulted in an increase in airway mucus secretion, development of subepithelial fibrosis, airway hyper-responsiveness (ahr), and eosinophilic airway inflammation-that is, several key features of the human disease [ ] . il is produced by a variety of cells, including th cd +, th cd +, cd + t cells, mast cells, basophils, and eosinophils. il mediates its effects by interacting with a complex receptor system comprising an il- rα/il rα heterodimer and the il rα receptor [ ] . the finding that genetic polymorphisms within il that determine levels and/or structure and are associated with the development of asthma make biological sense. the use of gwas approaches to identify asthma susceptibility genes has revolutionized our understanding of asthma genetics. while positional cloning showed great success with simple mendelian disorders, it did not perform well for complex diseases. similarly, candidate gene studies are useful but very inefficient. the capacity to interrogate the entire genome for , + common snps in cases and controls has provided an excellent platform for gene discovery. the first gwas for asthma used a discovery cohort of patients with childhood onset asthma and nonasthma controls, and identified significant association to a locus on chromosome q [ ] . this locus includes genes for zona pellucida binding protein (zpbp ), gasdermin b (gsdmb) and orm -like protein (ormdl ). the q locus has been reproduced as a locus associated with childhood onset asthma in many studies; however, the identification of the specific gene(s) underlying these effects remains to be resolved. zpbp , gsdmb, and ormdl have been implicated in gene transcription, cell apoptosis, and sphingolipid synthesis, respectively. this initial gwas has now been superseded by several larger-scale studies. in particular, the gabriel consortium study, involving , asthma cases and , controls, identified association between polymorphisms spanning il , il rl /il r , hla-dq, smad , and il rb [ ] . note: for candidate gene approaches, genes focused to replication in more than studies [ , ] . for gwas, genes focused to those meeting conventional genome-wide significance (p < × − ) and/or independent replication in the caucasian population. to date, two of the most reproducible association signals are to the il gene on chromosome p and the il receptor (il rl or st ) gene on chromosome , both of which highlight the significance of this ligand/receptor system in asthma. interestingly, these loci are associated with severe asthma as well [ ] . there is good evidence from biology that the il /st axis may be of relevance in asthma; mice induced to develop allergic airway disease were treated with an antibody that blocks il binding to its receptor. this treatment was shown to block many of the features of the disease, including reduced serum ige and airway eosinophil and lymphocyte counts. significantly, both induction and resolution of the disease were attenuated [ ] . in human studies, il has been shown to be elevated in the airways of asthma patients, particularly in the airway structural cells, including the bronchial epithelium, and is induced in these cells by relevant stimulations (e.g., hdm [ ] ). in addition, a soluble form of the st receptor has been shown to be elevated during asthma exacerbation [ ] . overall, it is thought that the il /st axis may be particularly important in attracting and activating inflammatory cells in the airways; however, the precise role(s) of this pathway remains to be resolved. as outlined in table . , other genes have been identified using gwas, including those involved in diverse roles such as inflammatory cell function and airway smoothmuscle contraction, again providing a unique insight into potentially altered mechanisms in asthma. however, most gwas determined single variants only confer a very modest odds ratio of . - . of developing asthma. for a more comprehensive review of gwas findings in asthma, see akhabir and sandford [ ] . over the last years, there has been great progress in identifying asthma susceptibility genes. current and future approaches include meta-analyses using cohorts of tens of thousands of asthma patients, the incorporation of gene expression/snp analyses (the expression quantitative trait loci (eqtl)), the investigation of asthma subphenotypes using gwas, and whole-genome sequencing. the real challenge is the translation of these genetic findings into a deeper understanding of the biology of asthma and the potential identification of therapeutic targets. already several of these newly identified genes (e.g., il and il receptor (il rl /st )) represent excellent pharmacological targets, and it is anticipated that genetics in these loci will be essential to identifying patients most likely to gain clinical benefit from targeting this pathway. the lack of concordance between approaches (e.g., linkage versus gwas) can be explained by the fact that the methodologies are designed to detect different types of variants, e.g., linkage analysis has good power to detect high-risk disease-causing alleles but is not effective at identifying common alleles of modest effect, as gwas does. it is reassuring that many of the genes identified in candidate gene approaches have been reproduced in gwas (e.g., the il /il locus on chromosome q , a region that has also been linked to asthma). while there has been much success in recent years in identifying genes for asthma using gwas, the overall genetic variation accounted for by these genetic polymorphisms is very small, leading to the concept of "missing hereditability." possible explanations for missing hereditability include: ( ) rare variants (< % frequency) with larger effect size not measured on existing platforms; ( ) structural variation (e.g., copy number variation); ( ) gene-environment contributions; ( ) gene-gene interactions; ( ) epigenetic mechanisms; and ( ) overestimation of initial hereditability. the aim of treatment is to achieve control of asthma and prevent exacerbations. most guidelines adopt a stepwise approach to the management of asthma and advise stepping up treatment as necessary and stepping down when control is achieved for at least three months. although there are several pharmacological options for asthma treatment (see figure . ), the two main classes remain bronchodilators (short-or long-acting) and corticosteroids. short-acting bronchodilators (e.g., salbutamol/terbutaline) are sympathomimetics (β -adrenergic receptor agonists) that relax airway smooth-muscle, enhance mucociliary clearance, decrease vascular permeability, and possibly modulate mediator release from mast cells. they are used for symptom control, acute exacerbations, and exerciseinduced asthma. frequent or regularly scheduled use of rapid-acting inhaled β agonists for long-term management of asthma does not adequately control symptoms, pef variability, or airway inflammation. long-acting bronchodilators (e.g., salmeterol/formoterol) activity is identical to that of short-acting β -adrenergic receptor agonists, but should be used in combination with inhaled corticosteroids because of concerns regarding increased mortality when used as a monotherapy. long-acting bronchodilators are normally introduced at treatment step . inhaled corticosteroids (ics) form the backbone of treatment for all but the very mildest asthma. ics improve lung function, symptoms, and quality of life, reduce exacerbations, and improve mortality. they have broad anti-inflammatory and immunosuppressive effects. corticosteroids enter the cell cytoplasm and bind with the inactive glucocorticoid receptor complex. the activated glucocorticoid receptor binds to dna at the glucocorticoid response element sequence and promotes synthesis of anti-inflammatory proteins (transactivation), and inhibits transcription and synthesis of many proinflammatory cytokines (transrepression). they also reduce the number of t-lymphocytes, dendritic cells, eosinophils, and mast cells in the airway, and reduce inducible nitric oxide production. ics (beclomethasone dipropionate/fluticasone propionate/budesonide/ flunisolide/ciclesonide/mometasone) all have side effects related to their transactivation properties, including local effects on the oropharynx (hoarseness, candidiasis, cough, and dysphonia) and systemic effects (cushing syndrome, osteoporosis, cataracts, dermal thinning and bruising, adrenal insufficiency, and growth suppression) in children [ ] . consequently, the lowest dose required to control symptoms and prevent exacerbations should be prescribed. other medications are normally reserved for second-or third-line treatment. these include methylxanthines (theophylline), leukotriene modifiers (montelukast), inhaled anticholinergics (ipratropium bromide/tiotropium), and sodium chromoglycate. a wide range of interventions have been assessed for both the primary prevention of asthma and the secondary prophylaxis of the disease once present. these treatments have varying effects, but may be recommended in individual cases. primary prevention measures include aeroallergen and food allergen avoidance, fish oil supplementation, avoidance of tobacco smoke and air pollutants, immunotherapy, and immunization. measures that have been assessed for secondary prophylaxis include hdm and allergen avoidance, smoking reduction, subcutaneous and sublingual immunotherapy, dietary manipulation, weight loss, breathing techniques, and exercise training. some investigators argue that severe asthma is an inflammatory condition separate from mild/moderate asthma, rather than simply the severe end of the disease spectrum. this is difficult to prove, as underlying asthma may be complicated by other aspects (obesity/reflux/smoking/poor adherence), making it more difficult to treat and control. there are different definitions of severe asthma, but all require high-dose inhaled corticosteroids or oral corticosteroids and include recurrent exacerbations and poor control. as high-dose ics and oral corticosteroids are associated with side effects attempts have been made to design treatments to either reduce the amount of corticosteroid prescribed (steroid-sparing agents) or treat the underlying asthma in a different manner. steroid-sparing agents trialled include methotrexate, gold, cyclosporin, and azathioprine. currently, none have been recommended in any asthma guidelines. box . lists other treatments used on an individual basis. the need for a stratified-medicine approach to asthma has become apparent over the last decade. this has followed the realization that the term asthma covers a whole range of disease phenotypes. as techniques for investigating and researching asthma have evolved, it has become clear that different disease processes are present that require different treatments and approaches. whether this represents disease phenotyping or just a better understanding of disease pathogenesis is unclear. over the years, research focus has moved from the airway smooth-muscle via airway inflammation to airway immunology, and on to the epithelial-mesenchymal trophic unit. future insight into disease pathogenesis and new treatment developments will follow with the use of the latest technologies, which include "omics" platforms and assessment of airway microbiota. improving our understanding of stratified medicine for asthma-targeting the right therapy to the right patientaims to reduce hospital admissions due to poorly controlled asthma and fatalities. although most asthma can be controlled by ics and bronchodilators, there remains a cohort of patients with persistent symptoms, recurrent exacerbations, and worse quality of life. the current stepwise management approach (see figure . ) is inefficient, particularly in light of heterogeneity in clinical presentation and response to existing therapies, making a stratified approach essential. early work conceptualized asthma as extrinsic (allergic) and intrinsic (nonallergic) [ ] . however, the recent use of unbiased approaches to classify disease using three large datasets and cluster analysis [ ] [ ] [ ] has highlighted the different types of disease under the umbrella term asthma ( figure . ). although these early studies were performed in different parts of the world and had statistical variations, their results were similar. age at disease onset was found to be a key differentiating factor; early-onset disease was associated with more atopic and allergic conditions, whereas later-onset disease was associated with eosinophilic inflammation and obesity. these studies were all limited by a cross-sectional design and by the fact that treatment may have determined clusters. longitudinal studies initiated in childhood are ongoing. for example, pacman (formally, "pharmacogenetics of asthma medication in children: medication and anti-inflammatory effects") aims to differentiate children with uncontrolled asthma, despite ics treatment, using a range of clinical and cellular markers [ ] . the role of allergy (an inappropriate and harmful immune response to a normally nonharmful substance which requires sensitization) and that of atopy (the tendency to develop ige antibodies to commonly encountered environmental allergens by natural exposure in which the route of entry is across intact mucosal surfaces) have been well studied in asthma. people with extrinsic asthma were thought to develop the disease earlier in life, be atopic, and have identifiable allergic triggers as well as other allergic diseases such as rhinitis or eczema. intrinsic asthma was thought to develop later in life (after years of age), and be associated with aspirin-exacerbated respiratory disease but not with allergic sensitization. when small studies in humans suggested that levels of th cytokines were similar in extrinsic anti-ige bronchial thermoplasty macrolide antibiotics antifungals (itraconazole/voriconazole) anti-il anti-il box . new and emerging therapies reserved for severe asthma and intrinsic asthma, and that treatment with ics was effective in the majority of mild to moderate asthma cases, the distinctions between extrinsic and intrinsic asthma fell out of favor [ ] . heterogeneity in response to existing medication is exemplified by malmstrom's study, which investigated individual response to leukotriene receptor antagonist (ltra) (montelukast mg once daily), and the inhaled steroid beclomethasone ( μg twice daily) over a -week period in chronic asthma patients [ ] . overall, there was a clear improvement in lung function (primary outcome fev ) for both treatments; however, when stratified based on individual patient data, there was a very large degree of heterogeneity for both treatment groups, with some patients showing up to % improvement in fev and others showing a decline in lung function with a % decrease in fev (figure . ). this interindividual variability, a component of clinical trials that is seldom appreciated, is thought to have a strong genetic component. the therapeutic regimen for the treatment of asthma has been well defined and was described earlier in this chapter. despite such well-defined treatment regimens, which are constantly updated and streamlined, as well as the availability of high-quality medications, in many cases asthma is still not sufficiently controlled. while current asthma therapeutics are able to successfully treat - % of the asthmatic population, around % still have daily symptoms and almost all patients report limitations of daily activities [ ] . this also leaves around - % of patients who do not respond to conventional treatments. this demonstrates that the current one-size-fits-all approach is not perfect, and emphasizes the need for a stratified medicine approach to asthma. variability in current therapy involves variation in drug efficacy and presentation of side effects, each of which presents challenges to successful maintenance of asthma. variability in drug efficacy, especially when occurring for β -adrenergic receptor agonists and glucocorticosteroids, and in the presentation of side effects, can result in patient therapy having to deviate from the standard therapeutic regimen for the treatment of asthma. this high degree of interpatient variability in treatment response and in side effects can be attributed to a number of factors: ( ) the interaction of genetic factors; ( ) individual patient characteristics, such as weight, gender, and pregnancy; and ( ) exposure to environmental insults, such as air pollution, allergens, and cigarette smoke. these factors interact, making the control of asthmatic episodes and the reduction of exacerbations more difficult. for example, asthma attacks can affect different age groups differently according to the season-children are more affected in summer while the older population suffers more in winter. when considering variation in treatment efficacy, one must also be mindful of individual patient factors such as inhaler technique and noncompliance (discussed earlier). currently asthma therapy is planned without consideration of genetic variance. however, genetic variance is arguably the most important of the factors listed previously-a claim supported by evidence gained from investigating the repeatability of response to therapy. this approach demonstrated that repeatability was between - %, with a substantial proportion of the variance due to genetic factors [ ] . one important form of variance in therapeutic efficacy is non-response. non-response is an important limitation in the maintenance and control of asthma, in that therapeutics expected to manage the disease at a certain level of severity fail outright, leading to the use of a trial approach in which different therapies are utilized with variable results until a suitable regimen is determined. a good example is severe asthma, where the patient is on permanent oral glucocorticosteroid therapy because of nonresponse to other therapies such as ics, leukotriene receptor antagonists, and β -adrenergic receptor agonists. non-responders present with longer duration of asthma exacerbations and worse morning lung function, as well as a more frequent family history of asthma, when compared to those individuals responsive to glucocorticosteroid therapy [ ] . the recognition that allergen specific ige activation of mast cells is central in driving allergic asthma lead to the discovery that the primary mast cell-signaling cascade could be inhibited by a monoclonal antibody toward the ige binding site to the high affinity receptor (fcer ). anti-ige (omalizumab) became the first specific biologic to be used in the treatment of severe allergic asthma. clinical trials revealed efficacy as well as almost total inhibition of early and late asthmatic responses to inhaled allergen [ ] . following a rigorous examination of trial data by the national institute for clinical excellence (nice), omalizumab is now recommended as an option for treating severe individual data persistent, confirmed allergic ige-mediated asthma as an add-on to optimized standard therapy in people aged six and older who need continuous or frequent treatment with oral corticosteroids (defined as four or more courses in the previous year) in the united kingdom. although the drug is prescribed for cases of therapy-resistant asthma associated with allergy, it is also licensed for patients with evidence of allergy but a normal ige. omalizumab represents the first stratified treatment for asthma, but there is evidence that its use can be further stratified by using biomarkers of th inflammation to predict response. in a recent paper studying the effect of omalizumab on uncontrolled severe persistent allergic asthma, treatment effect was analyzed in relation to f e no, blood eosinophils, and serum periostin (an epithelial protein that is induced by il ). after weeks of omalizumab, reductions in protocol-defined exacerbations were greater in high versus low subgroups for all three biomarkers, suggesting a potential prognostic ability [ ] . this biomarker approach has helped divide asthma into and th high and th low disease. asthma has traditionally been considered a th process linked to atopy and allergy, type i hyper-sensitivity reactions, eosinophilic inflammation, and response to corticosteroids. "th high disease," as evidenced by high f e no and a sputum eosinophilia of > %, has been associated with a better response to corticosteroids when compared to "th low disease" [ ] [ ] [ ] . studies on lebrikizumab, a novel monoclonal antibody to il [ ] , have suggested that serum periostin may be a biomarker for a more general th asthmatic phenotype. in one study a subgroup of individuals who had asthma and persistent elevation in serum periostin showed greater improvements in airway function and fewer exacerbations after lebrikizumab treatment than those with lower serum periostin. interestingly, levels of f e no, produced by inducible nitric oxide synthase, an enzyme induced in human airway epithelial cells by il , were as informative as periostin in identifying th -high individuals responsive to lebrikizumab. both the role of stratification based on phenotyping and the need for correct study end points in treatment trials have been informed by the salutary lesson of the anti-il antibody mepolizumab. although mepoluzimab effectively blocks eosinophilic inflammation, initial studies were disappointing in that no effect was demonstrated on spirometry or peak flow, despite a significant reduction in blood eosinophil numbers [ ] . when the effect of the drug was studied on an outcome measure related to eosinophilia, namely severe exacerbations, there were significantly fewer exacerbations with a concomitant improvement in asthma-related quality of life [ ] . tellingly, there were no significant differences with respect to symptoms, postbronchodilator fev , or airway hyper-responsiveness, suggesting that different physiological processes determine these asthma outcomes. the study also helps researchers reflect upon the need to measure the correct end point in a disease that has different pathological mechanisms that all respond differently to treatment. phenotyping asthma has led to the identification of new disease targets, but aside from the use of anti-ige, it has not yet led to a step change in management. other approaches assessing the separate aspects of asthma pathophysiologynamely, airway inflammation, airway hyper-responsiveness, and airflow obstruction have been trialled with differing results. the key determinant in the design of these stratified approaches was the recognition that although asthma is a disease defined by symptoms, with treatment response also assessed by symptom improvement, there is no clear correlation between pathophysiology and symptomatology/ exacerbation risk. in the facet study (designed to evaluate the benefits of adding a long-acting β -adrenergic receptor agonist to different doses of ics), higher-dose ics had a marked beneficial effect on exacerbation frequency, but relatively less effect on symptoms and peak expiratory flow. the opposite was true with the addition of long-acting β -agonists [ ] . this indicates that exacerbation frequency does not closely relate to symptoms and measures of disordered airway function, suggesting that the mechanisms responsible for these features are different [ ] . numerous studies have demonstrated that in asthma the features of airway inflammation, airway hyper-responsiveness, variable airflow obstruction and associated symptoms can overlap, occur independently or change over time, in response to treatment or other external factors such as allergen exposure, or viral infection. one obvious example of this is eosinophilic bronchitis, a condition characterized by corticosteroid-responsive cough and the presence of a sputum eosinophilia occurring in the absence of variable airflow obstruction or airway hyper-responsiveness [ ] . although asthma guidelines recommend the assessment of airflow obstruction (fev /pef) and related symptoms for their primary treatment response outcomes, the realization is that this approach looks only at one aspect of asthma pathophysiology which is not closely related to exacerbation risk. this has led to new approaches aimed at reducing asthma exacerbations and symptoms while maintaining corticosteroid burden at the lowest possible level. airway hyper-responsiveness is defined as increased sensitivity to an inhaled constrictor agonist and a steeper slope of the dose-response curve. two main forms of bronchoconstrictor stimuli exist: direct and indirect. direct bronchoconstrictors, such as histamine or methacholine, stimulate receptors on the airway smooth-muscle, while indirect ones cause bronchoconstriction by secondary release of bronchoconstrictor mediators from mast cells or activation of neural pathways. airway responsiveness is usually measured as the provocative dose of methacholine causing a % fall in fev by linear interpolation of the log dose-response curve (pc ). in the general population, the distribution of airway hyper-responsiveness follows a continuous unimodal log-normal distribution, with asthma sufferers representing the hyper-responsive part of the distribution curve. a pc is not usually measurable in non-diseased individuals, which suggests a large difference in airway responsiveness between non-diseased individuals and asthma patients. the cut-off used to identify asthma is normally a methacholine concentration of < mg/ml. this value had a sensitivity of %, a specificity of %, and a negative predictive value of % in a study on a population of college students with a diagnosis of current symptomatic asthma. the use of methacholine pc to diagnose asthma has been evaluated; one study demonstrated that when asthma is defined as consistent symptoms with objective evidence of abnormal variable airflow obstruction, a positive methacholine challenge is more sensitive than pef amplitude % mean and the acute bronchodilator response in diagnosis [ ] . the use of methacholine pc to guide treatment has also been assessed. although it was found that pc -guided treatment resulted in a reduction in asthma exacerbations, this was at the expense of increased inhaled corticosteroid use [ ] ; consequently, although pc is often used for asthma diagnosis, or as a study end point, it is not routinely used to guide treatment decisions. asthma has been traditionally viewed as a condition where airway inflammation causes airway hyper-responsiveness, which in turn leads to variable airflow obstruction and symptoms; however, cross-sectional and longitudinal studies of airway inflammation using sputum induction in large populations with a diverse range of presentations suggest that this hypothesis requires modification. a recent bronchoscopy study demonstrated that bronchoconstriction is independent of airway inflammation and can lead to airway remodeling [ ] . the development of noninvasive techniques to assess airway inflammation, including induced sputum and f e no, has made it possible to relate airway inflammation to objective measures of disordered airway function in larger and more heterogeneous populations than was possible with bronchoscopy studies. in general, these newer studies contradict findings in earlier studies and do not find a correlation between sputum eosinophil count and various markers of airway dysfunction. it has been observed that a subset of patients with symptomatic asthma do not have sputum evidence of eosinophilic airway inflammation [ ] . many have sputum neutrophilia. this sputum profile is evident in corticosteroid-naïve as well as corticosteroid-treated subjects, suggesting that it is not always an artifact related to treatment. importantly, patients with noneosinophilic asthma respond less well to inhaled budesonide than do a group with more typical sputum features [ ] . similar sputum findings have been reported in more severe asthmatics; a subgroup of patients with refractory asthma have been identified who have bronchoscopic evidence of neutrophilic airway inflammation, normal eosinophil counts, and a normal basement membrane thickness. these findings suggest the presence of a distinct asthma phenotype characterized by a predominantly neutrophilic airway inflammatory response and relative corticosteroid resistance. furthermore, there is evidence that neutrophilic asthma may result from activation of the innate immune system with the production of proinflammatory cytokines. the use of noninvasive measures to guide treatment decisions by stratification based on the presence or absence of airway inflammation has been assessed. the strongest evidence for effect relates to the use of induced sputum to guide corticosteroid dose in moderate to severe asthma. two studies have shown that using induced sputum differential counts to guide treatment results in fewer exacerbations for the same overall corticosteroid burden [ , ] . although the evidence is clear that this approach works, induced sputum is not widely employed, possibly because it is seen as time consuming, requiring expertise both to induce the sputum and to perform the differential counts. the discovery that levels of f e no, measured via a device similar to a breathalyzer, correlate well with sputum eosinophilia and relate to corticosteroid responsiveness, has driven the application of inflammometry (noninvasive measurement of inflammation in the airways). inflammometry using f e no measurements has also been used to guide and stratify treatment decisions. though one study was positive in patients with asthma in pregnancy, with a reduction in exacerbations, most have failed to show an improvement when compared to guideline-driven (fev /pef and symptoms) management. the hunt is still on for a reliable, inexpensive, and valid biomarker of airway inflammation. fev and pef measurements reflect changes in the caliber of the large airways. our knowledge of anatomical and physiological changes in the small airways of patients with asthma is based on small case series of resected lung tissue from patients with asthma undergoing surgery for cancer, or on cases of fatal asthma. these case series have demonstrated that there is significant inflammation present in the small airways (< mm diameter) in asthma. fatal asthma is associated with peripheral airway inflammation and differences in the number of activated eosinophils in the distal lung. other studies have revealed alterations in the epithelium and smooth-muscle, as well as mucous hypersecretion and distal airway plugging of the small airways. the presence of inflammation in the small airways in asthma may explain why small airways account for up to - % of total airflow resistance in asthma, but only % of airflow resistance in normal airways. recently, the development of "small-particle" ics, designed to target the peripheral lung, and the advent of new technologies-nitrogen washout, impulse oscillometry, and hyperpolarized noble gas magnetic resonance imaging, which allows assessment of peripheral lung function-have led to a resurgence of interest in the distal lung. studies of small-particle ics have been inconsistent; those comparing small-particle and standard-particle icss have failed to demonstrate improved asthma outcomes when administered in clinically comparable doses. future asthma treatment may yet be stratified by the presence or absence of small airway inflammation. there have been several attempts to base treatment decisions on measures of airway inflammation, including induced sputum and f e no in children. generally, they have been unsuccessful, although some experts do stratify treatment decisions on the response to oral or intramuscular steroids. while methods of stratifying asthma patients to specific treatments based on nongenetic factors such as clinical outcomes, cellular measures, or protein biomarkers have shown some success, a large body of work has investigated the potential of genetic markers as predictors of patient responses to existing therapies, i.e., pharmacogenetics. pharmacogenetics, the investigation of the effect of genetic polymorphisms on response to treatment or risk of adverse side effects, is one of the first steps in developing personalized prescribing. the use of genetic information to stratify patient prescribing is potentially more desirable compared to nongenetic stratification, as technology now allows a small amount of blood or saliva sample to be taken and a dna test can be completed within hours. to date, asthma pharmacogenetic studies have suffered from relatively small retrospective designs and a focus on only a few candidate genes; however, more recent, larger prospective studies have been completed that provide greater confidence in original findings and hypothesis-free approaches such as gwas. these studies are primarily focused on pharmacodynamic aspects (e.g., improvement in lung function post-treatment), and only limited information is available about the impact of pharmacogenetics on adverse effects. pharmacogenetics in asthma is relatively advanced compared to that for other diseases. genetic factors influencing the main treatment classes (i.e., β -adrenergic receptor agonists, corticosteroids, and leukotriene modifiers) have been identified with some confidence (table . ). an overview of these findings follows; however, for more in-depth analyses, see portelli and sayers [ ] . β -adrenergic receptor agonists carry out their function through the β -adrenergic receptor-a -amino-acid g-protein-coupled receptor encoded by an intronless gene (adrb ) located on chromosome q . . binding of agonists to the receptor activates adenyl cyclase through stimulatory gs proteins, which in turn activate protein kinase a. the latter phosphorylates several target proteins, resulting in a decrease in intracellular calcium that, importantly, causes smooth-muscle relaxation in the airways. it is not surprising that the majority of evidence of genetically driven effects on β -adrenergic receptor-agonist therapy stem from the adrb gene, which has therefore been extensively studied for pharmacogenetic effects on β -adrenergic receptoragonist responses. adrb is a highly polymorphic gene containing known and validated polymorphisms, of which are snps and are insertion/deletion variants. most studies have focused on the role of four nonsynonymous coding-region polymorphisms: arg gly (arginine-to-glycine substitution at position in the protein), gln glu, val met, and thr ile [ ] . in a caucasian population, the frequency of the polymorphisms at positions and were identified as % (arg glu) and %, (gln glu) [ ] . the remaining val met and thr ile polymorphisms are rare, having approximate frequencies of < . % and . %, respectively. the arg variant has been shown to have pharmacogenetic potential through association with; an enhanced acute response to β -adrenergic receptor agonists l a decline of asthma control following prolonged use of β -adrenergic receptor agonists l a subsensitivity of response for bronchoprotection by β -adrenergic receptor agonists however, several studies have failed to reproduce these effects, meaning that a common consensus on the contribution of arg has yet to be reached. this lack of consensus can be explained when we consider that there are at least different haplotypes (a specific combination of snps across the gene) in adrb . investigations into the effect of a genotype in isolation, without consideration of its haplotype, is likely to introduce confounding into the association. functional effects of coding-region polymorphisms in adrb have been identified through in vitro work carried out in cell lines. these have included the following: [ ] . in one of the larger studies, basu et al. identified an arg copy numberdependent increase in disease exacerbations in patients with asthma aged to years on daily exposure to β -adrenergic receptor agonist (regularly inhaled corticosteroid plus salbutamol on demand group, and regularly inhaled corticosteroid plus salmeterol and salbutamol on demand group) [ ] . it is important to note that this effect was driven by asthma patients who used salbutamol and/or salmeterol daily, supporting the suggestion that the arg polymorphism has an integral role in the effectiveness of β -adrenergic receptor-agonist therapy [ ] . however, as with other studies, study limitations did not allow a clear association to be made between the adrb gene (via its polymorphisms) and β -adrenergic receptor-agonist efficacy. namely, all participants were using β -adrenergic receptor-agonists as a reliever; a study arm of non-β -adrenergic receptoragonist reliever use would have provided a clearer interpretation of the detrimental effects of salbutamol/salmeterol in the arg subjects. large studies have failed to observe a clinically relevant effect of these polymorphisms. these include longitudinal studies of β -adrenergic receptor-agonist efficacy and the study of concomitant administration of corticosteroids. a good example is a study of asthma patients randomly assigned to: ( ) budesonide plus formoterol maintenance and reliever therapy; ( ) fixed-dose budesonide plus formoterol; or ( ) fixed dose fluticasone plus salmeterol for six months. no overall effect of the gly arg genotype on clinical outcomes was found [ ] . another contributing factor to the pronounced differences in the conclusions from different studies investigating these adrb polymorphisms is trial design variation. the majority of studies have focused on the adrb arg gly polymorphism; however, other potential pharmacogenetically relevant polymorphisms also affect β -adrenergic receptor-agonist efficacy. multiple polymorphisms in the gene's regulatory regions that have potential clinical relevance are present in adrb . by studying eight common haplotypes based on snps, a recent in vitro approach using "whole gene" transfection identified differential effects on receptor expression and downregulation that are haplotype-driven [ ] . this study identified four common haplotypes with elevated receptor expression and two haplotypes with enhanced receptor downregulation. another area recently investigated is polymorphisms occurring in the gene's untranslated regions, which may have an effect on gene expression and resultant drug efficacy. multiple genes are likely to be involved in the regulation of β -adrenergic receptor-agonist response and expression of side effects, due to the agonists' known complex and multifactorial mechanism of action. one gene that has been associated with patient response to these agonists is arginase (arg ), which was identified using a novel algorithm implemented in a family-based association test (fbat) [ ] . in this study of children and their parents, the arg snp, rs , was associated with bronchodilator response (bdr) when snps from candidate genes ( involved in β -adrenergic receptor-signaling/regulation, involved in glucocorticoid regulation, and from prior asthma association studies) were investigated for their association with acute response to inhaled β -adrenergic receptor-agonist in children and their parents. in agreement with this study polymorphisms spanning arg and influencing patient response to salbutamol have also been identified in a candidate gene study involving asthma subjects [ ] . the arg polymorphisms identified in both studies were in linkage disequilibrium (ld) (inherited together), suggesting a common causative mechanism involving potential transcriptional regulation due to the polymorphisms' location (predominantly ′ to the gene). this alteration in transcription has now been confirmed in promoter-reporter studies, which found that the key arg haplotype associated with improved bdr drives the highest level of arg promoter activity [ ] . in the study by vonk et al., arg snps were also associated with patient responses to salbutamol [ ] , suggesting an integral role for the arginase family in β -adrenergic receptor-agonist therapy. recently a s-nitrosoglutathione reductase (gsnor) snp (rs , promoter region) was associated with a decreased response to salbutamol in african-american children [ ] . in the same study, a post hoc multilocus analysis discovered that a combination of rs with adrb arg gly, gly glu, and the carbamoyl phosphate synthetase- (cps ) snp rs gave a % predictive value for lack of response to therapy [ ] , implying that pharmacogenetic regulation of β -adrenergic receptoragonist therapy may depend on several loci acting together via gene-gene interactions. in confirmation, / snps tested in gsnor were associated with asthma patient responses to salbutamol in puerto rican asthma patients [ ] . these snps were also associated with asthma susceptibility, and the key risk haplotype was associated with increased transcriptional activity based on promoter-reporter studies [ ] . gsnor is an alcohol dehydrogenase that breaks down gsno, an endogenous bronchodilator [ ] . in addition, gsno regulates nitrosylation of proteins, leading to alterations in function, including g-protein coupled receptor kinase (grk ), which phosphorylates and desensitizes the β -adrenergic receptor [ ] . other novel genes have recently been associated with β -adrenergic receptor-agonist therapy including the spermatogenesis-associated, serine-rich -like (spats l) and collagen (col a ) genes [ ] . the col a gene, through association with the intronic snp rs , was associated with acute bronchodilator response to inhaled salbutamol in a genome-wide association study in which ∼ , snps were tested in caucasian trios. this association was replicated in a pooled population of three asthma trial populations, as well as in three additional asthma populations [ ] . spats l was also identified as a modulator of β -adrenergic receptor-agonist function through a gwas. here, the snp rs , located near the spats l gene, was significantly associated with percentage change in baseline fev in caucasian asthma patients; this was replicated in two alternate caucasian populations (n ∼ each). molecular biology techniques confirmed these results and identified that spats l may be an important regulator of β -adrenergic receptor downregulation. the identification of patients at risk from potential adverse effects of β -adrenergic receptor-agonists remains a critical clinical question, as does the targeting of this class of drug to patients most likely to benefit from it. while there has been clear progress with respect to study design (e.g., examining haplotypes instead of genotypes in isolation) and adequately powered studies using thousands of individuals, there is still a need for large prospective studies of asthma patients with matched phenotypes and carefully controlled covariates that include environmental influences. similarly, these studies would benefit from gwa approaches and the identification of gene-gene interactions. while investigations of the effect of genetic polymorphisms on β -adrenergic receptor-agonist responses have predominantly focused on clinical end points (e.g., lung function parameters) in the different genotype groups, there has been recent interest in the real-life application of genetic knowledge. as outlined, the first-line treatment for asthma is a shortacting β -adrenergic receptor-agonist (e.g., salbutamol) as needed (step ); if symptoms persist, the addition of inhaled corticosteroid (e.g., beclomethasone) is considered (step ); for further control, a long-acting β -adrenergic receptor agonist (e.g., salmeterol) or a leukotriene receptor antagonist (ltra) (e.g., montelukast) is added (step ) (figure . ) . in a recent study, lipworth et al. set out to use genetic information on the adrb arg polymorphism to inform the choice of prescribing salmeterol or montelukast as addon therapy [ ] . children with persistent asthma and homozygous for the arg genotype (n = ) were randomized to receive salmeterol ( μg, bd) or montelukast ( or mg, once daily) as an add-on to inhaled fluticasone propionate for one year. the study tested whether carriers of the arg genotype were more prone to adverse effects (e.g., prolonged β -adrenergic receptor-agonist use associated exacerbations), and hence whether montelukast provided superior control for this preselected population. outcomes were school absences (primary outcome), exacerbation score, reliever use (salbutamol), morning dyspnoea, and asthma control questionnaire (acq) qualityof-life scores. montelukast provided superior benefit for all measures, with clinically relevant differences within three months. no significant difference in fev (% pred) was observed, providing further evidence of limited correlation between lung function and symptom-based scores [ ] . these results suggest that larger and longer prospective studies are warranted to provide more definitive data on the clinical utility of arg stratification including a gly study arm and the use of additional markers to define the population would provide clearer interpretation. as outlined, leukotriene synthesis inhibitors (ltsis) and leukotriene receptor antagonists (ltras) are commonly used as an add-on therapy in asthma to provide greater control or steroid-sparing effects. the cysteinyl leukotrienes (ltc , ltd , lte ) and dihydroxy leukotriene (ltb ) contribute to the inflammatory process in asthma and are synthesized from arachidonic acid via the -lipoxygenase pathway (figure . ) . cysteinyl leukotrienes have been implicated in bronchoconstriction, mucus secretion, vascular permeability, inflammatory cell infiltration and cytokine production. arachidonic acid is converted to -hydroperoxyeicosatetraenoic acid and leukotriene a (lta ) by -lipoxygenase ( -lo/alox ) and -lipoxygenaseactivating protein (flap/alox ap), which acts as an adaptor protein for this reaction. lta is then converted to leukotriene b (ltb ) by lta hydrolase (lta h) or, alternatively, is conjugated with reduced glutathione to form ltc via the actions of leukotriene c (ltc ) synthase (ltc s). ltc is next transported to the extracellular space via the multidrug resistance protein (mrp ) ( figure . ). leukotriene modifiers include ltsis, which act by targeting -lo (e.g., zileuton), resulting in a decrease in all leukotriene biosynthesis (ltc , ltd , lte , and ltb ) or ltras, which act by specifically blocking cysteinyl leukotrienes from binding to their primary receptor, cysltr , which is found on many cell types including inflammatory cells and airway smooth-muscle cells, (e.g., montelukast and zafirlukast). data demonstrating the large degree of heterogeneity of asthma patient responses to this class of drug are summarized in figure . . these data have led to extensive genetic studies investigating the roles of snps in a large number of candidate genes associated with leukotriene production and/or activity and acute responses to ltras and ltsis. associations have been described for polymorphisms in alox , alox ap, ltc s, cysltr , cysltr , and mrp ; however, many of these studies have been small and so have led to a lack of reproducibility in findings [ ] . more recent studies have looked at different aspects of leukotriene modifier functions, including the association of montelukast absorption (i.e., pharmacokinetics) with polymorphisms in oatp pb [ ] . however, others have failed to observe these associations [ ] . -lo (along with -lo-activating protein) is the major regulatory switch for leukotriene production, and extensive studies have demonstrated that the level of -lo can have dramatic effects on both cysteinyl and dihydroxy leukotriene production. -lo catalyzes the conversion of arachidonic acid to lta , one of the early stages in leukotriene production ( figure . ) . alox is found on chromosome q . and is a large gene composed of exons and introns; it spans approximately kb. multiple studies suggest that alox polymorphisms can influence clinical responses to ltras and ltsis-in particular, a functional repeat polymorphism in the promoter region (resulting in alterations in sp transcription factor binding) have been associated with altered gene transcription and with response to the ltsi, abt- [ , ] . these early studies have now been extended to investigate multiple polymorphisms, spanning the entire gene, and association with ltra and/or ltsi responses in asthma patients. for example, tantisira et al. observed an association between alox intronic snps-rs , rs , and rs -and change in fev post-ltsi(zileuton)-in a cohort of asthma patients [ ] . the rs snp was also a predictor of response to the ltra, montelukast, in a previous study of asthma subjects using percentage change in fev as the primary outcome [ ] . in both cases, the gg versus ga or aa genotype had the greatest improvement in lung function (fev ) post-drug [ ] . the lack of association for the rs , and rs snps in the montelukast study may be due to the reduced power of this study compared to the zileuton study. the functional significance of these snps remains to be resolved, and it is important to note that they may not be the causative genetic change, and that polymorphisms inherited at the same time (i.e., in ld) may be of relevance. two alox snps (rs (synonymous thr thr) and rs ( ′ region)) were also associated with improvement in pef on treatment with montelukast in a -week study in asthma patients. this further confirmed the relevance of alox snps and this key enzyme in leukotriene production [ ] . additional support for the relevance of alox in asthma control is provided by a recent study of children which found that the alox sp promoter polymorphism determined urinary lte levels and was associated with reduced lung function and, potentially, an acq score in non- copy carriers [ ] . in this study, there was an inverse relationship between urinary lte levels and fev . another important enzyme in the leukotriene pathway is leukotriene c synthase (ltc s), which specifically results in the generation of the first cysteinyl leukotriene, ltc , by conjugating glutathione to lta . there has been intense research into the role of ltc s in asthma susceptibility because this enzyme, like -lo, is thought to be a key regulatory switch for cysteinyl leukotrienes, which are thought to play a more prominent role in asthma (e.g., via bronchoconstriction) than the dihydroxy ltb . ltc s is relatively small, spanning . kb on chromosome q . in particular, a promoter polymorphism (a- c, rs ) has been intensely investigated for pharmacogenetic effects as it is thought to alter ltc s levels via transcription (higher levels in c allele carriers) and therefore alter ltc production [ ] . of the ten published studies investigating the role of ltc s- a>c, five showed an improvement in outcome measures, including fev post-ltra therapy for carriers of the c allele, as hypothesized, although results were not always statistically significant. in a more recent study of zileuton responses (fev change over time) in asthma subjects, the ltc s- polymorphism showed no effect; however, an alternative ltc s polymorphism (rs , intron ) was associated with improved response (mean fev ) [ ] . as these results were obtained in different populations and using different end points, the relative contribution of the ltc s polymorphism to ltra or ltsi therapeutic responses in asthma are still unclear. larger prospective studies using multiple ethnic groups are required. montelukast and other ltras target the cysteinyl leukotriene receptor , which is expressed in a variety of cells, including airway smooth-muscle and various inflammatory cells (e.g., eosinophils), therefore inhibiting the activation of this receptor by cysteinyl leukotrienes (figure . ) . cysteinyl leukotriene receptor is thought to be the main receptor mediating cysteinyl leukotriene receptor smooth-muscle contraction and inflammatory cell cytokine production in asthma. again, genetic variation in the target receptor for these compounds may influence how effective they are in carriers of these alleles by regulating receptor function/expression. the cysteinyl leukotriene receptor gene, cysltr , is intronless and found on chromosome xq - ; it generates a amino acid g-protein-coupled receptor (gpcr). a second receptor for cysteinyl leukotrienes has also been described and, while not the target of ltras, may modulate the effects of cysteinyl leukotrienes in vivo. the gene for cysteinyl leukotriene receptor is found on chromosome q and encodes for a amino acid gpcr. interestingly, in combination with several alox snps, polymorphisms spanning cysltr have been identified as determinants of responses to montelukast, suggesting snp-snp interactions may be important [ ] . however, direct evaluation of cysltr polymorphisms and responses to montelukast [ ] or zileuton [ ] have not identified a significant effect for them to date. studies of cysltr are limited; however, snps rs and rs ( ′utr) have been associated with an improvement in morning pef following administration of montelukast for weeks [ ] . while the majority of studies have focused on candidate polymorphism analyses in genes directly involved in leukotriene production or activity, several studies have identified additional genes of relevance to ltsi and ltra response. in addition to its effects on vasodilation and bronchoconstriction, the prostaglandin d receptor (gene: ptgdr) is thought to regulate, at least in part, levels of leukotriene c . in a study of asthmatic children prescribed montelukast ( mg/day), a modest effect of the ptgdr- t/c was observed. similarly, in a more extensive study of snps in candidate genes in asthma patients prescribed fluticasone, fluticasone propionate plus salmeterol or montelukast, a significant association between four snps in the cholinergic muscarinic receptor gene (chrm ) and montelukast response (change in fev ) over weeks was observed [ ] . chrm and chrm are thought to mediate airway tone via regulation of contractile/relaxation responses and targeting of chrm using antagonists; for example, tiotropium has shown clinical efficacy in multiple respiratory diseases. these data potentially suggest that the nature of the airway obstruction/tone determined by altered chrm expression and/or activity may influence responses to montelukast. while the majority of studies have investigated the association between gene polymorphisms and acute responses to asthma therapy, a recent study by mougey et al. identified that solute carrier organic anion transporter family, member b (slco b , alternative name: organic anion transported sb ; oatp b ), was able to mediate montelukast permeability using a model cell system engineered to express the oatp b protein [ ] . subsequently, the same group also showed that an alteration in the protein structure of oatp b that occurs naturallythat is, arg gln (rs )-was associated with reduced morning plasma concentrations of montelukast following an evening dose during one month or six months of treatment in asthma patients [ ] . more specifically, ga (arg/gln) genotype carriers had ∼ % lower montelukast concentration than the gg (arg) group at one month, and ∼ % lower concentration at six months, and of clinical relevance a allele carriers did not demonstrate benefit from montelukast using a symptom-based score [ ] . the same researchers have now replicated these findings on montelukast absorption [ ] ; however, another study did not replicate this finding and failed to identify any effect of the arg gln polymorphism on montelukast plasma concentrations, albeit in a different study design involving fewer subjects [ ] . these studies highlight the potential importance of genetic factors influencing drug transporters that may be anticipated to affect pharmacokinetics and the pharmacodynamics. similarly, several snps in another transporter protein, multidrug resistance protein (mrp ) (alternative name: atp binding cassette, subfamily c, member (abcc ))-for example, rs have been associated with montelukast response (change in fev % predicted) [ ] and zileuton response [ ] . this association potentially confirms the pharmacogenetic significance of this mrp snp marker. this snp is intronic, and the underlying functional mechanism, including which genetic variant explains these effects, remains to be resolved. overall, there has been good progress in the pharmacogenetics of leukotriene modifier therapy in asthma, with snps in multiple leukotriene-synthesising enzymes (e.g., alox ) showing robust association with lta and ltsi responses. multiple genes in the leukotriene synthesis pathway and/or receptors have polymorphisms that have been associated with asthma susceptibility, which implies that there may be a more leukotriene-driven asthma that is therefore more amenable to treatment with ltsis and ltras. of interest in this drug class, preliminary data suggest a significant contribution of snps in drug transporter genes, ultimately determining both the pharmacokinetics and the pharmacodynamics. these data set the scene for larger prospective studies to provide accurate effect sizes and determine clinical implications with greater confidence. corticosteroids are an important pharmacogenetic target in asthma. initial investigations into a pharmacogenetic approach for the corticosteroid element of asthma therapy focused on the glucocorticoid receptor gene (gr, alternative name: nuclear receptor subfamily , group c, member , nr c ), which maps to chromosomal region q -a region associated with multiple asthma phenotypes. as with other genes discussed in this chapter, several polymorphisms have been described in the gr gene that have functional consequences, such as a val asp polymorphism that has been shown to influence the binding affinity for dexamethasone. however, similar to most polymorphisms shown to have potential pharmacogenetic effects, several of these polymorphisms are rare and their functional significance is questionable [ ] . despite this, the gr remains a tantalizing target for asthma pharmacogenetics, and it is surprising that there have not been further recent investigations into the role of gr polymorphisms in corticosteroid responses. gr is not the only gene to be associated with geneticbased variance in corticosteroid efficacy in asthma treatment. a number of other genes have been implicated, including crhr , tbx , nk r, stip , dusp , and fcer , and these are discussed below. snps in the corticotrophin-releasing hormone receptor (crhr ) gene are associated with response to inhaled corticosteroid treatment, based on end point change in fev following weeks of treatment in three asthmatic cohorts using a candidate gene approach ( snps in genes, n = asthma subjects). for example, the intronic crhr snp rs exhibited genotype-specific changes in the percentage predicted change in fev in response to corticosteroid therapy in adult asthma subjects [ ] . this study was the first to show a pharmacogenetic effect for steroid efficacy in an asthmatic cohort, and it highlights the crhr gene to be at least one of a number of factors determining corticosteroid efficacy. crhr is thought to influence responses to exogenously administered corticosteroid through the regulation of endogenous levels of corticosteroid. the t box (tbx ) gene has been shown to be a predictor of improvement in bronchial hyper-responsiveness (bhr) ( year change) post-corticosteroid treatment in children, based on a genotype-dependant variation of the nonsynonymous snp rs (his gln), where the presence of the g allele gave greatest improvements [ ] . however, the association of this gene remains under question because of its low allele frequency in caucasians (maf ∼ . ), which resulted in only a few subjects (n = ) being available to contribute to this observation [ ] . additional data have given more confidence to the involvement of tbx because the his gln polymorphism was significantly associated with improved asthma control in the presence of corticosteroids, although the alternative allele to that described was associated with greater control in korean asthma patients during - weeks treatment [ ] . interestingly in the same study, ye et al. identified a novel gene, the neurokinin receptor (nk r), which was associated with improved asthma control in the presence of corticosteroids; the g allele (gly) of the nk r snp rs g/a, (gly glu) was associated with the greatest improvement. neurokinin a induces bronchoconstriction and inflammation, therefore modulation of the neurokinin receptor may influence the magnitude of these responses. multiple snps in the stress-induced phosphoprotein gene (stip ) were associated with variable fev responses to treatment with the inhaled corticosteroid flunisolide in asthma subjects [ ] . this study investigated snps spanning eight candidate genes and identified stip snps rs , rs , and rs as snps affecting percent change in fev in response to flunisolide at weeks and weeks [ ] . stip codes for an adaptor protein that coordinates functions with hsp and may be involved in formation of the glucocorticosteroid receptor heterocomplex. the dual-specificity phosphatase (dusp ) gene encodes a protein that has dual specificity for tyrosine and threonine and inactivates p mitogen-activated protein kinase (mapk). recently, several snps including a ′ region snp, rs , were associated with ( ) bronchodilator responses and ( ) asthma control in the presence of corticosteroid in a cohort of asthma patients (n = ) [ ] . the mechanism underlying these effects is unclear; however, it was suggested that corticosteroid induces dusp expression, which may be altered in carriers of the rs ′-region snp. this influences the ability of dusp to target the p mapk signaling pathway. in a cohort of asthmatic children, the low-affinity ige receptor gene (fcer ) has been highlighted as putatively involved in corticosteroid (budesonide) regulation of exacerbation by virtue of an association between the snp t c (rs , intronic) and the relative risk of severe exacerbations while taking budesonide [ ] . interestingly, the c allele was also associated with elevated ige levels in the asthma patients [ ] . importantly, this pharmacogenetic association has now been replicated in two other asthma cohorts (n = and , respectively), with the c allele being associated with increased hospital visits and uncontrolled asthma in patients receiving ics [ ] . it is not surprising that this polymorphic variation in an ige receptor influences receptor expression, which alters ige levels and is associated with more severe forms of asthma. in turn, these subjects cannot be adequately controlled by corticosteroid treatment. the first gwas in asthma, published in , identified snps in the glucocorticoid-induced transcript gene (glcc ) as determinants of glucocorticoid response in asthma subjects [ ] . this study examined , snps using the human hap v beadchip to identify snps of interest in an initial cohort of parent-child trios. the primary outcome was change in fev . of interest was a snp in the glcc gene promoter region (rs c/t) that was associated with attenuation in fev improvement post-corticosteroid in three of four cohorts tested [ ] . a subsequent gwas identified the t-gene as a novel player in lung function response (fev ) to inhaled corticosteroids. analyses were carried out in caucasian asthmatics and replicated in a secondary asthmatic population (n = ), where out of successfully replicated snps were associated under the same genetic model in the same direction, including of the top snps ranked by p value. these snps (rs c/ t and rs a/c) were in strong ld with a variant located in the t-gene, suggesting that this gene is a novel player in the pharmacogenetic regulation of corticosteroid efficacy. this was confirmed by a follow-up association, where the t-gene variant was associated with lung function response to inhaled corticosteroids in the initial gwas analysis (an average - -fold increase in fev response for homozygous wild-type subjects; rs tt; and rs cc) [ ] . not all recent developments have been due to gwas. for example, a recent hypothesis-driven study implicated the cytochrome p a enzymes in glucocorticosteroid efficacy through modulations in corticosteroid metabolism. in this study, roberts et al. showed, through mrnadriven studies confirmed via microsomes and recombinant enzymes, that cyp a and cyp a metabolize corticosteroids (budesonide) into inactive metabolites [ ] . they suggested that differences in the expression or function of these enzymes in the lung and/or liver could influence corticosteroid efficacy in the treatment of asthma. to date, the majority of pharmacogenetic studies have focused on one class of asthma therapy. however, several recent studies have investigated combination therapy, which more accurately reflects the clinical situation. we have summarized the main findings regarding the influence of adrb polymorphisms, including arg gly, on combination therapies, particularly fluticasone/salmeterol and budesonide/formoterol in section . . . . however, it is clear that reports on associations between adrb arg and increased exacerbation in asthma patients using regular salbutamol in addition to their maintenance combination are mixed, with some studies failing to report the association, potentially because of differences in study design. more recently, additional genes have been investigated for snp associations with patient responses to combination therapy. in a study of asthma patients regularly using an ics/laba combination, it was shown that polymorphisms in the nitrous oxide synthase (nos ) gene involving an amino acid change (g t, glu asp), were associated with post-drug improvement in lung function (change in fev (% pred)) [ ] . nos codes for a key enzyme in the production of no in the airways, and f e no has been shown to be elevated in asthma and is considered a marker of ongoing inflammation. importantly, earlier data identified that the glu asp polymorphism is functional in nos and influences levels of f e no with the tt genotype associated with lower f e no levels. these data suggest that lower nos activity and potentially lower f e no levels identify patients more likely to respond to combination therapy; however, larger prospective trials are required to validate these findings because they are potentially counterintuitive if no is a marker of inflammation and ics target inflammation. a recent -week study on the effect of genetic determinants ( snps in candidate genes) on asthma patient response to combined fluticasone propionate/salmeterol therapy identified three snps in the cholinergic muscarinic receptor gene (chrm ) that are associated with asthma acq scores, and found a single snp, rs in heat shock kd protein (hspa ), to be associated with change in fev [ ] . of interest, chrm snps were also associated with responses (lung function) to montelukast (see earlier), suggesting that the "tone" of the airways as determined by muscarinic receptors is important for therapies that target airway contraction. hspa is involved in protein folding in the cell, but it is important to note that hspa snps previously were not associated with response to ics [ ] . drug development in asthma has been slow to generate new classes, and the major advances in patient care have come from new compounds and/or indications for existing drug classes (e.g., improved duration of action for labas). one potential explanation for this limited progress is the design of and recruitment to phase i and phase ii trials for the evaluation of new compounds. we have already outlined how initial trials of anti-il demonstrated disappointing effects on lung function and asthma symptoms. only after careful selection of patients, based on clinical and cellular parameters, were clinically relevant improvements observed. researchers now understand that asthma is heterogeneous and, particularly for strategies targeting specific mediators, careful selection of patients is necessary to fully understand the therapeutic potential of new drugs. recent approaches to the treatment of asthma [ ] include the following: the specific targeting of single mediators is unlikely to provide a therapeutic option for asthma in its broadest definition. however, targeting specific subpopulations has shown clinical efficacy. very recently, a study investigating a human il -neutralizing monoclonal antibody (tralokinumab) used a carefully balanced population of atopic and nonatopic asthmatics, with exclusions for additional respiratory pathology, cigarette smoking ≥ pack-years, recent infection, or treatment with immunosuppressive medication, in a randomized double-blind study [ ] . here, tralokinumab was associated with improved lung function based on an increase in fev and a decrease in daily β -adrenergic receptor-agonist use [ ] . these studies overall demonstrate that careful selection of asthma subjects, in this case based on phenotype, is critical in asthma drug development. as novel asthma genes are identified, it is paramount that their evaluation as potential drug targets, particularly in the context of phase ii trials, takes into account pharmacogenetic factors. preliminary data support genetic testing in phase ii trials of newer compounds, as shown in a recent report on a il /il dual antagonist (pitrikinra). polymorphic variation in the target receptor for this antagonist (i.e., il rα) significantly influenced outcomes in allergic asthma subjects [ ] . pitrikinra is a recombinant form of il differing at two amino acid residues (i.e., a mutein, r d/y d). this has been shown to reduce late-phase antigen responses (lar) to inhaled antigen as defined by changes in lung function (fev ) over - hours postantigen exposure. in one study, subjects had been following a four-week period of twice daily active treatment of nebulized pitrikinra or placebo, with an increased lar ratio correlating with a reduced fev [ ] . stratification of subjects (pitrikinra n = , placebo n = ) into genotype groups for the nonsynonomous il ra snp rs (gln arg) identified arg/arg carriers as having an attenuated lar (p < . ) following pitrikinra treatment compared to gln/gln or gln/arg genotypes. similarly, stratification based on rs (glu ala) showed an attenuated lar in the glu/ala group but not the glu/glu group [ ] . interestingly, the arg variant (when in combination with val ) was shown to be a risk factor for allergic asthma and to lead to enhanced il rα signaling post il stimulation [ ] . these preliminary data suggest that selecting a subgroup of patients with a particular genotype when it is anticipated that the receptor/pathway may have a more dominant role in that individual's asthma is critical to interpreting phase ii clinical trials. approaches to further define asthma subphenotypes (e.g., using cluster analyses) have been useful and further confirm this heterogeneity in clinical presentation. while current medications have been extremely successful in asthma management (e.g., ics), it is clear that individual responses to these medications and to therapies in development are heterogeneous, with large variation in clinical benefits and/ or detrimental effects. the stratification of asthma treatment using clinical and/or genetic approaches therefore shows great potential for maximizing clinical outcomes and minimizing adverse effects, leading to improvement in the management of asthma. there has been excellent progress in this area of research over the last five to ten years with the introduction of anti-ige (omalizumab) therapy into mainstay asthma treatment, but only for those patients with clinical indications (i.e., severe allergic asthma) with an elevated serum ige level. this stratification in prescribing for anti-ige shows proofof-concept by targeting this expensive biologic to those patients most likely to show clinical benefit. similarly, there is accumulating evidence that stratification of patients in phase ii trials of newer therapies-for example, anti-il (mepoluzimab) and anti-il (lebrikizumab), again based on clinical and cellular patient profiles, is essential to adequately evaluate these therapies and will be important if these therapies are introduced into clinical practice. clear progress in asthma pharmacogenetics has come with completion of newer studies that interrogate multiple snps in large prospective cohorts. to date, predominantly candidate gene/pathway approaches have been used, but several genetic variants have now been identified with confidence (e.g., alox and leukotriene modifier response; fcer and glucocorticoid response). importantly, these studies show independent replication (the gold standard in genetic studies, including specific snps and direction of effect). also importantly, several gwass have now been completed that provide integration of common variation spanning the entire genome (typically testing , snps) identifying novel gene variants in a hypothesis-free approach (e.g., the collagen (col a ) locus and spats l and response to albuterol/salbutamol). these more recent findings still need further replication and validation; however, they potentially pave the way for a "responder" and "nonresponder" profile based on multiple snps in multiple genes, all measured on the same platform. a key question is the relative contribution of common variation (measured on these platforms) and rare variation (not currently measured to these outcomes). another remaining question relates to the magnitude of changes in clinical measures between genotype groups in asthma patients (e.g., fev ). data so far suggests that these differences can be considered clinically relevant; however, further work with larger populations is required to accurately estimate effect sizes. similarly, the genetic variation identified to date accounts only for a modest proportion of the overall variation of the trait (e.g., a recent estimate suggests the hereditability of bronchodilator response at ∼ . %). it is likely that studies so far have been confounded by factors including snp/haplotype analyses, gene-gene interactions, and the relative contribution of snps in different ethnic backgrounds. therefore, while our knowledge has dramatically increased, there is a need for prospective trials of these current compounds that involve large numbers of subjects and hypothesis-free approaches including gwas. as with gwas approaches to identifying disease susceptibility genes, "missing hereditability" will be an important issue in pharmacogenetics studies. this small proportion of hereditability currently assigned to identified loci (e.g., crhr < %) of corticosteroid response needs to be addressed because asthma medications cannot be successfully personalized unless we resolve a larger degree of genetic variability and/or environmental factors. as with clinical/cellular approaches to stratifying phase ii trials of newer asthma therapies, it is clear that genetic stratification has a role to play. for example, the recent phase ii trials of the il /il dual antagonist (pitrikinra) suggests patient selection is critical to evaluating the clinical efficacy of this compound, with specific il rα genotypes identifying responder groups. more pharmacogenetic integration is required for phase ii trials, which represents a clear challenge for drug development pipelines. as our understanding of the genetic basis of asthma increases other therapeutic targets for asthma will become apparent (e.g., il receptor antagonists). it is clear that the genetic variation associated with asthma susceptibility in these genes will influence pathways targeting methods, as they essentially identify individuals in which the pathway may be particularly important. it is also likely that existing therapies may be influenced by the genetic factors that underlie asthma susceptibility, particularly therapies that have multiple targets (e.g., ics). the impact of our genetic knowledge on asthma management and prescribing practice is limited at this time, as there is a need to further define the complex genetic basis of responders and adverse effects in large prospective studies. however, preliminary "real-life" studies with related outcome measures (e.g., days from school) have provided proof of concept that genetic information can have important implications for disease management (e.g., informing second-line therapy in asthma using adrb arg ) [ ] . there remain many unanswered questions regarding the potential of stratified approaches in the management of asthma. while progress has been made in identifying patient subgroups based on clinical measures, cell counts, or marker expression, more work is required using very large studies to extensively characterize asthma patients not only cross-sectionally, but also longitudinally over many years to identify the natural progression of the disease. the field of genetics is rapidly progressing, with great technological developments moving at a dramatic pace, e.g., gwas comprising a million common snps or several million rare snps, gene expression studies, and wholeexome or targeted resequencing. these approaches will allow the interrogation of genetic variation effect in patient subgroups. it is likely that a drug-specific genetic profile involving several genes (e.g., receptors, signaling mediators, transcription factors) will be a step toward personalized medicine in asthma, with associated benefits including avoidance of adverse side effects and adequate control of the disease. we anticipate that through these approaches many novel common variants/genes will be identified as underlying responses to current asthma medication. as the cost of genetic analyses continues to fall, the potential of a relatively simple test that will have an impact on asthma management costs becomes a real possibility. if genetic or clinical information is going to 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corticosteroids determining asthma treatment by monitoring sputum cell counts: effect on exacerbations genetic basis for personalized medicine in asthma mutations in the gene encoding for the beta -adrenergic receptor in normal and asthmatic subjects polymorphisms of the beta -adrenergic receptor correlated to nocturnal asthma and the response of terbutaline nebulizer adrenergic beta( )-receptor genotype predisposes to exacerbations in steroid-treated asthmatic patients taking frequent albuterol or salmeterol effect of adrb polymorphisms on response to longacting beta -agonist therapy: a pharmacogenetic analysis of two randomised studies common adrb haplotypes derived from polymorphic sites direct beta -adrenergic receptor expression and regulation phenotypes arg is a novel bronchodilator response gene: screening and replication in four asthma cohorts arginase and arginase variations associate with asthma, asthma severity and beta agonist and steroid response regulatory haplotypes in arg are associated with altered bronchodilator response genetic variants of gsnor and adrb influence response to albuterol in african-american children with severe asthma gsno reductase and beta -adrenergic receptor gene-gene interaction: bronchodilator responsiveness to albuterol endogenous nitrogen oxides and bronchodilator s-nitrosothiols in human airways regulation of beta-adrenergic receptor signaling by s-nitrosylation of g-protein-coupled receptor kinase a genome-wide association study of bronchodilator response in asthmatics tailored second-line therapy in asthmatic children with the arg( ) genotype leukotriene pathway genetics and pharmacogenetics in allergy absorption of montelukast is transporter mediated: a common variant of oatp b is associated with reduced plasma concentrations and poor response slco b c. g>a single nucleotide polymorphism has no effect on the pharmacokinetics of montelukast and aliskiren. pharmacogenet pharmacogenetic association between alox promoter genotype and the response to anti-asthma treatment the glucocorticoid receptor heterocomplex gene stip is associated with improved lung function in asthmatic subjects treated with inhaled corticosteroids influence of leukotriene pathway polymorphisms on response to montelukast in asthma pharmacogenetics of the -lipoxygenase biosynthetic pathway and variable clinical response to montelukast pharmacogenetics of asthma controller treatment variant ltc( ) synthase allele modifies cysteinyl leukotriene synthesis in eosinophils and predicts clinical response to zafirlukast asthma pharmacogenetic study using finite mixture models to handle drugresponse heterogeneity effect of citrus juice and slco b genotype on the pharmacokinetics of montelukast treatment heterogeneity in asthma: genetics of response to leukotriene modifiers corticosteroid pharmacogenetics: association of sequence variants in crhr with improved lung function in asthmatics treated with inhaled corticosteroids pharmacogenetic study of the effects of nk r g e g>a and tbx h q c>g polymorphisms on asthma control with inhaled corticosteroid treatment dualspecificity phosphatase as a pharmacogenetic modifier of inhaled steroid response among asthmatic patients fcer : a pharmacogenetic basis for severe exacerbations in children with asthma fcer t c variant associated with chronic symptoms and exacerbations in steroidtreated asthmatic children genomewide association between glcci and response to glucocorticoid therapy in asthma genome-wide association identifies the t gene as a novel asthma pharmacogenetic locus metabolism of beclomethasone dipropionate by cytochrome p a enzymes g t polymorphism of enos gene is a predictor of response to combination of inhaled corticosteroids with long-lasting beta -agonists in asthmatic children pharmacogenetics of asthma ancestry, ancestry-informative markers, asthma, and the quest for personalized medicine a phase ii placebo-controlled study of tralokinumab in moderate-to-severe asthma il- receptor alpha polymorphisms are predictors of a pharmacogenetic response to a novel il- /il- antagonist effect of an interleukin- variant on late phase asthmatic response to allergen challenge in asthmatic patients: results of two phase a studies v r il- receptor alpha is associated with allergic asthma and enhanced il- receptor function research in the authors' laboratory is funded by asthma uk, the medical research council, and the british medical association. we thank dr emily hodge for originally generating figure . . key: cord- -l dehit authors: diaz-arévalo, diana; zeng, mingtao title: nanoparticle-based vaccines: opportunities and limitations date: - - journal: nanopharmaceuticals doi: . /b - - - - . - sha: doc_id: cord_uid: l dehit infectious diseases are the tip of the iceberg in the economic burden of the developing countries, due to the resistance of the pathogens to antibiotics and the lack of vaccines. the vaccines have become a big challenge in the last decades, where the attention has been focused on scientific challenges such as new vaccine development and adjuvants or delivery systems. the classical vaccines were developed from live-attenuated or killed organisms, such as influenza, smallpox, and bcg, as well as subunits such as hepatitis b. the attenuated vaccines carry the risk of regaining their pathogenicity under immunosuppression conditions. the development of subunit vaccines without risk are considered as an essential need in combination with adequate delivery systems to obtain desired cell and humoral immune responses against infectious diseases. in the last decades, the use of nanoparticles as a delivery system in vaccines has received special attention to improve vaccine efficacy. these nanoparticles could be composed of lipids, metal and nonmetal inorganics, several polymers, and virus-like particles, which have been tested in research; some of them have already been approved for human and animal use. the characteristics of the nanoparticles have allowed targeting desired antigen-presenting cells to improve immunization strategies to induce protection. the main characteristics of the nanoparticles are to protect the antigens from early proteolytic degradation, control antigen release, and help antigen uptake and processing by antigen-presenting cells, and they should be safe for human and veterinary use. in addition, the nanoparticles could be modified in their physicochemical properties to target specific cells and improve vaccine efficacy. this chapter focuses on the nanoparticle-based vaccine formulations and the approaches used to realize efficient delivery of vaccines in order to induce host protective immunity against infectious diseases. nanomedicine has seen increased interest by the medical community in recent years, as it has enabled modification of engineering devices for delivery to and interaction with cell environments. in particular, this technology has enabled advances in the design of delivery systems for drugs and vaccines [ ] . nanoparticles (nps) are now being engineered from different biodegradable materials, including natural and synthetic polymers (poly[lactic-co-glycolic] acid (plga) and polylactic acid (pla)), metals (gold, copper oxide, aluminum oxide, zinc oxide, iron oxide, and silver), or lipids (phosphatidylserine, phosphatidylcholine, and cholesterol) [ ] . nps and microparticles (mps) have been widely used to deliver drugs, especially cytotoxic drugs or immune-suppression treatments for transplantation. these nps are effective in the distribution of medications to target specific organs and control drug delivery. these drug carriers not only transport drugs to sites of cancer or other target diseased organs, preventing damage to healthy cells, but also protect the drug from degradation [ ] . some of the metal nanoparticles (e.g., gold, silver, or plga) themselves may have dual functions, working both as carriers and as targeted delivery systems using the membranes of cancer cells, which contain tumor-associated markers. in a recent study, fang et al. used a membrane from mouse melanoma cancer cells in the outer layer of plga nanoparticles. these nps were stable and persisted in the structure during cellular endocytosis, activating the maturation of dendritic cells (dcs) and presenting especially to t cells that have tcr binding to gp and inducing the production of ifn-g. moreover, the authors found that the plga covering membrane had receptors that allow interaction with cancer cells and delivery of the drug [ , ] . furthermore, the nps were used to deliver drugs to the specific site of organ transplantation to prevent rejection. systemic immunosuppression is a high risk for recipients of organ transplantation, as it uses high doses of immunosuppression agents that make them more susceptible to infections and in some cases cause death. previous studies found that diabetic mice were transplanted with islets into the eye to prevent islet graft immune rejection in vivo using rapamycin mps. the islets transplanted with the immunosuppressed drug mps survived more than month compared with the control (empty mps), which rejected the islets in the second week [ ] . work in last few decades has increased our knowledge of infectious diseases and the mechanisms of evasion of immune responses. however, new variants of antibiotic-resistant pathogenic microorganisms have emerged and are becoming a challenge for designing new vaccines and adjuvants. until now vaccines have been developed from live-attenuated microorganisms or killed pathogens (first-generation vaccines) [ ] , dna vaccines (third-generation vaccines), subunit vaccines [ ] , and synthetic peptides (second-generation vaccines). the last three vaccine types eliminate the risk of developing the disease, but they must be used in conjunction with an adequate adjuvant or delivery system (table . ). the combination of the vaccine with the adjuvant or delivery system should be safe, stable, and have the ability to induce long-lived memory b and t cell responses, preferably with a single dose and a maximum of two doses and be free from strict storage requirements [ ] . dna and rna vaccines are safe but need a second boost with recombinant protein or dna from another vector. nps have become an alternative to targeting vaccine delivery to immune cells, improving vaccine efficacy with slow release, easy antigen uptake, and induction of humoral and cellular responses [ ] . nps have played an important role in the activation of antigen-presenting cells (apcs), especially dcs, which may determine vaccine efficacy. although there is some cytotoxic effect of the nps [ , ] , the risk is low compared with the benefits of vaccine delivery [ ] . in this chapter, we will summarize the different nanocarrierbased vaccine formulations that achieve the desired host immunity against infectious diseases and cancer, and at the end, we will discuss on the limitations of the respective carriers. dcs are specialized apcs that coordinate the innate and adaptive immune responses to until now there have been various types of nps, composed of gold, dendrimers, carbon, polymers, and liposomes, that have been used to deliver vaccines. all can stimulate the production of cytokines and antibody responses [ e ]. the cargo is not limited to vaccines, and it is possible to add adjuvants and immune stimulatory molecules, including silica and iron, tlr agonist, and cytokines, to improve immunogenicity [ , ] . several vaccines have been tested on the different types of nps (table . ). liposomes are the second-most common type of np, and they self-assemble in water under special conditions. they are composed of lipids, which have a hydrophilic head and a hydrophobic tail that maintain hydrophilic inner and outer membranes, in lamellar lipid bilayers or in multilayers that simulate vesicles found within cells [ ] . liposomes can induce cellular responses or humoral responses, depending on the charge, size, and lipid composition. a previous study showed that charge had the main role in activation of the cellular or humoral immune responses. the authors' approach was to investigate the importance of the antigeneliposome interaction in immunogenicity and depot formation. they used subunit antigens ag be esat- (pi ¼ . ) from tb, and "cth " (pi ¼ . ), from chlamydia vaccines and a model antigen, lysozyme (pi ¼ ). the injection of cationic dimethyldioctadecylammonium using np platforms such as liposomes, emulsions, nanogels, and other substances. (b) nanovaccines can access the lymphatic drainage system for lymph node delivery while protecting their cargoes from environmental degradation. once at the lymph nodes, the nanocarriers deliver their cargoes to apcs for immune processing. (c) nanovaccine properties can be tuned to efficiently deliver their cargoes for maximum immune activation. for example, nps can be modified to target specific subsets of immune cells. they can also be delivered to specific intracellular compartments, where receptors for immune pathways can be triggered. to migrate to the secondary lymph nodes to activate the t and b cells, which the authors did not analyze in this manuscript [ ] . previous studies have shown that dda induces proinflammatory cytokines and chemokines after infiltration of monocytes and macrophages in vivo [ ] . one of the advantages of liposomes is mimicking properties of the pathogens, inducing humoral and cellular immune responses. the antigen's presentation to apcs depends on the membrane characteristics, size, and ligand-receptor binding. liposomes could induce th responses if the lipids are unsaturated whereas saturated lipids promoted th -type immune responses [ , ] . liposomes could be modified according to the needs of delivery to produce th , th , th , or tfh immune responses. cationic liposomes can increase the uptake of subunit vaccines as synthetic peptides and recombinant proteins; this can be explained by the interaction between positively charged liposome with the apcs that have a negative charge in their membrane [ , ] . another modification is the ph-sensitive fusogenic liposomes, which are stable in neutral ph . , but in acidic conditions, the antigen is released and presented by mhc class i or ii. these liposomes containing phosphatidylethanolamine and amphiphilic stabilizers allow pe-containing liposomes to form aggregates, due to the poor hydration of their headgroups, which can explain their high affinity to adhere to cell membranes [ , ] . another study designed a unique structure composed of inter-bilayer-crosslinked multilamellar vesicles (icmvs), which is stable in the extracellular environment but rapidly released in endosomes/lysosomes, thereby enhancing vaccine immune responses. the icmvs carried the antigen ova mixed with the adjuvant monophosphoryl lipid a (mpla). this mixture amplified vaccine responses and upregulated the costimulatory cells on splenic and bone marrow dcs. in addition, splenic dcs incubated with the ova þ vaccine mpla triggered proliferation of na € ive ot- cd þ t cells in vitro, suggesting that the icmvs enhanced cross-presentation of the antigen. the same results were obtained in vivo after the vaccination of mice with the mixture, and the icmvs elicited robust antibody titers that were w -fold greater than simple liposomes. these results could be attributed to activation enhancement of dcs and antigen cross presentation [ ] . the first use of liposomes as a delivery system for a malaria vaccine was in the s. ballou et al. synthetized peptides derived from the repetitive region of the circumsporozoite (cs) protein of plasmodium falciparum sporozoites. the synthetic peptides were conjugated to keyhole limpet hemocyanin (klh) proteins and were incorporated into liposomes. immunized mice and rabbits produced antibodies against the repeat region of the protein with biologic activity correlated with protection [ ] . rts, s is the only vaccine against malaria that is in phase three clinical trials in africa. the rts, s vaccine has the central repeat region of csp, and t cell epitopes localized in the c-terminal region are fused to hepatitis b surface antigen (hbsag) and expressed in saccharomyces cerevisiae yeast. this virus-like particle (vlp) vaccine also contains mpla and qs- , which enhances humoral and cd þ t cell responses in the first years, with a level of protection of %e %. protection decreased rapidly thereafter, with negative efficacy in some children. the problem is not rts, s nor the adjuvant system, as both induce potent immune responses. however, the csp part of rts, s is an antigenic polymorphism, and t cell epitopes present on the cs protein that are incorporated into the vaccine are also polymorphic. to address this problem, the newest generation of this vaccine is coformulated with matrix-m, a saponin-based liposomal adjuvant [ ] . however, the vaccine protection against p. falciparum has not been achieved due to the short-lived memory cells and the evasion mechanisms of the parasite [ ] . the first malaria vaccine was tested in in animals using plasmodium berghei s radiation-attenuated sporozoites, with a % of protection [ ] . hoffman et al. reported in that human volunteers were vaccinated with irradiated sporozoites of p. falciparum strain nf , and all of them were protected between to weeks [ ] . this vaccine will be used to vaccinate , children a year in three african countries (ghana, malawi, and kenya). the vaccine could prevent in cases according to previous clinical trials. virus-like particles (vlps) are composed of a self-assembling viral membrane maintaining viral surface proteins. vlps can be modified to express additional proteins of other microorganisms, which could be engineered by fusion of the proteins with membrane antigens or by endogenous expression of other antigens [ ] . gardasil is a four-component vlp-type vaccine specific against hpv. it contains the l major capsid protein of hpv types , , , and and is administered along with an aluminum adjuvant. this vaccine was administered to women and was followed up for incidence of persistent associated infection for months. the efficacy was % for preventing clinical disease. the hpv combination vaccine was immunogenic, inducing the production of long-lived antibodies [ ] . however, some problems with the vaccine in teenage girls were reported. the adverse events included dermatologic/ mucosa-allergic reaction ( %), rash ( %), and local/injection-site reaction ( %). however, some serious adverse events following immunization were reported ( . % of reports), including two incidents of anaphylaxis, two seizures, one incident of thrombocytopenia, and one death [ ] . inorganic metal nps are frequently used in drug delivery and bioimaging, especially in treating cancer patients. dna vaccines are more stable and protected from degradation when carried by a gold, silica, or silver np delivery system. the covalent attachment of chito to gnps increases the np molecular weight, enhancing dna binding and stability without compromising dna release and transfer. chito egnpedna (hbsag) complexes induce effective antibody and t cell responses after immunization of balb/c mice. by contrast, naked dna-primed hbsag induces antibodies after a series of four immunizations with mg of naked dna. hbsag-specific cd þ t cells eliminated p /balb target cells that had been sensitized with an hbsag ctl epitope peptide in vitro. these chimeric nps, employing a minimal amount of dna, induce effective immune responses when compared with naked dna [ ] . biodegradable polymers are of significant interest in the delivery of drugs and vaccines against infectious diseases. these polymers consist of either natural or synthetic monomers that are biodegradable, are nonimmunogenic, have low cytotoxicity, and are easy to prepare. there are several polymers, such as chitosan, plga, polyethylene glycol (peg), polycaprolactone, and dextran used as delivery systems [ ] . plga is approved for human use by the us food and drug administration (fda) and european medicines agency (ema), while plga has been used to deliver drugs for long periods. dcs play an important role in the activation of adaptive immune responses, in which b cells and t cells activate and differentiate into subpopulations that determine which humoral or cellular immune responses eliminate the microorganism. cruz et al. studied the delivery of nps and mps to dcs to create a biocompatible and biodegradable slow-release vaccine and effectively target the cells. the nps and mps (plgaepeg) were loaded with tetanus toxoid peptideefitc linked to a lyselys cathepsin cleavage site and an anti-dc-sign antibody. the dcs targeted plgabased vaccine nps but not mps. the nps were efficiently taken up with the help of anti-dc-sign antibodies and induced proliferation of t cells (fig. . ) . however, the mps were taken up for all apcs, including the dcs (fig. . ) . new research is needed to understand the biology, antigen processing, and presentation needed to induce long-lived memory b and t cells [ ] . botulism is a lethal neuroparalytic disease produced by clostridium botulinum toxins (a-h). ruwona et al. demonstrated that cationic plga nps can carry plasmid dna encoding the bont heavy-chain (hc) fragment and that its product is nontoxic. immunized mice produced high titers of antibodies after to weeks (fig. . ) . after four immunizations, specific igg had decreased, but igg a had increased, compared with the first immunization. after challenge, % of the mice vaccinated with plgae pvax/opt-bont/c-hc survived, while only % of the mice immunized with naked plasmid were protected (fig. . ) [ ] . modifications of polymeric nps have been used to deliver synthetic peptides or recombinant proteins to dendritic cells and macrophages. salvador et al., modified plga ms, : lactideeglicolide ratio, to produce cationic nps using polyethylene imine (pei), and bsa as antigen. in addition, the nps were modified encapsulating monophosphoryl lipid a (mpla) or polyinosinic-polycytidilic acid poly(i:c) and a-galactosyl ceramide to address their adjuvant effect. coumarin was incorporated into the oily phase for the preparation of fluorescent nps to evaluate in vitro assays. the nps were tested in vitro using monocytes and dendritic cells, and mice were immunized with the different np modifications to determine the immune responses in vivo. the cationic nps showed a noticeable enhancement of their uptake by the monocytes, mdcs, and pdcs in comparison to the classical nps. in addition, the cationic nps increased immunostimulatory effect since they induced the production of antibodies and th cell responses producing ifn-g [ ] . the interest of companies in nanoparticlebased vaccine delivery dramatically increased in the last decades. pevion biotech ltd., a swiss company founded in , developed the virosome-based technology platforms to make efficient and safe prophylactic/therapeutic vaccine candidates. for candida albicans that can cause vulvovaginal mucosal infections, pevion used the aspartyl-proteinase (sap ), which is an immunodominant antigen and virulence factor; this recombinant protein was assembled with virosomes (pev ). the evaluation in mouse model and the initial clinical trial on women showed that this candidate vaccine, intravaginally administered, has a therapeutic potential for the treatment of recurrent candidiasis [ ] . in addition, this company tested a malaria vaccine in africa. this vaccine had combination of ffm me-trapþpev a (ama- ) and was tested in phase i/iia clinical trials. although the vaccine did not show sterile protection, it induced responses on blood stage parasites and lower rates of parasite growth in human volunteers vaccinated with pev a, compared to unvaccinated controls [ ] . glaxos-mithkline plc is working to improve the coformulated rts, s vaccine using matrix-m, a saponin-based liposomal adjuvant [ ] . novavax is developing respiratory syncytial virus (rsv) f nanoparticle vaccine with aluminum. the purpose of the reported study was to evaluate the safety and efficacy of maternally transferred antibodies in preventing rsv in infants. the vaccinated healthy third-trimester pregnant women showed significant protection to newborn children from rsv challenge and reduced pulmonary inflammation, and the vaccine was safe and effective for maternal and . nanoparticle-based vaccines: opportunities and limitations adult vaccination [ ] . furthermore, this company is working in recombinant trivalent nanoparticle influenza vaccine with matrix m- . the vaccine can induce responses to one or more conserved ha head and stem epitopes. the antibody responses are able to neutralize against both homologous and heterologous strains [ ] . the nanoparticles have been used in recent years in only a few of advanced clinical trials. the vaccine against rsv infection has three studies using rsv f nanoparticle vaccine with aluminum in phase and trials. the first clinical study of rsv was rsv f dose-ranging study in women, which started october and finished may . the study showed that all formulations were well tolerated, without treatment-related serious adverse events. antibodies anti-f igg and palivizumab-competitive antibody responses were correlated and increased after both doses, while microneutralization assays increased significantly after the first dose, then plateaued [ ] . the second study was rsv f vaccine maternal immunization study in healthy third-trimester pregnant women, which started september and finished june . this study demonstrated that the vaccine is safe to infants and pregnant women. the third clinical study is in phase three trials and will finish in july . the vaccine is safe and effective for infants and pregnant women [ ] . the study "evaluation of the safety and immunogenicity of a recombinant trivalent nanoparticle influenza vaccine with matrix m- adjuvant (nanoflu)" was used to test protection in people older than age , but no results have been publicly released. the other study is the phase two trial entitled "dose and formulation confirmation of quad-niv in older adults." in this study, subjects were randomized to seven treatment groups to receive the vaccine or an active comparator. table . summarizes the clinical trials [ ] : phase ieiv clinical trials/vaccines/nanoparticles. engineered nps have revolutionized the delivery of drugs, monoclonal antibodies, and vaccines to target cells. nps have been designed from metals and nonmetals, polymeric materials, lipids, vlps, and bioceramics, giving distinctive physicochemical and electrical characteristics that specifically interact with a targeted cell or organ. however, nps can enter easily into the human body by crossing lipid bilayers and may interact with sensitive organs [ ] . the clearance and excretion of nps is mediated through the mononuclear phagocytic system, the renal urinary system, and by biliary clearance. macrophages phagocytize the nps and keep them in the secondary lymph organs and/or liver using the mertk (mer) receptor (the same receptor tyrosine kinase family as axl and tyro- ), which is responsible for promoting apoptotic cell engulfment and supports platelet aggregation and clot stability in vivo [ ] . in addition, kupffer cells, which are liver macrophages, sequester -nm nanoparticles [ ] . when the particles are nm in diameter, they are rapidly cleared from the circulatory system via renal filtration [ ] . on the other hand, some studies have shown that metallic nps (menps) have effects on innate immunity. specifically, in vitro assays showed that menps have some cytotoxicity and genotoxicity and interfere with cytokine production and gene expression due to receptor modifications. the response of the innate immune system to threats is mediated by inflammation and the production of cytokines, chemokines, free radicals (nitric oxide) [ ] , and other reactive oxygen species (ros). in this process, there is an accumulation of oxidized glutathione (gssg), generating stress from the proinflammatory signal (involving tlrs) on the menps and causing cell death and cancer [ ] . other studies have shown that adenovirus vlps combine with a gene to control gelsinger's ammonia metabolism (encoding ornithine transcarbamylase), which invades all the organs and induces severe reactions that can lead to death [ ] . other severe side reactions have been reported as carcinogenesis or germline alterations observed in animal models, in which the vlps can become widely dispersed in the body, and the viral vector could end up in the ovaries and testes [ ] . eudragit is a drug delivery polymer: poly (ethyl acrylate-comethyl methacrylate-cotrimethylammonioethyl methacrylate chloride) : : . . it has been reported that using nr macrophages cocultured with np/ers, the nps were close to the inner membrane of the mitochondria, as observed by transmission electronic microscopy. the authors also analyzed genes responsible for mitochondrial function by microarray and found that opa was reduced in expression. this gene helps to maintain network morphology and dynamics and in the regulation of the signaling pathways for cell death, and np/ers-treated cells reduced glutathione (gsh), stimulating ros production. due to unbalancing of oxidanteantioxidant homeostasis and changes of proteins implied in activation and autophagy, the mitochondria decay through phagophore and autophagosome formation, and mitophagy can occur [ ] . the nps are characterized for their size (< nm) with greater surface area per mass compared with larger-sized particles of the same chemistry causing nps more active biologically. the physicochemical properties of the material could increase the uptake by the antigen-presenting cells, but they can go to other tissues that can generate adverse biological effects such as apoptosis or necrosis. there are some materials that can be degraded easily. however, the nps (plga) had been modified to release antigens slowly to immunize only once and to increase the efficiency of protective adaptive immune responses. nevertheless, intranasal immunization with nps had been reported to cause lung injury through oxidative stress, which induced the production of cytotoxic cellular responses and inflammatory cytokines. another problem with the nps is the aggregation that may block the blood vessels in the host. modifications of polymeric nps with pei have been used to prevent the recognition to other cells such as epithelial cells in the lung. other technologies could be used as the singlechain fragment region of antibodies binding to nps, which recognizes the specific receptor on dendritic cells or macrophages that may deliver the antigens to target organ. other modifications in the nps have been used as plga covered with peg (plgaepeg) and formulated with tetanus toxoid peptideefitc linked to a lyse lys cathepsin cleavage site and an anti-dc-sign antibody, which help the delivery to dcs but not macrophages, with enhanced efficient proliferation of t cells [ ] . negatively charged nps prevent the activation of the immune system and induction of immunotolerance, therefore preventing the effect of exacerbated inflammatory responses. nanoparticles can also be engineered to be porous to increase the diffusion of intracellular proteases, resulting in earlier processing of apcs and presentation t cells [ ] . in addition, some nps carrying antigen and adjuvant in the same np are less efficient to induce the immune response than the ones with adjuvant separated from antigens in different nps. this may because of the competing effect of coactivation in several pathways [ ] . nps are widely explored in vaccine delivery and targeting apcs, especially dcs. despite advances in immunology, it is critical to understand the mechanisms of entry of nps into cells and activation of the adaptive immune response and related toxicity. some approaches inhibit the cytokine and chemokine cascade with dcs and t and b cells. nps have great potential in immunology, although in-depth study of toxicity is necessary. in addition, nps require molecules, such as antibodies or ligands, that bind to receptors on target cells to ensure that nps only adhere to those cells. any modification should be tested in cell lines or animals to prevent side effects or death. the synthesis of nps can be shifted to methods that generate reproducible nanoparticles in terms of size (< nm, allowing elimination by the kidney), shape, and composition (biodegradable materials) to reduce cytotoxicity. dna mediated vaccines delivery through 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specifically deliver antigen to human dendritic cells via dc-sign in vitro candida vaginitis: virulence, host response and vaccine prospects evidence of blood stage efficacy with a virosomal malaria vaccine in a phase iia clinical trial efficacy of a respiratory syncytial virus vaccine candidate in a maternal immunization model novel hemagglutinin nanoparticle influenza vaccine with matrix-mÔ adjuvant induces hemagglutination inhibition, neutralizing, and protective responses in ferrets against homologous and drifted a (h n ) subtypes a phase randomized, observerblind, placebo-controlled, dose-ranging trial of aluminum-adjuvanted respiratory syncytial virus f particle vaccine formulations in healthy women of childbearing age current opinion on nanotoxicology a soluble form of the mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation can controversial nanotechnology promise drug delivery? sub- -nm pd nanosheets with renal clearance for efficient near-infrared photothermal cancer therapy nitric oxide. a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells engineered metal based nanoparticles and innate immunity gene therapy death prompts review of adenovirus vector trial halted after gene shows up in semen conformational changes and catalytic competency of hydrolases adsorbing on fumed silica nanoparticles: ii. secondary structure improving vaccine and immunotherapy design using biomaterials delivering adjuvants and antigens in separate nanoparticles eliminates the need of physical linkage for effective vaccination nanoparticle-based vaccines: opportunities and limitations key: cord- -zpn h mt authors: chaffee, mary w.; oster, neill s. title: the role of hospitals in disaster date: - - journal: disaster medicine doi: . /b - - - - . - sha: doc_id: cord_uid: zpn h mt nan when the first rain began to fall in houston,texas, in june , did hospital staff know they would soon be providing care for hundreds of patients without electrical power or running water in flooded hospital buildings? on april , , did the emergency department staff arriving for the day shift at oklahoma city hospitals know that a former soldier was driving a rented van filled with pounds of ammonium nitrate toward the murrah federal building and that they would soon be faced with bombing victims? in , did restaurant patrons in wasco county, ore., have any idea, as they selected food from salad bars, that they would soon be evaluated in hospitals for profuse, watery diarrhea from intentional food contamination by a religious cult? in march , did the toronto healthcare workers who were caring for patients with respiratory symptoms know they would soon become infected with severe acute respiratory syndrome (sars) ? we can be quite certain that none of them knew. the capricious nature of disaster implies victims and responders are generally caught unaware. but we do know some things. we know there will be hurricanes, typhoons, tornadoes, earthquakes, mudslides, fires, and blizzards this year. we know people will pick up firearms, make bombs, and inflict pain and suffering on others. we know there will be casualties from train accidents, cars crumpled in chain reactions, building collapses, and explosions.we know infectious diseases will do what they do best: spread, sicken, and kill. we know terrorists have not given up their violent assaults. we know there will be mental health symptoms in accident survivors and the caregivers who respond to their needs. it is the hospital, at the heart of the health system, that will receive the injured, infected, bleeding, broken, and terrified from these events. we know the victims will seek life-saving care, comfort, and relief at hospitals, but many u.s. hospitals continue to prepare for disaster as though it will not happen to them. there are more than hospitals in the united states that form a diverse patchwork of healthcare services. u.s. hospitals vary greatly by geographic location (urban, suburban, and rural); financial and management structure (for profit, not-for-profit, private, public); type of care (general medical services or specialty care, such as psychiatric or pediatric); and government affiliation (department of defense,veterans health administration, or public health service). any of these hospitals may be called on to respond to the next disaster or may be the victim of a disaster. many experts believe that these hospitals are not adequately prepared to respond effectively ( the hospital was of little significance in american healthcare before the civil war. only hospitals existed in when the first survey was conducted-a time when no proper gentleman or lady would venture into a hospital by choice. the murky medical practices of the s offered little that couldn't be found in homes, and physicians had little in their armamentarium to change the course of disease and injury. however, discovery and scientific advance changed that. effective anesthesia, surgical antisepsis, antibiotics, the x-ray, and other advances turned the hospital into a place of comfort, hope, and healing. the th-century hospital became a sophisticated financial institution, the core of medical education, and the site of dazzling technological display. medical advances offered aid not only to the chronically ill but offered hope to those who suffered acute trauma or medical or psychiatric emergency. past events illuminate the variety and complexity of demands placed on a hospital in a disaster: the potential impact of disaster is staggering. the release of tons of methyl isocyanate from the union carbide factory in bhopal, india, in december exposed more than , to the deadly gas and killed about in the first week after the release. in september , workers scavenging a dismantled cancer clinic in goiania, brazil, took home a source containing cesium- . they sold it to a junkman who showed the glowing item to friends and neighbors. once radiation exposure victims presented to hospitals, and the release became well known, hospitals were overwhelmed. although were actually exposed and showed signs of radiation sickness, , people were evaluated. when the aum shinri kyo cult placed sarin on five trains in the tokyo subway system on march , , people made their own way to hospitals, were transported by authorities, and hospital staff and rescue personnel were contaminated due to poor or nonexistent decontamination procedures. on sept. , , when u.s. hospitals and healthcare professionals were confronted with the worst attack on american soil, and again during the anthrax attacks along the eastern seaboard, individuals and organizations responded heroically. a powerful change in thinking, also called a paradigm shift, occurred after the terror attacks: the health system came to be viewed as a foundation of national security. another perspective has changed as well. in the event of a disaster, emergency medical services (ems), police, and fire have long been recognized as first responders. however, just recently, hospitals also have been designated as first responders-and first receivers. the value of the hospital in national security has been increased, and hospitals are recognized as safe havens in communities, the public expects hospitals to be prepared to care for their needs, and the hospital is now recognized as a first responder in emergencies. however, hospitals remain significantly underprepared to respond as effectively as the public expects. most importantly, preparedness is at direct odds with productivity. daily operating requirements stretch most hospitals' resources. allocating funds to improve emergency response capabilities that may never be used could be viewed as foolhardy. community integration is now seen as necessary, but hospitals (other than those in networks or that are government facilities) have had few reasons to build healthy relationships with other hospitals in their communities. to meet the needs of communities in a disaster, business competitors must work as partners. hospitals play a vital role in the health,social structure,and economic life of a community. patients expect hospitals, and health system workers, to be available to provide care for them in all circumstances. a level of preparedness that was viewed as adequate in the past is no longer seen as acceptable. to be more highly prepared and to be able to respond effectively, hospitals must make substantial investment in equipment, training, facilities improvements, and supplies. hospitals depend on public trust; poor performance during a disaster could be financially crippling to a facility. rubin writes that hospitals are expected to handle whatever they receive and do it right the first time. hospitals are vulnerable to the stresses of disaster responses due to a number of inherent characteristics: • complexity of services: hospitals are facilities that provide healthcare but must also function as laundromats, hotels, office buildings, laboratories, restaurants, and warehouses. • dependence on lifelines: hospitals are completely dependent on basic public services: water, sewer, power, medical gases, communications, fuel, and waste collection. • hazardous materials: the hospital environment contains toxic agents and poisonous liquids and gases. • dangerous objects: heavy medical equipment, storage shelves, and supplies can fall or shift during an event such as an earthquake. multiple forces have placed hospitals in a precarious preparedness posture. the capacity of the health system has been scaled down to a bare minimum to cut operating costs. emergency departments are crowded with the uninsured and the underinsured who have no other access to care. the nursing workforce has withered, and physicians have left practice due to uncontrolled liability insurance costs. many hospitals determine their surge capacity by the number of patients they could comfortably care for using standard spaces, quality care standards, and additional teams of personnel to help. in reality, a disaster is not going to comply with the limits of hospital capacity. if bombing victims arrive at a -bed community hospital, spaces will need to be converted and used that planners may have never imagined, such as chapels, hallways, and offices. nurses accustomed to a certain nurseto-patient ratio may find the ratio in a disaster much higher and have to adapt practice accordingly. surge capacity must not be viewed only as the number of beds or spaces that can be allotted to care for patients, but it must include all supporting hospital services that are involved in patient care. if hospital services fail during a disaster, the hospital fails the population depending on it. the population includes not just the victims of the disaster, but the others presenting for needed care-women preparing to give birth, patients with chronic disease exacerbation, and children with lacerations that need sutures. a vital hospital emergency management program acts as an insurance policy that increases the chances of continued operations under difficult circumstances. an effective hospital emergency management program guides the development and execution of activities that mitigate, prepare for, respond to, and recover from incidents that disrupt the normal provision of care. the program should include the following components: • emergency manager: the emergency manager is the primary point of leadership in the development, improvement, exercise, and execution of the hospital's emergency management plan. • emergency management plan: the plan identifies the hospital's response to internal and external emergencies. deliberate (advance) planning permits the development of strategies while the organization is not under pressure to react. • executive leadership: hospital executive leadership charts the course for an organization. a hospital that lacks executive leadership committed to emergency preparedness will be significantly hampered in its efforts. • strategic planning: the hospital's strategic plan is the blueprint that guides all efforts to achieve its mission. it is critical that emergency management and preparedness efforts are woven into strategic planning. • emergency management committee: extremely broad membership is desired to ensure all hospital operations that will be stressed in a disaster are integrated and well prepared. • hazard vulnerability analysis (hva): the hva is a tool used to assess the risks in a specific environment. the emergency management plan can be tailored to address the hazards most likely to affect hospital operations. • vulnerability analysis: every aspect of hospital operations that will be depended on in a disaster should be assessed to determine whether there are weaknesses present that fail when stressed. hospitals in the u.s. navy medical department and a number of civilian hospitals in new york have had their level of preparedness assessed using the hospital emergency analysis tool (heat). the heat examines more than factors that contribute to effective emergency preparedness and response. after the systematic analysis by a team of experts, the hospital receives an after-action report that documents strengths and weaknesses and permits the development of a strategic plan to improve preparedness. • staff training, exercise, and continuous improvement: the joint commission on accreditation of healthcare organizations requires hospital staff members involved in the execution of the emergency management plan to receive orientation and education relative to their role in an emergency. exercise of the emergency plan is also required. lessons learned should be integrated into plans to continuously revise them. a commitment to the following philosophies will enhance hospital emergency preparedness: • imagine the unimaginable: when flood waters rise in a community, when a tornado touches down and demolishes an elementary school, when a disgruntled hospital employee opens fire with an automatic weapon in the emergency department, when a passing train derails and spills toxic chemicals, or when a wildfire closes in, it is too late to update an old plan, train staff to respond effectively, check phone numbers, and stock disaster supplies. disaster complacencybelieving a problem won't happen to you or your hospital-is a significant threat to effective planning and response. • protect the staff: only a true obsession with self-protection will ensure that staff members are not injured or become ill during disaster response. adequate stockpiles of gloves, masks, and other equipment must be available, along with training and leadership commitment to self-protection policies. • build in redundancy: expect the primary plan to fail and build in alternatives to every emergency measure. • rely on standard procedures whenever possible: people perform best in unusual situations when they perform activities that closely mirror what they do under normal conditions. • maintain records: patient care records are critical to obtaining reimbursement for disaster care provided. • plan to degrade services: normal levels of services cannot be maintained during disaster response. identify services, such as elective surgery, that can be temporarily curtailed or minimized so that personnel and resources can be reassigned. the federal government has implemented programs to augment local and state capabilities when they are overwhelmed. the united states has a well-established emergency medical safety net: the national disaster medical system (ndms). the ndms has two primary capabilities designed to enhance disaster medical response. the first is specialized disaster response teams who augment the medical emergency response at the site of disaster. the second ndms capability is a plan to share the inpatient bed capacity of the civilian and federal health systems in the event either system is overwhelmed with patients requiring inpatient care. ndms federal coordinating centers (fccs) play a regional role in maintaining a supply of ndms hospital members and providing training and exercises. when the ndms is activated, fccs coordinate patient reception and distribution of patients being evacuated. hospitals enter into a voluntary agreement to participate in the ndms. they must be accredited and generally have more than beds. the agreement commits a hospital to provide a certain number of acute care beds to ndms patients; however, it is recognized that hospitals may or may not be able to provide the agreedupon number of beds. hospitals that receive ndms patients are reimbursed for care by the federal government. the strategic national stockpile the strategic national stockpile (sns) was established in as the national pharmaceutical stockpile. it is now managed by the u.s. department of homeland security and serves as a national repository of antibiotics, chemical antidotes, antitoxins, intravenous therapy, airway management equipment, and medical/surgical items. the stockpile is designed to supplement local agencies that are overwhelmed by a health emergency. the noble training center in anniston,ala., (on the site of the former fort mcclellan army base) is the only hospital facility in the united states that trains healthcare professionals in disaster preparedness and response. the department of homeland security operates the noble training center, which offers a variety of training programs, including one for hospital leadership. more information is available online at: http://training.fema. gov/emiweb/ntc/. even though the federal government has many emergency response assets that can help in the response to an emergency, experience has shown that hospitals must be prepared to be self-sufficient for to hours after an event. a comprehensive hospital emergency management program must address a number of critical elements to adequately protect patients and staff and permit the facility to continue to operate. these are discussed in the following. just as one team leader is necessary for a controlled response to a cardiac arrest, an organized approach is essential to a successful hospital-wide emergency response. the hospital emergency incident command system (heics) is designed to provide that coordination. developed and tested in orange county, calif., in , it provides structure to response. heics uses: • a reproducible, predictable chain of command • a flexible organizational design that can be scaled to the scope of the problem • checklists for each position to simplify response and carefully define each task • a common language that permits communication with outside agencies the eoc will serve as the command post for operations during an emergency response. it should be fully operational and integrated into local and county emergency operations (box - ). hospital disaster drills have often been treated as annoyances and are planned in ways to render them futile. exercises are generally announced (unlike actual events), planned during regular business hours, and rarely include all hospital operations that will be affected by an actual event. hospitals are encouraged to drill individual units-frequently and during nights and weekends-and then build up to full, functional exercises involving management of moulaged "casualties." community participation is critical to identify elements that work or that need fine-tuning. only through exercise will the plan be adequately stressed so that failure points are identified. the facility's structural integrity and essential services are an often overlooked part of preparedness. box - recommended equipment and supplies for a hospital eoc (hvac) system so that it can be shut down and, ideally, so that specific zones can be manipulated to control airflow in the building in case of contamination • maintain a fuel source for full-load demand for to days' duration • develop a plan for the management and disposal of increased volumes of contaminated waste maintaining the physical security of the structure is important on a daily basis but becomes more of a challenge during a disaster. to ensure that the environment remains safe, egress must be controlled. additional elements of the physical security plan should include the following: • a security force with full-time security responsibilities; the force should have undergone criminal background checks and professional law enforcement training. • all entrances and exits should be controlled, monitored, and capable of being locked. • the hospital should be able to perform perimeter security protection ("lockdown") within minutes of notification. • hospital staff should be trained and drilled on the performance of lockdown. • hospital leadership should know what triggers the execution of a lockdown procedure. • a plan should exist for supplementing security staff in a disaster. it is critical that a hospital be able to rapidly assess the impact of a disaster on its operations and communicate the status to leadership in a situation report (often referred to as a "sitrep"), or a rapid needs assessment (ran). the assessment should, at a minimum, include the following: • the extent and magnitude of the disaster and the scope and nature of casualties • the status of operations and any disrupted critical services • the impact of disruptions on operations and the ability to sustain operations hospital staff must be able to receive timely and accurate notifications in a disaster, including when and where to report and for how long and other essential information. contact information for all staff members must be continuously updated and tested. additionally, the facility must be able to receive warnings and notifications from external agencies and be able to send warnings. triage is performed daily in emergency departments, where the most critical are treated first. but during a dis-aster, triage procedures must adapt to become like what is used on the battlefield, where the greatest good is offered to the greatest number. multiple disaster triage systems exist, including start (simple triage and rapid treatment), id-me (immediate, delayed, minimal, expectant), and mass (move, assess, sort, and send). it is important that a hospital use a system that is consistent with what is being used by services delivering patients to the facility. whatever system is selected, there must be predisaster training and exercises. when casualties present to an emergency department in numbers that overwhelm the facility, an alternative area must be available to manage overflow. the alternative triage area should be lit so that it can be used at night, weatherproofed, and temperature-controlled. a plan for working with the media will be needed. it is not recommended that media personnel be permitted access to a hospital during a disaster, but rather be provided regular, factual updates on activities and the status of the facility at a predetermined meeting place. risk communications involve using credible experts to deliver carefully worded messages to communicate most effectively in a high-stress, low-trust environment, such as a disaster. preparing hospital leaders in risk communications principles will ensure that they are able to communicate effectively to the public via the media. there is conflicting evidence about the value of certain types of mental health services in the wake of disaster, but it is clear that every disaster creates emotional trauma victims. primary victims are those who have been directly affected by the disaster. secondary victims are rescue workers in whom symptoms develop, and tertiary victims are relatives, friends, and others who have been affected. the critical incident disrupts a victim's sense of control as daily life is abruptly changed. hospitals must plan for providing mental health services to disaster victims but must also consider the needs-acute and long-term-of the hospital staff who attempt to respond to an overwhelming event. it is recommended that hospitals have trained crisis intervention teams that are well integrated into the emergency management plan. in the event of an intentional act that results in mass casualties, not only must a hospital care for the victims, but it has a critical role in bringing perpetrators to justice. hospital staff members require training in proper management of potential evidence-in both collection and preservation. evidence collection containers, including -gallon drums for patient decontamination run-off, should be available as well as bags to preserve other types of evidence. law enforcement agencies and forensic departments can provide training and guidance. staff members should be familiar with and follow procedures for maintaining chain of custody for evidence that is collected during patient care activities. a disaster will place significant demands on the food service system of a hospital. the adequacy of food supplies for patients and staff should be evaluated. because a hospital may need to be self-sufficient for several days in a disaster, a -to -day supply of food products is advisable. food service personnel should be included in disaster exercises. volunteers may or may not be of assistance, depending on their relationship with the hospital and their background. a volunteer pool that consists of individuals who serve regularly at the facility, are familiar with standard procedures, and participate in exercises can add valuable manpower to a disaster response effort. on the other hand,disasters will draw volunteers who wish to assist,a phenomenon known as "convergent volunteerism," in which unexpected and uninvited healthcare workers arrive and wish to render assistance at a large-scale incident. these "freelancers" may cause problems or may even be impostors. despite "just-in-time" supply schedules and empty warehouses, hospitals should maintain dedicated disaster supplies and arrangements for rapid resupply in the event of a disaster. disaster response will rapidly deplete critical supplies-administrative as well as clinical. conducting realistic exercises will help with the determination of the adequacy of stock and can be done without opening actual supplies so they can be restocked. disaster supplies can be rotated into the daily-use stream to ensure stock does not expire. experience with disasters has demonstrated a number of predictable pitfalls that occur in hospital disaster response. because immediate on-scene control of a disaster is chaotic and communication is often problematic, patients will present to the closest hospital available. this often leaves other nearby facilities with capacity and personnel that go unused. hospital personnel must be experts in protecting themselves, or they will become part of the problem and fur-ther stress the facility. some controversy exists over the level of protection needed in certain environments, but it is clear that masks (n ) and gloves (latex or nonlatex) will prevent transmission of biological agents. communications failure has often been identified as a predictable failure in disaster response. hospitals need to examine both internal communications systems (with staff and patients) and with external agencies. multiple layers of redundancy are essential to deal with expected failures and include the use of -mhz radios, dedicated trunk lines in the emergency operations center, two-way communications for hospital units and essential personnel, communications-on-wheels (cows), and access to amateur radio (ham) operators. the last resort is using runners who carry messages. hospitals must be able to identify and decontaminate patients who have been exposed to radiation or a compound that poses a threat to the patient's health and the safety of the facility. if the hospital depends on an external agency or has decontamination equipment that requires time to set up, an immediate alternative must be in place, such as a hose and hose bib outside of the emergency department. consideration should be given to patient privacy, managing patient valuables and clothes, and handling weapons brought into the hospital. a trained, exercised, and well-equipped team will be the foundation of successful efforts. hospitals will benefit from having a plan to care for children and other dependents of staff. in a disaster, staff will be called on to work extended hours, and usual family care arrangements may be unavailable. the creation of emergency patient admission packs that are maintained with disaster equipment will facilitate the admission of a large number of patients. if an automated patient tracking system is used, a back-up manual system should be available. all systems should be able to manage unidentified (john and jane doe) patients. many hospitals have wholly inadequate or nonexistent plans to manage mass fatalities. morgue space is generally limited in most facilities, so additional surge capacity must be identified in advance. arrangements for refrigerated storage trucks, refrigerator space, and other alternatives, including ice rinks, should be addressed with socially sensitive plans. complex cultural and religious issues may come into play in the event that there are contaminated remains and should be examined in advance. emergency drugs must be available at the point of care. often they are secured in pharmacy departments or warehouses, resulting in precious minutes of life-saving time being lost as personnel try to locate and obtain critical medications. in addition to drugs needed to respond quickly to nerve agents and other emergency situations, stockpiles of antibiotics should be maintained to provide prophylaxis to patients and staff. in a disaster, patients converge on the place they know they can obtain care-the hospital-and they arrive using any means possible. furthermore, with the victims of disaster, come their families, loved ones, and the media-all who have very important needs that must be addressed. hospitals can no longer approach disaster planning with a minimalist attitude that relies heavily on luck and belief that it will be someplace else that gets hit by the disaster. the hospital that received the most patients from the rhode island nightclub fire got lucky-the victims began arriving during a change of shift so there were two shifts of nursing staff available. however, the hospital also attributes its effective response to having drilled critical departments and procedures. emergency planning is the backbone of preparedness, but events will occur in each disaster that demand creative responses under pressure. this ability to respond flexibly is known as planned innovation. good plans will use general "all hazards" templates for disaster management but will permit independent initiative and a tailored response to a specific situation. the u.s. health system appears to be emerging from the dark ages of emergency planning. a minimalist attitude of preparedness was acceptable in the past despite the regular occurrence of natural disasters. the threat of terrorism and the resulting health system impact have stimulated investment in research, a resurgence of disaster training in nursing and medical schools, and visionary projects such as er one. er one is a national prototype for a next-generation emergency department. located in washington, d.c., it is developing new approaches to the medical consequences of terrorist attacks, natural disasters, and emerging illnesses. more information is available online at: http://er .org. the next phase of hospital emergency management will be a renaissance if creative planning prevails over naysayers, if resources are applied to priority preparedness activities, and if healthcare leaders are committed to ensuring that all who depend on hospitals will receive the care they need in a disaster (box - ). the care of strangers-the rise of america's hospital system combined external and internal hospital disaster: impact and response in a houston trauma center intensive care unit implications of hospital evacuation after the northridge, california, earthquake lessons learned from the activation of a disaster plan: / two new york city hospitals' surgical response to the a test of preparedness and spirit emergency department impact of the oklahoma city terrorist bombing tragedy and response-the rhode island nightclub fire the station nightclub fire and disaster preparedness in rhode island mass decontamination: why re-invent the wheel? meeting new challenges and fulfilling the public trust: resources needed for hospital emergency preparedness recurring pitfalls in hospital preparedness and response pan american health organization. principles of disaster mitigation in health facilities health care at the crossroads-strategies for creating and • auf der heide e. principles of hospital disaster planning department of health and human services. (includes recommendations on prehospital and hospital care preparing for the psychological consequences of terrorism-a public health strategy. this publication of the national academies of science includes an examination of current infrastructure and response strategies guide to emergency management planning in health care regional care model for bioterrorist events • the hospital emergency incident command system • the international critical incident stress foundation box - hospital preparedness and response resources sustaining community-wide emergency preparedness systems dvatex: navy medicine's pioneering approach to improving hospital emergency preparedness advanced disaster medical response-manual for providers. boston: harvard medical international trauma and disaster institute critical incident stress making room for outside the box thinking in emergency management and preparedness key: cord- - yiuuye authors: mims, cedric a.; dimmock, nigel j.; nash, anthony; stephen, john title: mechanisms of cell and tissue damage date: - - journal: mims' pathogenesis of infectious disease doi: . /b - - - - . - sha: doc_id: cord_uid: yiuuye nan the impact on the host of microbial damage depends very much on the tissue involved. damage to muscle in the shoulder or stomach wall, for instance, may not be serious, but in the heart the very existence of the host depends on a strong muscle contraction continuing to occur every second or so, and here the effect of minor functional changes may be catastrophic. the central nervous system is particularly vulnerable to slight damage. the passage of nerve impulses requires normal function in the neuronal cell membrane, and viruses especially have important effects on cell membranes. also a degree of cellular or tissue oedema that is tolerable in most tissues may have serious consequences if it occurs in the brain, enclosed in that more or less rigid box, the skull. therefore, encephalitis and meningitis tend to cause more severe illness than might be expected from the histological changes themselves. oedema is a serious matter also in the lung. oedema fluid or inflammatory cell exudates appear first in the space between the alveolar capillary and the alveolar wall, decreasing the efficiency of gaseous exchanges. respiratory function is more drastically impaired when fluid or cells accumulate in the alveolar air space.* the effect of tissue damage is much less in the case of organs such as the liver, pancreas or kidney, which have considerable functional reserves. more than two-thirds of the liver must be removed before there are signs of liver dysfunction. cell damage has profound effects if it is the endothelial cells of small blood vessels that are involved. the resulting circulatory changes may lead to anoxia or necrosis in the tissues supplied by these vessels. here too, the site of vascular lesions may be critical, effects on organs such as the brain or heart having a greater impact on the host, as discussed above. rickettsiae characteristically grow in vascular endothelium, and this is an important mechanism of disease production. by a combination of direct and immunopathological factors there is endothelial swelling, thrombosis, infarcts, haemorrhage and tissue anoxia. this is especially notable in the skin, and forms the basis for the striking rashes in typhus and the spotted fevers. these skin rashes, although important for the physician are less important for the patient than similar lesions in the central nervous system or heart. it is damage to cerebral vessels that accounts for the cerebral disturbances in typhus; involvement of pulmonary vessels causes pneumonitis, and involvement of myocardial vessels causes myocardial oedema. in q fever, rickettsiae sometimes localise in the endocardium, and this causes serious complications. sometimes an infectious agent damages an organ, and loss of function in this organ leads to a series of secondary disease features. the signs of liver dysfunction are an accepted result of infections of the liver, just as paralysis or coma is an accepted result of infection of the central nervous system. diabetes may turn out to be caused by infection of the islets of langerhans in the pancreas. coxsackie and other virus infections of the islets of langerhans can certainly cause diabetes in experimental animals, and coxsackieviruses have been associated with juvenile diabetes in man. there are many diseases of unknown aetiology for which an infectious origin has been suggested. sometimes it is fairly well established that an infectious agent can at least be one of the causes of the disease, but in most instances it is no more than a hypothesis, with little or no good evidence. for conditions as common and as serious as multiple sclerosis, cancer and rheumatoid arthritis it would be of immense importance if a microorganism were incriminated, since this would give the opportunity to prevent the disease by vaccination. accordingly, there is a temptation to accept or publicise new reports even though the evidence is weak or the observations poorly controlled. as if to warn us about this and remind us of possibilities from environmental toxins, parkinson's disease, a chronic neurological condition in which there is a loss of neurons in a sharply defined region of the brain (substantia nigra), can be caused by exposure to the chemical mptp. one example which raises the possibility that subtle cns disturbances may be caused by viruses is experimental infection with borna disease virus. this virus was used to infect tree shrews (tupaia glis) which are primitive primates. there is little overt disease, but afterwards the male is no longer able to enact the ritual courtship behaviour which (as students well know), is an essential preliminary to mating in all primates, and the frustrated male usually ends up bitten by the female. thus it can be said that infection with borna disease virus renders the male psychologically sterile. presumably the virus in some way alters the functioning of neurons concerned in this particular pathway. all other behavioural and physiological aspects appear normal. borna disease virus is not known to occur in man, but speculation about an analogous human situation is fuelled by the finding of borna disease virus-specific antibodies in patients with psychiatric/ behavioural disorders. in an entirely different clinical context, infection of a particular strain of rats with borna disease virus causes immense obesity, the underlying physiological basis of which is not understood. since the aetiology of such diseases raises interesting problems in pathogenesis, the present state of affairs is summarised in table . , which includes some of the human diseases whose infectious origin is probable, possible, conceivable, or inconceivable. causal connections between infection and disease states are particularly difficult to establish when the disease appears a long time after infection. it was not too difficult to prove and accept that the encephalitis that occasionally occurs during or immediately after measles was due to measles virus. but it was hard to accept that a very rare type of encephalitis (subacute sclerosing panencephalitis or sspe), occurring up to years after apparently complete recovery from measles, was also due to measles virus and this was only established after careful studies and the eventual difficult isolation of a mutant form of measles virus from brain cells. 'slow' infections, in which the first signs of disease appear a long time after infection are now an accepted part of our outlook. the disease kuru occurred in new guinea and was transmitted from person to person by cannibalism. the incubation period of the disease in man appears to be - years, and was caused by a nonisolatable infectious agent that grew in the brain. this was established when the same disease appeared in monkeys several years after the injection of material from the brain of kuru patients. a similar agent called scrapie (see ch. ) infects sheep, mice and other animals and also has an incubation period representing a large portion of the life span of the host. in both kuru and sspe the agent was eventually shown to be present in the brains of patients. so far this has only been demonstrated indirectly as, despite strenuous efforts, the causative agent has yet to be isolated. if in a slow infection the microorganism that initiated the pathological process is no longer present by the time the disease becomes manifest, then the problem of establishing a causal relationship will be much greater. this may possibly turn out to be true for diseases like multiple sclerosis and rheumatoid arthritis. liver cancer in humans and leukaemia in mice, cats, humans and cattle can be caused by slow type virus infections. cancer or leukaemia appears as a late and occasional sequel to infection. the virus, its antigens or fragments of its nucleic acid are detectable in malignant cells. one important factor that often controls the speed of an infectious process and the type of host response, is the rate of multiplication of a microorganism.* different infectious agents show doubling times * every infection is a race between the spread and multiplication of the microbe and the generation of an antimicrobial response by the host. a day or two's delay in this response may let the microbe reach the critical levels of growth that give tissue damage and disease. varying from min to weeks, and some of these are listed in table . . often the rate of multiplication in the infected host, in the presence of antimicrobial and other limiting factors and when many bacteria are obliged to multipy inside phagocytic cells, is much less than the optimal rate in artificial culture. clearly a microorganism with a doubling time of a day or two will tend to cause a more slowly evolving infection and disease than one that doubles in an hour or less. it is uncommon for an infectious agent to cause exactly the same disease in all those infected. its nature and severity will depend on infecting dose and route, and on the host's age, sex, nutritional status, genetic background, and so on. many infections are asymptomatic in more than % of individuals, clinically characterised disease occurring in only an occasional unfortunate host, as 'the tip of the iceberg'.* asymptomatically infected individuals are important because they are not identified, move normally in the community, and play an important part in transmission. this chapter deals with demonstrable cell and tissue damage or dysfunction in infectious diseases. but one of the earliest indications of illness is malaise, 'not feeling very well'. this is distinct from fever or a specific complaint such as a sore throat and although it is difficult to define and impossible to measure, we all know the feeling. it can precede the onset of more specific signs and symptoms, or accompany them. sometimes it is the only indication that an infection is taking place. almost nothing is known of the basis for this feeling. toxins', of course, have been invoked and the earliest response to pyrogens (see pp. - ) before body temperature has actually risen, may play a part. interferons may have something to do with it because pure preparations of human a or ß interferons cause malaise and often headaches and muscle aches after injection into normal individuals. if interferon is eventually recognised as an important antimicrobial force we may have to regard these side effects as unfortunate but acceptable. soluble mediators of immune and inflammatory responses, such as interleukin- (see glossary) or other cytokines may also play a part. in some infectious diseases weakness and debility are prominent during convalescence. this can be especially notable following influenza and hepatitis, but its basis is as mysterious as in the case of malaise. the infections that matter are those causing pathological changes and disease. before giving an account of the mechanisms by which these changes are produced, it is important to remember that many infectious agents cause little or no damage in the host. indeed, it is of some advantage to the microorganism to cause minimal host damage, as discussed in ch. . virus infections as often as not fall into this category. thus, although infection with rabies or measles viruses nearly always causes disease, there are many enterovirus, reovirus and myxovirus infections that are regularly asymptomatic. even viruses that are named for their association with disease (poliomyelitis, influenza, japanese b encephalitis) often give an antibody response as the only sign of infection in the host. tissue damage is too slight to cause detectable illness. there is also a tendency for persistent viruses to cause no more than minor or delayed cellular damage during their persistence in the body, even if the same virus has a more cytopathic effect during an acute infection, e.g. adenoviruses, herpes simplex (see ch. ). a few viruses are remarkable because they cause no pathological changes at all in the cell, even during a productive infection in which infectious virus particles are produced. for instance, mouse cells infected with lcm or leukaemia virus show no pathological changes. a mouse congenially infected with lcm virus shows a high degree of immune tolerance, and all tissues in the body are infected. throughout the life of the animal, virus and viral antigens are produced in the cerebellum, liver, retina etc. without discernible effect on cell function. but sometimes there are important functional changes in infected cells which lead to a pathological result. for example, the virus infects growth hormoneproducing cells in the anterior pituitary. although the cells appear perfectly healthy, the output of growth hormone is reduced, and as a result of this, suckling mice fail to gain weight normally and are runted. when bacteria invade tissues, they almost inevitably cause some damage, and this is also true for fungi and protozoa. the extent of direct damage, however, is sometimes slight. this is true for treponema pallidum, perhaps because the lipopolysaccharide-protein components that might have induced inflammatory responses, are not exposed on the surface of the bacteria. it produces no toxins, does not cause fever, and attaches to cells in vitro without harmful effects. leprosy and tubercle bacilli eventually damage and kill the macrophages in which they replicate, but pathological changes are to a large extent caused by indirect mechanisms (see below). in patients with untreated lepromatous leprosy, the bacteria in the skin invade blood vessels, and large numbers of bacteria, many of them free, may be found in the blood. in spite of the continued presence of up to bacteria m l - of blood there are no signs or symptoms of septicaemia or toxaemia. mycobacterium leprae can be regarded as a very successful parasite that induces very little host response in these patients, even when the bloodstream is invaded. the resident bacteria inhabiting the skin and intestines of man and animals do not invade tissues and are normally harmless; indeed, as discussed in ch. , they may benefit the host. bacteria such as meningococci and pneumococci, whose names imply pathogenicity, spend most of their time as harmless inhabitants of the normal human nasopharynx: only occasionally do they have the opportunity to invade tissues and give rise to meningitis or pneumonia. cell and tissue damage are sometimes due to the direct local action of the microorganism. however, it is not at all clear how viruses cause the death of cells. many virus infections result in a shutdown of rna synthesis (transcription), protein synthesis (translation) and dna synthesis in the host cell, but usually these are too slow to account for the death of the cell. after all, cells like neurons never synthesise dna, and the half life of most proteins and even rnas is at least several hours. a possible alternative mechanism is the alteration of the differential permeability of the plasma membrane. this is important as the cell has a high internal k + concentration and low n a + concentration, while the reverse is true of body fluids. viruses do alter membrane permeability, but the unresolved question is whether or not this is responsible for the death of the cell or is merely an after-effect. it now appears that at least some virus infections (hiv, adenovirus, influenza virus, sindbis virus) cause the cells to commit suicide by a mechanism called 'programmed cell death' or 'apoptosis'. this is the natural process by which the body controls cell numbers and rids itself of superfluous or redundant cells during development. a familiar example is a tadpole 'losing' its tail. cells do not disintegrate but round up, and are then removed by phagocytes. a possible example of apoptosis is seen in the common cold when caused by the rhino virus group of picorna viruses. rhinoviruses infect nasal epithelial cells, and at an early stage the cells round up, fall off the mucosal surface and are carried away, often with their cilia still beating, in a stream of fluid induced by the infection.* this leaves areas of raw mucosa, with the exposed underlying tissues inflamed, oedematous, and susceptible to infection by the normally harmless resident bacteria. the gross pathology of cells infected with viruses appears generally nonspecific, very like that induced in cells by the toxins of diphtheria bacilli and streptococci, or by physical and chemical agents. the most common and potentially reversible change, the oedema seen as 'cloudy swelling' by routine histology, is associated with membrane permeability changes. changes in the endoplasmic reticulum, mitochondria and polyribosomes are seen by electron microscopy at this stage. later the nuclear chromatin moves to the edge of the nucleus ('margination' of chromatin) and becomes condensed (pycnosis), but the cell has already died by this time. there are two more characteristic types of morphological change produced by certain viruses, and these were recognised by histologists more than years ago. the first are inclusion bodies, parts of the cell with altered staining behaviour which develop during infection. they often represent either cell organelles or virus factories in which viral proteins and/or nucleic acids are being synthesised and assembled. herpes group viruses form intranuclear inclusions, rabies and poxviruses intracytoplasmic inclusions, and measles virus both intranuclear and intracytoplasmic inclusions. the second characteristic morphological change caused by viruses is the formation of multinucleate giant cells. this occurs, for instance when hiv 'fusion' proteins (gpl -gp ) present in nascent virus particles budding from an infected cell attach to cd receptors in the plasma membranes of neighbouring cells; membranes then fuse and multinucleate cells are formed. it also happens in measles and certain herpes virus infections. before leaving the subject of direct damage by viruses, one supreme example will be given. here the direct damage is of such a magnitude that the susceptible host dies a mere six hours after infection. if rift valley fever virus, an arthropod-borne virus infecting cattle, sheep and man in africa, is injected in very large doses intravenously into mice, the injected virus passes straight through the kupffer cells and endothelial cells lining liver sinusoids (see ch. ) and infects nearly all hepatic cells. hepatic cells show nuclear inclusions within an hour, and necrosis by four hours. as the single cycle of growth in hepatic cells is completed, massive liver necrosis takes place, and mice die only six hours after initial infection. the host defences in the form of local lymph nodes, local tissue phagocytes etc. are completely overcome by the intravenous route of injection, and by the inability of kupffer cells to prevent infection of hepatic cells. direct damage by the replicating virus destroys hepatic cells long before immune or interferon responses have an opportunity to control the infection. this is the summit of virulence. the experimental situation is artificial but it illustrates direct and lethal damage to host tissues after all host defence mechanisms have been overwhelmed. most viruses, rickettsiae and chlamydia damage the cells in which they replicate, and it is possible that some of this damage is due to the action of toxic microbial products. this action, however, is confined to the infected cell, and toxic microbial products are not liberated to damage other cells. mycoplasma (see table a . ) can grow in special cell-free media, but in the infected individual they generally multiply while attached to the surface of host cells. as studied in culture and on the respiratory epithelium they 'burrow' down between cells, inhibit the beat of cilia and cause cell necrosis and detachment. the mechanism is not clear. if a complete lawn of mycoplasma covers the surface of the host cell, some effect on the health of the cell is to be expected, but it is possible that toxic materials are produced or are present on the surface of the mycoplasma. dental caries provides an interesting example of direct pathological action. colonisation of the tooth surface by streptococcus mutans leads to plaque formation, and the bacteria held in the plaque utilise dietary sugar and produce acid (see p. ). locally produced acid decalcifies the tooth to give caries. caries, arguably the commonest infectious disease of western man, might logically be controlled by removing plaque, withholding dietary sugar, or vaccinating against streptococcus mutans. however, fluoride in the water supply or in toothpaste has been the method of choice, and has been very successful. it acts by making teeth more resistant to acid. bacteria generally damage the cells in which they replicate, and these are mostly phagocytic cells (see ch. ). listeria, brucella and mycobacteria are specialists at intracellular growth, and the infected phagocyte is slowly destroyed as increasing numbers of bacteria are produced in it. bacteria such as staphylococci and streptococci grow primarily in extracellular fluids, but they are ingested by phagocytic cells, and virulent strains of bacteria in particular have the ability to destroy the phagocyte in which they find themselves, even growing in the phagocytes, as described in ch. . many bacteria cause extensive tissue damage by the liberation of toxins into extracellular fluids. various toxins have been identified and characterised. most act locally, but a few cause pathological changes after spreading systemically through the body. this is a huge and growing part of our subject and we need to define the term toxin, a task which is more difficult than one might think. an attempt was made by bonventre who in defined toxins as a 'special class' of poisons which differ from, for example, cyanide or mercury by virtue of their microbial origin, protein structure, high molecular weight, and antigenicity. this view is too embracing, because it includes proteins of doubtful significance in disease, and also too restrictive, because it excludes nonprotein toxic complexes such as endotoxin. another suggestion is that toxin must include all naturally occurring substances (of plant, animal, bacterial or whatever origin) which when introduced into a foreign host are adverse to the well-being or life of the victim. this, too, is unsatisfactory because some substances-potent toxins within the scope of this definition-are being used in some contexts as therapeutic agents! perhaps it is pointless to strive for an all-embracing definition, although the obvious differences between bacterial and fungal toxins warrant the continued use of the appropriate prefix. for example, bacterial toxins are usually of high molecular weight and hence antigenic, whereas fungal toxins tend to be low molecular weight and not antigenic. the problem of definition is compounded because there are substances (aggressins) which help to establish an infective focus as well as those whose action is uniquely or largely responsible for the disease syndrome. also there are substances known to be produced by bacteria in vitro, whose properties on a priori grounds make them potential determinants of disease, but which have not been shown to play a role in vivo. we will concentrate on those toxins known to be, or likely to be, involved in some aspect of disease causation. it is not difficult to demonstrate the production of'toxins' by bacteria in culture, as judged by some bioassays. primary consideration will be given to those substances which are produced under ecologically significant conditions (i.e. in the natural host or relevant animal model) and cause (also in biologically relevant systems) damage to cells or tissues thereby contributing to disease. exotoxins are produced and then either secreted by, or released upon lysis from, both gram-positive and gram-negative bacteria. they are proteins, some of which are enzymes. when liberated locally they can cause local cell and tissue damage. those that damage phagocytic cells and are therefore particularly useful to the microorganism have been described in ch. . those that promote the spread of bacteria in tissues have been referred to in ch. . a description of some of the more interesting exotoxins follows. proteases and hyaluronidases, which help the spread of bacteria through tissues have already been mentioned in ch. . here we consider toxins which act on extracellular substances and are responsible for many of the main features of the diseases caused by the infecting organism. pseudomonas aerwgmosa-elastase, and one of at least six proteases of legionella pneumophila, both induce fibrinopurulent exudation in the rat lung (a model for p. aeragmosa-induced pneumonia in human cystic fibrosis) and the guinea-pig lung (a model for legionnaires' disease) respectively. these characteristics almost certainly arise from the release of oligopeptides from extracellular matrix components of the host which are chemotactic for leucocytes and fibroblasts. the l. pneumophila protease is the same major secretory protein (the m r zinc metalloprotease) already considered in ch. in relation to survival within macrophages. staphylococcal exfoliatin is important in staphylococcal 'scalded skin syndrome' (ssss), a disease of newborn babies. the disease is characterised by a region of erythema which usually begins around the mouth and, in - days, extends over the whole body. during this period, small yellowish exudative lesions often appear. the most striking feature of the disease, however, is that the epidermis, although apparently healthy, can be displaced and wrinkled like the skin of a ripe peach by the slightest pressure. soon large areas of the epidermis become lifted by a layer of serous fluid and peel at the slightest touch. large areas of the body rapidly become denuded in this way and the symptoms resemble those of massive scalding. while the toxin causes cleavage of desmosomes (specialised cell membrane thickenings through which cells are attached to each other) in the stratum granulosum, it may also act intracellularly. despite numerous attempts to characterise the biological activity of exfoliatin, the genetically predicted serine protease and/or lipase activity has never been demonstrated. some enterotoxigenic e. coli elaborate families of low molecular weight heat stable (st) peptides as well as heat labile (lt; cholera-like) toxin. sts bind to a receptor which then activates a tightly coupled membranebound guanylate cyclase in gut cells, resulting in the transmission of a signal to the inside of the cell, thereby elevating cgmp, or some other second message. as described later in the section on diarrhoea this gives rise to efflux of ions, and hence water, from enterocytes. we know that these toxins are able to traverse membranes since their targets are intracellular; we still know very little about the details of how they achieve this. these proteins have common features: one component (usually described as the b fragment or subunit) binds to and interacts with a cell receptor and promotes the uptake of an active component (a fragment or subunit) in which resides the biological activity which confers toxicity. there are several ways in which one can classify these toxins. ( ) biologically-some kill cells (cytotoxic toxins) whereas others (sometimes called cytotonic toxins) 'deregulate' cells. ( ) biochemically-some belong to a well defined biochemical group recognised by their ability to cleave nad + into nicotinamide and adp-ribose moieties. they are designated adp-ribosyl (adpr) transferases since they transfer adpr to different target proteins; this group straddles groups and . ( ) genetically-the third possibility reflects the genetic origin of these toxins: (i) production from a single gene of a single peptide which undergoes post-translational modification into a and b fragments which are covalently linked; (ii) production from separate genes of a and b subunits which noncovalently associate into stable complexes; (iii) production from separate genes of different proteins which do not associate into stable complexes but which must act in concert to express toxicity. these are known as binary toxins; again, group straddles group . examples of each of these groups will be given illustrating how they work, their importance in disease, and surprisingly (!) how some of the most toxic substances known to man are being used or may be used for therapeutic purposes. diphtheria toxin (dt). corynebacterium diphtheriae organisms multiply on the epithelial surfaces of the body (nose, throat, skin) but do not penetrate deeply into underlying tissues. the infection on the body surface causes necrosis of mucosal cells with an inflammatory exudate and the formation of a thick 'membrane' (hence the name c. diphtheriae: gr., diphthera = membrane) and if the infection spreads into the larynx there may be respiratory obstruction. the toxin probably assists colonisation of the throat or skin by killing epithelial cells and polymorphs. active immunisation with diphtheria toxoid has made diphtheria a clinical rarity in developed countries. dt is disseminated from the infection site and has important actions, especially on the heart and nervous system. dt is encoded by lysogenic phage ß but its expression is controlled by the host bacterium under conditions of iron stress. it is synthesised and processed as shown in fig. . . there is intense interest in seeking to understand the mechanisms of uptake of the active moieties of diphtheria and other toxins whose target is intracellular. this is driven by the desire to understand fundamental mechanisms in cell biology and to develop selective cytotoxic therapies' in clinical medicine. the mechanism of internalisation of the active a under the acidic conditions of the endosome, some dt-a is released by reduction, dt-b undergoes conformational change and interacts with the endosomal membrane resulting in the opening of cation channels. the latter are not absolutely necessary, but their formation is associated with times greater uptake of dt-a. in contrast, most other toxins which enter the cytosol appear to do so by more complicated routes which initially involving endocytosis. shiga toxin (and ricin, a toxic plant lectin believed to have been located in the tip of an umbrella and used to poison a bulgarian spy!) are not translocated across the endosomal membrane but are transported to the trans-golgi network and all the way back to the endoplasmic reticulum. somewhere in the exocytotic pathway conditions exist for the translocation of shiga toxin (sht) into the cytoplasm. sht acts by modifying s ribosomal rrna. for cholera toxin, it used to be thought that a l ( fig. . ) moved down through the central hole of the ring-shaped complex of ct-b which was anchored to the membrane by five ganglioside gm binding sites. structural work on this family of proteins shows that this is not physically possible. the finding that the c-terminal sequence lysine-aspartateglutamate-leucine (the kdel* motif) in the a subunit of cholera toxin and related sequences in e. coli heat labile toxin and pseudomonas exotoxin a raises the possibility that transport to the endoplasmic reticulum may also be necessary for these a subunits to reach the cytoplasm. a new and exciting development is beginning. for decades, attempts have been made to make immunotoxins by coupling native toxins to antibodies specific to some surface antigen on tumour cells, with little practical success, as yet. however, scientists have now genetically engineered dt by substituting that portion of the dt structural gene encoding the native toxin receptor-binding domain with modified cdna encoding one of a variety of cytokines and growth factors including il- , il- , il- , egf and α-msh. the resultant fusion toxin bears a new 'cellular address', but retains all of the other biological properties of the native dt molecule as well as a three-dimensional structure nearly identical with native dt. the il- receptor-binding domain fusion toxin has undergone phase i/ii clinical trials for the treatment of il- receptor positive haematological malignancies (and other il- related conditions) and has been shown to be safe and well tolerated. such constructs are potent against a murine model of il- receptor positive lymphoma, and the hope is that a durable remission of disease will be achieved in humans. (an era of new 'magic bullets'?) p. aeruginosa exotoxin a. p. aeruginosa, already alluded to above, is common in soil and water and can occasionally be isolated from the faeces of normal, healthy individuals. it is virtually harmless for healthy adults, but its ability to multiply in almost any moist environment and its resistance to many antibiotics have made the bacterium a major cause of * the kdel motif is normally found in proteins which having been processed in the golgi are trapped by the endoplasmic reticulum which recognises the kdel motif. this prevents the protein being lost to the cell via exocytotic trafficking and results in its translocation into the cytoplasm. the schema shows a round of peptide elongation and illustrates the key role played by two enzymes, ef and ef . dta and pea each adp-ribosylates diphthamide (a modified histidine) in ef -gtp which can no longer translocate the newly elongated peptide from the a site to the p site. sht a fragment is a specific n-glycosidase which cleaves an adenine residue from near the '-prime end of the s ribosomal rna. this depurination results in failure of ef -dependent binding of aminoacyl-trna to site a and hence inhibits protein synthesis. poliovirus achieves selective inhibition of host protein synthesis at an earlier stage than is depicted here. host mrna is first modified (capped) then bound to the small ribosomal subunit; poliovirus mrna is not capped. the function of a cap-binding protein, which recognises and binds host mrna to the ribosome, is inhibited by a poliovirus virion protein thereby allowing differential translation of virus messenger rna. ef-Ια: nucleotide-binding protein. hospital-acquired infection, particularly among patients with impaired host defence mechanisms such as those with chronic illness, genetic immunodeficiencies, those under treatment with immunosuppressive drugs, or patients suffering from extensive burns. p. aeruginosa causes localised infection in the urinary tract, respiratory tract, burns and wound infections. in severely debilitated patients these localised infections may develop into general septicaemia, with mortality in such cases approaching %. the mechanisms of pathogenicity of p. aeruginosa are complicated and unclear because (unlike c. diphtheriae) the organism elaborates several potentially toxic extracellular products, including a phospholipase, several proteases, lipase, haemolysin, enterotoxin, lipopolysaccharide endotoxin, elastase, exoenzyme s (pes), and exotoxin a (pea); evidence definitely implicates the last three. elastase has already been mentioned above (p. ). pes is without question a virulence determinant, but at present its biological mode of action is not wholly understood. pea is in many ways like dt. it inhibits protein synthesis by cleavage of nad + and subsequent adp-ribosylation of ef at the same diphthamide residue, is synthesised predominantly during the decline phase of cell growth, and its production appears to be dependent upon the concentration of iron in the medium. its m r is , and it can be activated in cell-free systems by proteolysis, or by reduction with thiols in the presence of urea and other chaotropic agents. all the enzymic activity of the toxin resides in a proteolytically derived fragment (m r approximately ). however, dissimilarities exist between the two toxins. firstly, the active a fragment resides in the cooh-terminal portion of the molecule, whereas the diphtheria a fragment is at the nh -terminal end. secondly, the b fragment of pea recognises a different receptor to that of dt, and there are no sequence homologies or serological crossreactivity between the two toxins. shiga toxin. this toxin is a subunit protein with an ab type structure ( a subunit; b, an oligomer of subunits). certain biotypes of e. coli make vero toxins (vts) or shiga-like toxins (slts) which are identical (slt ) or near identical (slt ) with shiga toxin. the role of shiga toxin in bacillary dysentery is discussed below. slt-producing strains of e. coli are responsible for haemorrhagic colitis and haemolytic urea syndrome. very recent crystallographic studies have shown a remarkable similarity in organisational structure of the b subunits of shiga toxin and those of the cholera toxin family discussed below, despite the near complete absence of sequence homology. the b subunits attach to a ganglioside structure gb or gb (cholera toxin attaches to gm ) on susceptible cell surfaces. sht-a fragment is a specific n-glycosidase which cleaves an adenine residue from near the '-prime end of s ribosomal rna. this depurination results in failure of efl-dependent binding of aminoacyl-trna to site a and hence inhibits protein synthesis ( fig. . ). this is based on studies on cultured cells, and how such cytotoxic activity gives rise to the enterotoxic activity-watery diarrhoea seen in cases of dysentery preceding the bloody mode of action of actin-adp-ribosylating toxins. c. botulinum c toxin component binds to the cell membrane followed by c i. the latter is internalised and upsets the equilibrium between polymerisation and depolymerisation of actin. adp-ribosylation of actin inhibits its polymerisation and turns g-actin into a capping protein which binds to the fast growing (concave) ends of actin filaments. capping of the concave ends increases the critical concentration for actin polymerisation. since the slow-growing (pointed) ends of actin filaments are free, depolymerisation of actin occurs at these ends. released actin is substrate for the toxin and will be withdrawn from the treadmilling pool of actin by adpribosylation, i.e. trapped. both reactions will finally induce the breakdown of the microfilament network. (taken with permission of authors (k. aktorieseia/.) and publisher (springer-verlag gmbh & co. kg, heidelberg, germany) from botulinum c toxin. clostridium botulinum c toxin is not a neurotoxin but belongs to a family of cytotoxins {clostridium perfringens iota toxin, clostridium spiroforme toxin, clostridium difficile adp ribosyltransferase) whose common features are that they are binary toxins and that they target cytoskeletal actin. of these c. botulinum c is the best studied at present. component c i is the equivalent of the a subunit and c ii, the b subunit. they are produced in parallel although different strains produce different ratios. more c is produced during sporulation than in the vegetative phase of growth. c ii is activated by proteolysis to produce a m r protein which binds to cell surfaces and thereby exposes a binding site for c i (m r ca. ) either on the cell surface or on attached c ii. once inside, c i adp-ribosylates monomeric actin and not polymerised f actin. the substrate specificity is high. the toxin will modify a specific arginine residue present in certain isoforms of actin. neither subunit is toxic on its own, but together the ld dose for a mouse is ca. - ng. (other examples of such 'binary' activity include anthrax toxin and staphylococcal leucocidin). the two components may be injected into different sites or by different routes but toxic effects always occur at the site of injection of c ii. this mode of action, summarised in fig. . , is believed to underlie the biological manifestations of toxicity which, in rats, include hypotension, haemorrhaging in and fluid accumulation around the lungs as a result of vascular permeability changes. the toxin is currently being used as a tool in cell biology to study cytoskeletal systems and dissect mechanisms of cell signal transduction. cholera and related toxins. there is a growing number of toxins in this category. easily the most studied is cholera toxin (ct), a major research area for three decades, which was originally stimulated by the desire to make a more effective vaccine against cholera. ironically, effective immunity is best mediated by antibacterial antibodies which prevent colonisation of the gut with v. cholerae rather than antitoxin-neutralising antibodies. cholera toxin is an ab subunit toxin ( fig. . ). enterotoxigenic e. coli (etec) strains produce two heat labile toxins: lti and ltii. ltii exists as two minor variants, ltiia and ltiib, the latter requiring trypsin treatment for full activation in the y- adrenal cell assay. the b subunits of ct, ltiia and ltiib recognise and are bound to cell surface gangliosides gm , gdlb and gdi a respectively and mediate the translocation of a l subunits into the cytoplasm; lti binds to either gm or a glycoprotein. the same reaction occurs as was described above for dt: nad + is cleaved into nicotinamide and adp-ribose (adpr) moieties, with two important differences: ( ) adpr transferase is greatly enhanced by a group of proteins (both membrane bound and soluble) of ca. m r designated adpribosylation factors (arfs; of as yet unknown physiological significance); ( ) adpr is transferred to the a s subunit of the g s regulator of adenylate cyclase as illustrated in fig. . . this results in the elevation of camp levels in the cell with the consequences described below in the section on diarrhoea. bordetella pertussis toxin (pertussigen) and adenylate cyclasehaemolysin (ac-hly). whooping cough (pertussis) is a severe respiratory tract infection characterised by prolonged paroxysmal coughing, attacks of which continue long after infection has cleared. the disease is capable of striking all ages but is particularly prevalent and severe in young children, where hospitalisation is required in about % of cases. the causative agent of pertussis, bordetella pertussis, is transmitted aerially from the respiratory tract of an infected individual to that of a susceptible host. the organism attaches (probably via its filamentous haemagglutinin, pili, and a m r outer membrane protein) to, and colonises the mucosal surface between the cilia, and multiplies there during the incubation period of the disease which is commonly around seven days. the infection then manifests as a slight fever and catarrh which is often indistinguishable from a common cold. however, one to two weeks later bouts of uncontrollable coughing begin. it is this paroxysmal coughing, along with the notorious 'whoop' as the child attempts to draw breath, which characterises the disease. the paroxysmal coughing stage often lasts for several weeks and no treatment fully effective in controlling the symptoms. the only proven means of controlling whooping cough is vaccination but, in the uk at least, sporadic reports of vaccine-induced brain damage in infants has diminished public acceptance of the vaccine. it should be noted that permanent encephalopathy (brain damage) is a recognised though rare consequence of whooping cough infection. much current work is being devoted to producing immunogenic, completely nontoxic preparations of pertussis toxin by genetic manipulation of t h e gene encoding t h e s i s u b u n i t ( fig. . ). in clinical trials in italy, such engineered vaccines h a v e been shown to be both safe a n d effective a s j u d g e d by antibody titres to pertussis toxin. several toxic substances h a v e been isolated from bordetella pertussis including a h e a t labile toxin, tracheal cytotoxin, endotoxin, adenylate cyclase-haemolysin (ac-hly), a n d pertussis toxin. t h e latter h a s m a n y biological activities-histamine sensitisation, leucocytosis promotion, islet (a) the production of cyclic amp by adenyl cyclase. cyclic amp is an important second messenger invoked in the intracellular amplification of many cellular responses to external signals including hormones. the nature of the physiological response reflects the biochemical differentiation of the cell responding to the stimulus. for example, in gut cells the response would be altered ion transport and hence fluid secretion; in muscle cells it would be glycogen breakdown in response to the call for more energy. the production of camp is controlled both positively and negatively at two different levels. the central cycle represents a normal membranebound hormone receptor and the heterotrimeric (aß ) g protein regulator complex which is activated upon binding of hormone to the receptor. there are two receptors, each with regulatory g proteins: one system responds to stimulatory and the other responds to inhibitory stimuli; only one system is shown. in gut cells these receptors would be on the basolateral (nonluminal) side of enterocytes enabling responses to stimuli from the circulation. (b) the second level of control involves endogenous gtpase properties of both stimulatory (a s ) and the inhibitory (a a ) subunits of the g protein regulator which may be outlined as follows. stimulation: agonist stimulation of its receptor results in the dissociation of the a s ß g protein from the receptor and of the subunits from each other, and the binding of gtp to a s . a s -gtp will productively interact with the catalytic unit of adenyl cyclase (not shown) and stimulate the production of camp from atp. endogenous gtpase activity in a s results in a delayed conversion of a s -gtp to inactive a s -gdp which can no longer stimulate the cyclase but allows the reassociation of the trimeric g protein and loss of gdp. inhibition: antagonist stimulation of its receptor results in an analogous situation for inhibiting cyclase activity via c^: inhibitory cti-gtp inactivates the cyclase; cti-gdp does not inactivate the cyclase. (c) the level of camp may be affected by physiological stimuli (which stimulate removal and modification of the g-protein complex) or by perturbation of the normal regulatory cycle as illustrated, by cholera toxin (ct) or pertussis toxin (ptx). (d) cholera toxin acts first by interacting with its receptor resulting in the internalisation of the active a l subunit. a l adp-ribosylates a s -gtp which promotes continued dissociation of the heterotrimer; also the endogenous gtpase is no longer functional hence the stimulation of the cyclase continues. lt toxins act in a similar manner. (e) pertussigen acts first by interacting with its receptor resulting in the internalisation of the active si subunit. si adp-ribosylates the ai-gdpß -heterotrimer which can no longer associate with the receptor (or lose gdp) to undergo another cycle of gtp activation; activated cyclase can no longer be turned off. (adapted, with kind permission of author (gierschik, p.) and publisher (springer-verlag gmbh & co. kg, heidelberg, germany) from figure in 'adp-ribosylation of signal-transducing guanine nucleotide-binding proteins by pertussis toxin', current topics in microbiology andlmmunology ( ) , , edited by klaus aktories. activation and adjuvanticity-and is believed to be the most important but by no means the only virulence determinant of b. pertussis: it is called pertussigen and is the basis of the vaccine against whooping cough. pertussigen is a complex subunit toxin whose biochemical mode of action is identical to that of ct except that the target is the aj subunit of the gj regulator of adenylate cyclase (see fig. . ). it is not yet clear how such biochemical activity relates to the clinical syndrome. ac-hly has two functional domains. one confers adenylate cyclase activity and another haemolytic activity. its haemolytic activity arises from its pore-forming properties in cell membranes. pore formation is probably responsible for the translocation of the adenylate cyclase portion of the complex. ac-hly is also known as cyclolysin and belongs to the group of toxins whose common feature is the presence of an array of a nine amino acid repeats; they have been designated rtx (repeats in toxin) toxins. the prototype is the haemolysin of e. coli which is important in extraintestinal infections caused by this organism. other toxins of this group include the haemolysin from proteus vulgaris, and the leukotoxin from pasteurella haemolytica. anthrax toxin. anthrax is a disease of animals, particularly sheep and cattle, and to a lesser extent man, caused by infection with bacillus anthracis. infection takes place following the ingestion of spores, the inhalation of spores, or by the entry of spores through abraded skin. the spores germinate and then the bacteria form a toxin that increases vascular permeability and gives rise to local oedema and haemorrhage. infection of the skin in man leads to the formation of a lesion (malignant pustule; a black eschar, hence b. anthracis; gr. anthrakos = coal) consisting of a necrotic centre surrounded by vesicles, blood-stained fluid and a zone of oedema and induration. in severe infections there is septicaemia with toxic signs, loss of fluid into tissues, with widespread oedema and eventually death. anthrax toxin, the discovery of which by smith and keppie in the early s was a major milestone in our subject, consists of three components, none of which are toxic by themselves: factor i (oedema factor or ef), factor ii (protective antigen or pa) and factor iii (lethal factor or lf). the toxin is largely responsible for local lesions, kills phagocytes, enters the circulation and accounts for the toxicity, oedema and death caused by b. anthracis. virulent strains also have a capsular polypeptide composed of d-glutamic acid, which inhibits opsonisation and phagocytosis (see ch. ). anthrax in man occurs mainly in those whose work brings them into contact with infected animals. it is not a common disease in the uk, and the usual source of infection is imported bones, hides, skins, bristles, wool and hair, or imported fertilisers made from the blood and bones of infected animals. most of what has just been described was discovered more than three decades ago but more recently new insights have been obtained as to the biochemical basis of the mode of action of anthrax toxin. ef was shown to be an inactive form of adenylate cyclase which is activated by calmodulin; pa and lf were not active. however combinations of pa and ef (but not pa and lf) caused an elevation of camp. thus anthrax toxin (at least the pa/ef combination) is a binary toxin. the response observed is more rapid and greater than that induced by cholera toxin. in contrast to cholera toxin, the anthrax complex is the cyclase, acts without a lag phase and its effects are instantly reversible upon washing toxin-treated cells. this suggests that the enzyme is either rapidly degraded after internalisation, or that it is not completely internalised but wsfhiw ii camp atp fig. . mode of action of anthrax toxin. factor ii (pa) interacts with the cell membrane. after proteolytic cleavage sites are exposed which bind factor i (ef) and facilitate internalisation of factor i (ef). this internalisation takes place via an endocytic step, with factor i being released into the cytosol. factor i must be rapidly inactivated since washing toxin-treated cells results in a rapid loss of adenylate cyclase activity. factor i interacts with calmodulin (cal) to become an active adenylate cyclase enzyme. interaction of factor i with factor ii and subsequent internalisation is blocked by prior binding of factor iii (lf) to factor ii. associated with the cell membrane in a manner allowing it to be readily removed. these facts together with the observations that lf would block the activity of ef are summarised in fig. . . this explains how lf blocks the effect of ef. they either compete for the same sites on pa, in which case the explanation is self-evident, or they bind to separate sites on pa which are situated such that occupancy of one occludes access to the other. one can extend these observations made in vitro to the in vivo situation. the model provides a basis for understanding the protective function of pa. antibodies to pa could either block the initial attachment to the cell membrane of target organs in vivo, or prevent the binding of ef to pa, or both. the fact that camp is known to be a potent secretagogue could explain how the oedematous reaction occurs when pa and ef are injected into test animals, i.e. ef is, after activation, an oedema factor. moreover, in biological tests, addition of lf (factor iii) depresses oedema production as would be predicted by this model. the observation that pa was probably fixed first may now be placed on a firmer basis since binding of pa is a prerequisite for the expression of ef (and by implication) lf activities. the case for the view that, in experimental anthrax in guinea-pigs, animals died (as do humans) of secondary shock, is also placed on a firmer basis with perhaps one important reservation. smith and coworkers claimed that factor iii (lf) decreased oedema but increased lethality; the former but not the latter observation is explicable by the model which is therefore too simplistic to explain the effects of the holotoxin. it is likely that lf exerts its activity together with pa on other cells in vivo. it has in fact been shown that lf in combination with pa is cytolytic for macrophages and macrophage-like cell lines-the probable basis of the earlier observation that anthrax toxin kills phagocytes and perhaps justification for talking about a plurality of anthrax binary toxins. clostridium tetani spores germinate in an infected wound and produce their toxin. spores are ubiquitous in faeces and soil, require the reduced oxygen tension for germination provided locally in the wound by foreign bodies (splinters, fragments of earth or clothing) or by tissue necrosis as seen in most wounds, the uterus after septic abortion, or the umbilical stump of the newborn.* the site of infection may be a contaminated splinter just as well as an automobile or battle injury. all strains of clostridium tetani produce the same toxin. it also reaches the central nervous system by travelling up other peripheral nerves following bloodborne dissemination of the toxin through the body. the motor nerves in the brain stem are short and therefore the cranial nerves are among the first to be affected, causing spasms of eye muscles and jaw (lockjaw). there is also an increase in tonus of muscles round the site of infection, followed by tonic spasms. in generalised tetanus there is interference with respiratory movements, and without skilled treatment the mortality rate is about %. botulism* caused by clostridium botulinum, a widespread saprophyte present in soil and vegetable materials. c. botulinum contaminates food, particularly inadequately preserved meat or vegetables, and produces a powerful neurotoxin. the toxin is destroyed at °c after min-of great importance to the canning industry-and there are at least seven antigenically distinct serotypes (a-g) produced by different strains of bacteria but which have a pharmacologically similar mode of action. it is absorbed from the intestine and acts on the peripheral nervous system, interfering with the release of acetylcholine at cholinergic synapses of neuromuscular junctions. somewhere between and hours after ingestion there are clinical signs suggesting an acute neurological disorder, with vertigo, cranial nerve palsies and finally death a few days later with respiratory failure. * botulus (latin) = sausage. in a large sausage was eaten by people in wildbad in germany; all became ill, and six died. the disease was subsequently referred to as botulism. t in recent years a less typical form of botulism has been described in small infants. the spores, present in honey applied to rubber teats, appear to colonise the gut, so that the toxin is produced in vivo after ingestion. tetanus and botulinum toxins are two of the most toxic substances known to man: g of botulinum toxin will kill mice. a great deal is now known about the genetics of both these toxins. tetanus toxin and botulinum toxin type g are encoded by plasmids, botulinum toxins types c and d are encoded by phages that infect clostridia, and botulinum type a is encoded in the bacterial genome; the genes have been sequenced. both toxins are synthesised as single peptides and are released from bacteria upon lysis. they are proteolytically activated by endogenous proteases to yield dichain derivatives consisting of -s -s -linked l (m r ) and h (m r ) chains. the recently adopted nomenclature reflects our current understanding of their peptide structures ( fig. . ). very recently, huge strides have been made in our knowledge of the mode of action of these toxins ( fig. . ). there is very little overall sequence homology between bonts and tetx; this could account for their immunological characteristics and the animal types normally affected. however, there is one structural feature common to all these neurotoxins located in the middle of the light chain and that is a zincbinding motif. in fact these neurotoxins are zinc endopeptidases. their targets are protein constituents of the synaptic fusion machinery responsible for the exocytotic release of neurotransmitters. the intracellular target cleaved by bonts b, d, and f and tetx is synaptobrevin (vamp, vesicle-associated membrane protein) present in small synaptic vesicles. bonts a and e cleave snap- (synaptosomal associated protein of m r ) a highly conserved protein known to be involved in exocytosis. bont c acts on syntaxin, a synaptic membrane protein also involved in exocytosis. however, some puzzles remain to be resolved. tetanus and botulinum toxins enter neuronal tissue preferentially at motoneuronal endplates, but the nature of the 'physiologically relevant ectoacceptors' is still not known. it remains unclear why botulinum toxin acts directly at the site of uptake and not, as observed with tetanus toxin, in the central nervous system, although a considerable amount of botulinum toxin (like tetanus toxin) is retrogradely transported. botulinum toxin blocks the release of acetyl choline at neuromuscular junctions to cause flaccid paralysis. likewise, one might ask why tetanus toxin fails to act at the motoneuronal junction at concentrations which would completely block release of neurotransmitter from gabaergic synapses. to reach inhibitory interneurons-its principal site of action-tetanus toxin must leave α-motoneurons after the primary uptake step, traverse the synaptic cleft to interneurons, leave those again in order to become finally internalised again from presynaptic membranes-a route identical to that of several neurotropic viruses. it acts by blocking the release of inhibitory transmitters (glycine or gab a) resulting in a failure to relax the affected muscle-pathophysiological 'tetanus'. only in rare cases does it act peripherally like botulinum toxin. the most toxic substances known to man are now being used as therapeutic agents to treat focal dystonias such as neck twists and eye squints, or eyelid closure and, more recently, some childhood palsies. the preparations are made from bont a and consist of toxin-haemagglutinin complex-the form in which the toxin is usually produced by the organism. this complex is less toxic than purified neurotoxin when administered parenterally but relatively more toxic when given orally; the haemagglutinin apparently protects the neurotoxin from proteolytic degradation in the gut. the effects of bont in relieving muscle spasms are not permanent but last for several months. treatment has to be repeated. to date the successes significantly outweigh the failures and no long-term adverse effects have been reported. antibody to the toxin has not so far been detected in the sera of the majority of patients. some toxins destroy membranes by virtue of their proteolytic activities, and some by their ability to degrade lipid components, while others are pore-forming or detergent-like in their mode of action. in addition to their action on protein components of lung connective tissue referred to above, pseudomonas aeragmosa-elastase and the zinc metalloprotease of legionella pneumophila are believed to destroy cell membranes by their proteolytic activity. this is the probable reason for the haemorrhage associated with lung infections caused by these pathogens, i.e. effects on type i alveolar epithelial and endothelial cells. clostridium perfringens α-toxin. a large number of bacterial enzymes are phospholipases some of which, but by no means all, are important toxins. the best example is the α-toxin of clostridium perfringens, the organism most commonly associated with gas gangrene. it is strictly anaerobic and occurs as a normal inhabitant in the large intestines of man and animals; its spores are ubiquitous in soil, dust and air. c. perfringens does not multiply in healthy tissues, but grows rapidly when it reaches devitalised and therefore anaerobic tissues. this could be after contamination of a natural wound with soil or dust, particularly on battlefields or in automobile accidents, or after contamination of a surgical operation site with clostridia from the patient's own bowels or skin. after abortions, particularly in the old days before antibiotics, intestinal clostridia often gained access to necrotic or devitalised tissues in the uterus and set up life-threatening infections. invasion of the blood was common and soon resulted in death, the clostridia localising and growing in internal organs such as the liver after death. c. perfringens has various enzymes that enable it to break down connective tissue materials, including collagen and hyaluronidase, thereby facilitating spread of the infection along tissue planes. most of the enzymes are toxic (a) reflex arc (top). mechanism for inhibiting the antagonists to a muscle contracting in response to stretch. muscles are reciprocally innervated with sensory and motor neurons, although for clarity this is shown only for the protagonist muscle. on stretch, the stretch receptors generate an impulse which is transmitted along the afferent sensory (s) neuron of the protagonist (p) muscle. this sp neuron enters the spinal cord by the dorsal root and synapses with the motor neuron supplying the protagonist muscle (mp) and with an interneuron (i) which in turn synapses with the motor neuron supplying the antagonist muscle (ma); the efferent motor neurons leave the spinal cord by the ventral root. at the sp/mp synapse an excitatory transmitter is released which induces an impulse in mp which leads to contraction of protagonist muscle. however, excitation of i causes release of an inhibitory transmitter at the i/ma synapse which leads to relaxation of the antagonist muscle. note that the basic reflex arc has been shown for simplicity but tetx acts mainly on voluntary muscles. (b) a simplified version of the biochemical events occurring in synapses (lower left). excitatory and inhibitory synapses, neurotransmitter release and action. gly, glycine; r, receptors of neuro transmitters; x, hitherto uncharacterised (candidates include glutamate, dopamine, atp, substance p, and somatostatin). to host cells and tissues, but α-toxin is easily the most important one. it is dermonecrotic, haemolytic (a feature seen mainly in tissues close to the focus of infection but sometimes responsible for large-scale intravascular haemolysis in infected patients), causes turbidity in lipoprotein-rich solutions and is lethal. while it is still true that these activities are all due to one molecular species, they are not different expressions of the one enzymic activity. historically, c. perfringens α-toxin was the first bacterial toxin to be characterised as an enzyme: it is a phospholipase c (plc) which removes the head group, phosphoryl choline, from phosphatidyl choline and from sphingomyelin. it is of undoubted importance in gas gangrene. toxoid prepared by formalin-treated toxin will protect sheep against infection caused by c. perfringens. however, one might ask why all enzymes with such biochemical specificity are not equally toxic or important in determining virulence; there are several reasons which can be put forward. by the combined use of monoclonal antibodies and molecular genetic analyses we now know that there are at least two functional domains in this c. perfringens α-toxin. if one compares c. perfringens α-toxin with the phosphatidyl choline-preferring, nontoxic phospholipase of bacillus cereus, one finds that two-thirds of the n-terminal sequence shows homology with the entire sequence of b. cereus plc. this portion of c. perfringens α-toxin retains its plc activity but not its haemolytic and lethal activities. the c-terminal part is not haemolytic, not enzymatically active and not cytotoxic for mouse lymphocytes, but is necessary for conferring toxicity on the n-terminal part of the protein. in fact, the c-terminus is a potent immunogen that will solidly protect mice -and hopefully man-against experimental infection with c. perfringens. surprisingly, the c-terminal domain of the nontoxic c. bifermentans enzyme shows sequence similarity with that of its c. perfringens α-toxin counterpart. the nontoxicity of this enzyme is ascribed to its comparatively much lower turnover rate, i.e. it is a much less efficient enzyme. recently, attention has been drawn to the distinct possibility that while haemolysis may be initially induced by the plc activity of c. perfringens α-toxin, it is possible that actual disruption of the membrane is due to the activation by the toxin of other phospholipases (phosphatidylinosotol- , -biphosphate (pip ) specific phospholipase and phospholipase d) present in the erythrocyte membrane. some now believe (c) sites of neurotoxin action (lower right). the predominant site of action of tetx is the intermotor neuron synapse; the exocytotic machine is interfered with by the endopeptidase action of tetx on vamp. bont acts at the neuromuscular junction inhibiting the release of acetyl choline (ach) by its proteolytic action on vamp (types b, d and f), or snap (types a and e), or syntaxin (type c). (amplified from figs and , 'bacterial toxins', nd edition, by j. stephen and r. a. pietrowski ( ), pp. and , van nostrand reinhold (uk).) that the basis of toxicity is not simply a cytolytic one, but rather a consequence of its ability, in sublytic doses, to release inosotol triphosphate (ip ) and activate the arachidonic acid cascade. there are other pathogenic clostridia that cause gas gangrene and produce similar toxins. infected tissues show inflammation, oedema and necrosis, not necessarily with the formation of gas, and the illness can be mild or very severe according to the extent of bacterial spread and the nature and quantity of toxins that are formed and absorbed. since the bacteria grow and produce their toxins only in devitalised tissues, the most important form of treatment is to remove such tissues. clostridia are strictly anaerobic, and exposure of the patient to hyperbaric oxygen (pure oxygen at - atmospheres in a pressure chamber) has been found useful in addition to chemotherapy. staphylococcal ß-toxin. staphylococcal ß-haemolysin is known to be produced in vivo. in ch. studies with isogenic mutants were described which indicate that it is important in killing neutrophils. it probably has the narrowest substrate specificity among the phospholipases, and is a hotr-cold haemolysin: lysis of erythrocytes occurs only on cooling after incubation at °c. the phenomenon, though of doubtful significance in vivo, has attracted attention and generated speculation about its mechanism. perhaps the most likely explanation is that, when cooled below their phase-transition temperature, the remaining phospholipids undergo quasi-crystalline formation, thereby generating intramembranous stresses incompatible with structural integrity. these proteins (made by some species, not all of which are pathogens) have been called oxygen-labile haemolysins because they are reversibly inactivated on standing in air, and their most studied property is their ability to lyse red cells. sh-activation of crude preparations is necessary for the expression of haemolytic activity but they are also active towards a variety of cell types. thiol-activated cytolysins share the following properties: they are cross-neutralised by hyperimmune sera, lyse a wide range of species of erythrocyte, exhibit similar ph and temperature optima for cytolytic activity, are lethal and cardiotoxic, and lose activity on incubation with erythrocyte ghosts. they are inactivated irreversibly by small amounts of cholesterol. interaction with cholesterol is the key primary event in their interaction with susceptible membranes which leads to the impairment of the latter; cholesterol plays no further part in the subsequent damage process. a schematic outline of how they damage cell membranes is shown in fig. . . several facts have recently reopened considerable interest in this group. first, the recognition of what these substances do to host defence cells when presented in sublethal doses. for example, pneumolysin from s. pneumoniae inhibits the respiratory burst in neutrophils, inhibits antibody synthesis in b cells and activates the classical complement pathway in the absence of antibodies. since complement is an important defence mechanism against the pneumococcus in vivo this could lead to depletion of complement levels and abrogate protection. in fact, immunisation of mice against pneumolysin affords some protection against challenge infection. second, in addition to its role in promoting invasive bacteraemia, pneumolysin has also been implicated in causing sensorineural deafness associated with meningitis caused by the pneumococcus; this perception is based on very recent work with a guinea-pig other pore-forming toxins t h e r t x toxins h a v e a l r e a d y b e e n referred to above in connection w i t h b. pertussis a c -h l y . b u t t h e r e a r e o t h e r pore-forming toxins. staphylococcal δ-toxin. this toxin acts in a manner similar to that of the sh-activated cytolysins, with an important difference: there is no initial specific binding. δ-toxin initially forms small pores and then islands of membrane or large micelles; this gives rise to its perceived detergent-like properties. there is a family of closely related δ-toxins which inhibit the growth of gonococci. it is not often that one can ascribe a positive function to a toxin which is beneficial for the organism producing it. in this case -toxin(s) could have important ecological significance in the mixed culture situation that is characteristic of the real microbial world. of great interest is the synergy that δ-toxin displays. sublytic amounts of δ-toxin causes release of cell constituents without lysis. but, only . haemolytic units of staphylococcal ß-toxin will cause lysis of cells in the presence of . lytic units of δ-toxin. this synergistic interaction could be the way in which staphylococcal toxins, which rarely exert their lethal effects in the majority of infections, exercise important cytolytic effects. of less obvious significance is the fact that δ-toxin is a poor antigen; for a long time its antigenicity was controversial. if δ-toxin were to prove of crucial importance as a cytolytic potentiator, then this could also partly explain why natural acquired immunity to staphylococcal infection is either non-existent or sufficiently low as to be easily overcome. staphylococcal a-and -toxins. staphylococci produce a range of toxins several of which we have already met. the α-toxin is considered as the main cytolysin produced by s. aureus. like streptolysin- and staphylococcal δ-toxin, it is secreted as a water-soluble protein and undergoes self-induced oligomerisation on cell membranes to form pores. it has recently been suggested that transmembrane channels formed by the hexameric form allow the penetration of the monomeric form which interferes with certain steps in translation. in systemic staphylococcal infections death is most probably due to the potent α-toxin but in localised pyogenic infections-such as mastitis in cattle, goats, rabbits and mice-its role is most likely one of killing phagocytes or conferring survivability on intracellular bacteria. -toxin is a binary toxin with a haemolytic action on rabbit erythrocytes. there is evidence that α, β and haemolysins and leucocidin are produced in vivo during natural and experimental infections. this is based on the presence of antibodies to these haemolysins in subjects undergoing staphylococcal infection and the extraction of toxin from peritoneal abscesses in mice. however, the precise roles that these or any other staphylococcal toxins play in localised infections is not clear. atrophie rhinitis is a disease of pigs in which the principal clinical sign is atrophy of the nasal turbinate bones and shortening of the snout. scientists have isolated and purified a toxin believed to be important in the causation of this disease. this is a remarkable toxin on at least two counts. it shows an incredible predilection for the target tissue in that it causes turbinate atrophy in gnotobiotic pigs, even when injected intraperitoneally; it also causes necrosis of spleen and thymus. perhaps even more surprising is the fact that it is a potent mitogen, hardly the kind of activity that one would associate with atrophy! one can only assume that it upsets the dynamic balance between the laying down and resorption of mesenchymal bone tissue by osteoblasts and osteoclasts respectively. the remainder of this section deals with other important (or potentially important) toxins which have not already been referred to in earlier chapters or in the section above. several staphylococcal toxins have already been referred to above. staphylococci are ubiquitous and responsible for a large group of diseases which range from the relatively harmless skin pimple through abscesses, impetigo, food poisoning, osteomyelitis, mastitis, primary pneumonia, staphylococcal scalded skin syndrome (ssss) and toxic shock syndrome (tss) to fatal septicaemias. in the case of ssss a toxin is known and is described on p. . toxic shock syndrome toxin (tsst- ). tss is seen characteristically in menstruating women whose tampons harbour multiplying staphylococci. it is due to a toxin called toxic shock syndrome toxin (tsst- ; one of the so-called staphylococcal enterotoxins, see next paragraph). toxic shock syndrome is characterised by sudden onset of fever, vomiting, diarrhoea, an erythematous rash followed by peeling of the skin, hypotensive shock, impairment of renal and hepatic functions and occasionally death. the main symptoms of the disease have been reproduced in rabbits by implanting chamber-enclosed tss-strains in the rabbit uterus or peritoneum or by injection of tsst- into rabbits. complex changes are observed including: haemorrhage in kidney and liver; congestion and haematomas in the lungs; leakage of blood into the thymus; and fluid in the pericardial sac and in the gut lumen. these effects in rabbits are very similar to those seen in humans and would certainly explain the shock and diarrhoeal syndrome so characteristic of the disease. all of this indicates that the primary effect of tsst- is on the integrity of capillary walls. the lethal effect of tsst- is enhanced considerably by endotoxin (see below). such synergy is also known for streptococcal erythrogenic toxin and lps, but the mechanism(s) involved in this complex synergy is not clear. although relatively uncommon in the uk, staphylococcal food poisoning accounts for over % of all cases of food poisoning in the united states. the disease is characterised by vomiting and diarrhoea commencing - h after consumption of contaminated food, especially dairy produce. symptoms usually last no longer than h. like botulism, staphylococcal food poisoning is commonly caused by the ingestion of food containing preformed toxins, known collectively as the staphylococcal enterotoxins. they are serotypically heterogeneous single-chain globular proteins, of molecular weight between and kda, secreted by certain strains of s. aureus. one of these, serotype f is identical to tsst- and staphylococcal pyrogenic exotoxin c. these toxins are not classical enterotoxins acting on intestinal cells but 'neurotoxins' activating receptors on the abdominal viscera, the stimulus reaching the vomiting centre via the vagus nerve. several of the staphylococcal toxins referred to above (tss- and enterotoxins), and also the erythrogenic toxins a and c of streptococcus pyogenes (see below) have an additional important action. they are among the most powerful t cell mitogens known, acting at picomolar concentrations. they bind to mhc class ii molecules on antigenpresenting cells and interact with the vß chain of the t cell receptor (see chs and ). this causes proliferation and cytokine release by the entire subset of t cells bearing that particular vß chain- - % of all t cells are affected, whereas only . - . % of t cells respond to a given regular antigen. this represents an important interference with a coordinated immune response, and the widespread polyclonal activation and cytokine release can be regarded as a microbial strategy, a 'diversion' of host immune defences. in addition, if the superantigen reacts with developing t cells (with the correct vß chain) in the thymus, these cells are deleted. it seems probable, therefore, that this is a more important biological function of these toxins than the one responsible for the characteristic disease, which may be no more than an 'accidental' phenomenon. it turns out that similar molecules are formed by mycoplasma and by certain retroviruses (e.g. the mis antigen of mouse mammary tumour virus). scarlet fever is one of the many conditions caused by streptococcal infection and may accompany a streptococcal sore throat or occasionally a streptococcal wound infection. there is a generalised erythematous rash together with fever and a sore throat. the rash is due to an erythrogenic toxin produced by certain strains of streptococcus pyogenes. erythrogenic toxin is a low molecular weight protein produced in a complex form with hyaluronic acid, which acts as a carrier. the protein consists of two parts. one is heat labile, carries the determinants of immunological specificity which give rise to three serotypes, a, b and c, and is responsible for primary toxicity manifestations, including pyrogenicity, low lethality, cytotoxic effects on cultured spleen macrophages, and suppression of the reticuloendothelial and immune systems. the second part is heat stable, antigenically common to types a, b and c, and is responsible for secondary toxicity; that is, hypersensitivity effects, including skin hyperreactivity (the basis of the rash), myocardial necrosis, enhancement of pyrogenicity and lethality, and enhanced host response to other injurious agents. thus, it is not possible to define the biological activity of 'erythrogenic' toxins without considering the immunological state of the host. an individual may suffer no reddening of the skin upon intradermal injection of erythrogenic toxin (negative dick test) because a high neutralising titre of antibody blocks the primary toxiphore or because of a lack of hypersensitivity to the common antigen. streptolysins o and s are dealt with in ch. . there is a plethora of toxins produced by numerous clostridial pathogens important in both human and veterinary medicine. the clostridial genus comprises a large number of toxigenic species, some of which are known to produce several toxins, extracellular enzymes, and other factors which are as yet recognised only by a letter of the greek alphabet. all the α-toxins are dermonecrotic and the others have haemolytic, enzymatic properties. toxin production in vitro is used as the basis of typing clostridia, and the picture is complex. no attempt will be made to describe every disease in man or animals associated with clostridia; only those are selected which best serve to illustrate the involvement of some recognisable toxins. in the case of sheep diseases-lamb dysentery, struck, enterotoxaemia, black disease, braxy, black quarter-some of the best evidence for implicating relevant toxins comes from field studies using multivalent vaccines (based on toxoids of these toxins). clostridium perfringens type c causes pig bei in man, essentially due to the production of ß-toxin. this is a rare disease in developed societies but a public health hazard in papua new guinea. four factors are responsible: the ubiquity of c. perfringens type c in the soil and faeces of man and pigs; the relatively low immunogenicity of ß-toxoids in young children, the group most at risk; the high carbohydrate, low protein nature of the staple diet; and the sporadic consumption of large quantities of pork on occasions of celebration. the latter dietary change promotes a proliferation of clostridia in the intestine which may lead to intestinal gangrene and death. ß-toxin damages the mucosa, reduces mobility of villi and causes more bacteria to become attached to the villi. more toxin is absorbed and the mucosa and underlying the intestinal wall become necrotic, leading to death in many cases. the influence of diet is additionally important in t h a t low protein diets cause decreased secretion of pancreatic proteolytic enzymes and sweet potato contains a trypsin inhibitor. these conditions promote the survival of ß-toxin which is highly sensitive to proteolytic inactivation. immunisation with ß-toxoid preparations has dramatically lowered the incidence of this fatal disease in children. gas gangrene in man may be caused by several bacterial species separately or in concert. these include clostridium perfringens type a, c. novyi types a and b, and c. septicum; c. perfringens and its a-toxin have already been discussed. far less is known about c. novyi and c. septicum and their toxins in gas gangrene in man. much more is known about the role these organisms play in diseases of animals, and multivalent vaccines confer a very high degree of immunity (particularly to sheep) against several clinically identifiable but separate diseases. a few of these diseases are described below. clostridium perfringens type b causes lamb dysentery. this is an acute, fatal disease of young lambs occurring during the first week of life and caused by absorption of toxin(s) generated by c. perfringens type b in the small intestine. clostridium perfringens type c causes struck in sheep, a disease occurring in the romney marshes of kent, but rare in other areas in the world. the pathological changes observed differ markedly from other enterotoxaemias and include enteritis. clostridium perfringens type d enterotoxaemia in sheep is another acute fatal disease. the most constant lesion is subendocardial haemorrhage around the mitral valve. c. perfringens type d e-toxin or its protoxin are recoverable from intestinal contents. clostridium novyi type b causes black disease of sheep or infectious necrotic hepatitis. this is an acute infectious disease of sheep (occasionally cattle) caused by the absorption of the α-toxin elaborated by the organism in necrotic foci in the liver, and is nearly always associated with invasion of the liver by immature liver flukes. how c novyi gets to the liver in the first place is not known but it is readily demonstrable in livers of normal sheep in areas where the disease is prevalent. experimental reproduction in guinea-pigs of a similar disease is possible by the combined action of c. novyi spores and liver fluke infestation. clostridium novyi type d causes a rapidly fatal disease in cattle (redwater disease) similar to, and regarded by some as an atypical manifestation of, black disease. the characteristic lesions include jaundice, various haemorrhagic manifestations, and anaemic infarcts in the liver; active liver fluke infestation may or may not be present. in culture this organism produces ß-toxin, which explains the haemoglobinuria, but no a-toxin. clostridium septicum causes braxy in sheep. the role of c. septicum in this acute, fatal disease is assumed because of its association with the characteristic haemorrhagic inflammatory lesion in the abomasum. the disease has not been reproduced experimentally with c. septicum but can be prevented by immunisation with sterile toxoids derived from this organism. clostridium chauvoei causes black quarter in sheep and cattle. this is a gas gangrene type infection of muscles and associated connective tissues in cattle and sheep; c. chauvoei is also the causative agent of parturient gas gangrene in sheep. the initial stimulus which activates the infection in cattle is not known, since the disease is hardly ever associated with any overt wounding. washed spores alone do not cause disease when injected, but do in conjunction with a tissue-necrotising agent. in sheep, wounding caused by parturition, castration, tailing, shearing, vaccination, as well as accidental damage will create a focus within which c. chauvoei can multiply. there are numerous examples now known of synergistic reactions between toxins of the same or different species. bacillus cereus makes a phosphatidyl choline-preferring phospholipase and a sphingomyelinase which are separately nontoxic; in concert they are haemolytic and termed cereolysin a-b. the examples of staphylococcal a-and δ-toxins and of staphylococcal tsst- and endotoxin, have already been alluded to above. streptococcal erythrogenic toxins increase sensitivity to other streptococcal factors (for example, streptolysin-o) and also to gramnegative endotoxin. the susceptibility of rabbits to endotoxin is increased times, and adult cynomolgus monkeys die within h, when injection of low levels of steptococcal toxin is followed h later by an otherwise sublethal dose of endotoxin. this may be because streptococcal toxin creates a state of hypersusceptibility to a wide variety of stressful agents. perhaps, in view of the high incidence of exposure of man and his domestic animals to streptococci, we should actively investigate possible toxin-mediated synergy in mixed infections involving streptococci. another entirely different type of example is that of the increase in toxicities of staphylococcal a-and -toxins, diphtheria toxin and endotoxin for neonatal ferrets preinfected with influenza virus. increases were -, -, -and -fold respectively. no increase in viral replication was observed. neonates died suddenly without clinical symptoms as in human babies dying from sudden infant death syndrome (sids). pathological examination showed inflammation of the upper respiratory tract, lung oedema and collapse, and early bronchopneumonia in animals receiving the dual challenge but not those receiving either toxin or virus on their own. thus, some bacterial toxins in conjunction with influenza virus could be one of the several causes of sids. many fungi contain substances that are harmful when taken by mouth, and there are two diseases that result from the ingestion of food containing preformed fungal toxins. as with c. botulinum, the disease is caused without the need for infection. aspergillus flavus infects ground nuts (monkey nuts) and produces a very powerful toxin (aflatoxin). contaminated (badly stored) ground nuts used to prepare animal feeds caused the death of thousands of turkeys and pigs in the uk in and the survivors of intoxication nearly all developed liver cancer. human disease has not yet been associated with this toxin. clavicepspurpurae is a rust fungus affecting rye, and it produces toxins (ergotamine especially) that give rise to ergot poisoning when contaminated grain is eaten. mushrooms and toadstools have long been recognised as sources of poisons and hallucinogens. unlike the toxins already discussed in this chapter, there is a group of toxins which are distinct structural components and are not released into the surrounding medium in any quantity except upon death and lysis of the bacteria. several protein toxins (e.g. clostridium difficile toxin a, tetanus toxin) are released during the decline phase of batch culture-probably on autolysis-and these are classified as exotoxins. here we deal with toxins which are known to comprise well-recognised structural entities which on a priori grounds must have key functions in the organism: they are found in the outer membranes of gram-negative organisms. there are two chemically distinct types of toxin considered: lipopolysaccharide (endotoxin; lps) and protein. the bulk of this section is taken up with endotoxin. many pathogenic organisms, however, are pathogenic by virtue of possessing various types of surface structure important in conferring virulence. these include, for example, adhesins which are important in colonising body surfaces or a variety of surface molecules (which may or may not be inside capsules) which render them resistant to phagocytosis. but the majority of adhesins and anti-phagocytic determinants are themselves nontoxic. the gram-negative bacterial cell wall is subject to considerable variations in both the composition of lps and in the number and nature of the proteins found in the outer cell membrane. apart from the examples given in ch. in relation to the gonococcus, such phenotypic variation in lps has rarely been examined in the context of pathogenicity. however, the examination of cell-bound proteins of yersinia pestis from organisms grown in vivo led to the discovery of a toxin lethal for mice and guinea-pigs. plague is one of the most deadly diseases of man and has, over several thousands of years, claimed millions of lives. in the fourteenth century, 'the black death' wiped out a quarter of the population of europe before spreading through the middle east and asia. fortunately, however, the last years or so have seen a drastic decrease in outbreaks of plague, though the threat of another epidemic is still with us. the causative organism of plague, yersinia pestis, is primarily a parasite of rodents in which it is endemic in many areas of the world. only when man comes into close proximity with infected rodents do outbreaks of human plague occur. the disease is spread from rat to rat and from rat to man by fleas. rodents in the terminal stages of infection with y. pestis suffer massive bacteraemia and so when a flea sucks the blood of an infected animal it swallows large numbers of bacteria. when the rodent eventually dies, the flea leaves the corpse and awaits a new host. in the meantime, however, y. pestis multiplies rapidly in the alimentary tract of the insect, often completely blocking the proventriculus. the flea becomes voraciously hungry and feeds on any suitable host, which sooner or later is man. however, because the flea's alimentary canal is blocked by bacteria it cannot feed efficiently and usually succeeds in imbibing blood only to regurgitate it, now contaminated with y. pestis, back into the wound. during the next - days, organisms become localised and multiply in the regional lymph nodes, causing considerable swelling. these swellings, which are often in the axillary and inguinal regions because the most common sites for flea-bites are the arms or legs, are referred to as primary buboes (greek boubon = groin); hence the name bubonic plague. secondary buboes may develop, followed by necrosis of the lymphoid tissue and vascular damage giving rise to haemorrhaging in many organs and tissues. these pathological changes are accompanied by prostration, high fever and delirium, followed in the terminal stages of the disease by shock and death. it is also possible for the disease to be transmitted from human to human by the aerosol route: exhaled organisms from heavily infected patients are capable of infecting others giving rise to pneumonic plague. the principal features of human plague can be reproduced in guineapigs and mice. monkeys show shock-like signs only during the terminal period of - h, when they become quiet, progressively weak, prostrate and hypothermic; for the previous - days infected animals are lively and vigorous. in the terminal stages blood pressure drops rapidly but there is no evidence of oligaemia (low blood volume) caused by haemorrhage, or of oedema, suggesting that vascular collapse must be associated with a vasodilatory factor(s), resulting in pooling of blood. in this respect monkeys differ from humans and guinea-pigs. the symptoms of plague-high fever and vascular damage -are characteristic of intoxication with endotoxin. however, it is extremely unlikely that endotoxin alone is the main toxin involved in plague. it is much more likely to act in conjunction with one or more other potentially toxic fractions from y. pestis. plague murine toxin is a protein which, although highly lethal for mice and rats, is relatively nontoxic for guinea-pigs, rabbits, dogs and monkeys. a completely separate guinea-pig toxin complex exists comprising at least two cell wall/membrane protein components, one of which will kill mice, although both are needed to kill guinea-pigs. however, the nature of the toxin or toxins of y. pestis and their role in the human disease syndrome are still far from clear. the classical approach to such a problem is to prepare a specific toxoid and to determine whether injection of this confers immunity to the disease. needless to say, the severity of human plague renders such experiments impossible. until such questions can be answered, we are left to argue whether, in the context of plague, man resembles a mouse, a monkey or a guinea-pig. endoxotins are part of the outer membrane of gram-negative bacteria. it has been known for many years that the cells (alive or dead) or cell extracts of a wide variety of gram-negative bacteria are toxic to man and animals. the literature on this subject is vast, sometimes confusing and often controversial; here we can give no more than a brief outline. some of the diseases in which endotoxin may play an important role include typhoid fever, tularaemia, plague and brucellosis, and a variety of hospital-acquired infections caused by opportunistic gram-negative pathogens which include escherichia coli, proteus, pseudomonas aeruginosa, enterobacter, serratia and klebsiella. in addition, endotoxin has been intensively studied as a possible causative agent of shock arising from post-operative sepsis or other forms of traumatic injury in which the normal flora of the gut is often the source of endotoxin. the toxins we have considered so far have been protein (or at least part protein) in nature but, in contrast, endotoxin is a complex lipopolysaccharide. it is also much more heat stable than protein toxins and much less easily toxoided. in addition to lethality, endotoxin displays a bewildering array of biological effects. the complex nature of the multi-layered gram-negative bacterial envelope is shown in fig. . (see also fig. . ) . the outer membrane is composed of a bimolecular leaflet arrangement as are other membranes but has a different composition from the cytoplasmic membrane. the lipopolysaccharide (lps) is unique in nature, only found in gramnegative bacteria, and is, or contains within it, what we designate endotoxin. immunoelectron microscopy indicates that lps exists in the outer leaflet of the membrane and extends outward up to nm; it is on, rather than in, the cell. thus it is evident that the term endotoxin is a misnomer which derives from the era when toxins were considered to be either exotoxins, which were synthesised and secreted by the viable organism, or endotoxins, which were intracellular and released only upon lysis. moreover, extraction with edta shows that approximately % of lps is held noncovalently linked in the membrane. extraction with a variety of different solvents yields material which is highly heterogenous and of apparent molecular weight - x . however, treatment with pyridine or addition of detergents reduces the polydispersity. the endotoxic glycolipid from the rough mutant of salmonella minnesota, r , has an m r of for the basic unit, from which complex aggregate structures are derived. lipopolysaccharide consists of three regions: polysaccharide side chains, core polysaccharide, and lipid a which consists of a di-glucosamine backbone to which long chain fatty acids are linked (fig. . ). the relationship of this type of molecule to the outer membrane is also shown in fig. . . the long chain fatty acids interdigitate between the phospholipids in the outer leaflet and may also be linked (or interact) with lipoproteins, which in turn may or may not be covalently anchored to the rigid peptidoglycan (pg). the polysaccharide side chains project outwards. this structure is not invariant. for example, many organisms when first isolated give rise to colonies with a smooth appearance on agar but on subculture produce colonies with a rough appearance. in general, 'smooth' strains of pathogenic species are more virulent than rough strains. this s->r conversion is accompanied by a loss of region i side chains, which contain the deoxy and dideoxy sugars found in these lps complexes. in addition to these somewhat drastic changes involving loss of side chains, it is possible to induce major compositional changes by manipulating the growth rate of these organisms in a chemostat. thus the lps of salmonella enteriditis, when grown with a mean generation time of min is nearly totally deficient in tyvelose (a dideoxy sugar), possesses % of the galactose and % of the glucose contents of lps obtained when the generation time is min. these genotypic s organisms exhibit an r-phenotype in terms of their vastly reduced o-agglutinability (see below); such observations are potentially very important in the context of the in vivo phenotype and pathogenicity, since it is well known that the growth rate of salmonella typhimurium in mice is - times lower than in vitro. examples have already been given in ch. of changes in lps structures in vivo in relation to neisseria gonorrheae and neisseria meningitidis. the extent to which lipid a is common between different genera is uncertain, but it is not likely to vary tremendously. the core polysaccharide structure is the same or very similar within groups of the enterobacteriaciae: thus polysaccharides from salmonellae are similar to each other, but differ from those of e. coli strains. however, within a group such as the salmonellae, there is a wide variation in the composition and detailed structures of the side chains, a fact which is exploited in the kauffman-white scheme for classifying salmonellae, giving rise to several thousand serotypes. the side chains carry the o-somatic antigen specificities of which there are far more than can readily be accounted for on the basis of the known number of sugars involved in the basic repeating units. in the side chains are found a range of depxy and dideoxy sugars. the general principles governing the relationship between the various chemotypes and serotypes are now well understood; the multiplicity of antibody specificities evoked may be explained in terms of antibodies which can recognise different aspects of one three-dimensional structure. lipid a is the primary toxiphore, but the polysaccharide plays an important part in conferring solubility upon, and optimising the size of micellar aggregates of lps, hence affecting biological activity. however, the immune status of the test animal may affect toxicity: as normal animals produce antibodies to the antigenic determinants on the surface of normal gut organisms (including o-somatic antigens), some of the biological effects of endotoxin may be mediated by hypersensitivity mechanisms. the most powerful evidence that lipid a is the primary toxiphore comes from studies on smooth (s) and rough (r) mutants whose biosynthetic capabilities are blocked at various points. this established that neither the o-side chains, nor the core polysaccharide are necessary for endotoxicity. pure lipid a can be made toxic by complexing it to a hydrophilic carrier like bovine serum albumin. one can make synthetic lipid a preparations which are toxic and assume space-filling configurations, which make it easy to see how they could fit into and be part of a bimolecular leaflet arrangement in the outer membrane. the range of biological properties of endotoxin is quite bewildering and the mode(s) of action very complicated. included among those effects which might play a role in gram-negative bacterial infections, are abortion, pyrogenicity, tolerance (not immune tolerance), the schwartzmann phenomenon, hypotension and shock, and lethality, but the precise part played by lps in these phenomena in gram-negative infections is far from clear. lps causes the release of vasoactive substances, activates the alternative pathway of the complement cascade, and also activates factor xii (hageman factor), the first step of the coagulation cascade, which sometimes results in disseminated intravascular coagulation (p. ). many, perhaps nearly all, the actions of lps are due to the stimulation of cytokine release from macrophages and other cells. there is an effect on the circulation, leading ultimately to vascular collapse. the vascular regions most affected differ from species to species; in man and sheep the main changes are found in the lungs. lps has powerful immunological actions, which is surely no accident; as well as activating the complement system, it induces il- production and is a potent b cell mitogen. man is one of the most sensitive of all species to the pyrogenic action of endotoxin. a dose of n g per kg of body weight injected intravenously into man causes the release of an endogenous pyrogen (interleukin- , see glossary) and tumour necrosis factor (tnf) from macrophages, which act on the hypothalamus to give an elevation of body temperature within an hour. it is possible that the pyrogenic action of lps helps to generate fever in gram-negative bacterial infections, but lps is not the only bacterial factor capable of inducing a febrile response. in spite of all these toxic actions, there have been suggestions that some of the responses to lps (by macrophages, polymorphs) could be advantageous to the host, possibly assisting in the recognition and destruction of bacteria. could it be that host responses to lps are, like the complement or the clotting systems, useful in moderation but harmful in excess? there are reports that when animals with less vigorous responses to lps are infected they suffer fewer symptoms, but permit greater growth of bacteria. very large numbers of gram-negative bacteria are normally present in the intestines (see ch. ), their continued death and exit in the faeces being balanced by multiplication in the lumen. there is a continuous, inevitable low-grade absorption of endotoxin from the intestine.* absorbed (endogenous) endotoxin enters the portal circulation and is taken * in addition, various antigens are absorbed in small quantities from the intestine, and in normal individuals antibodies are formed against various food proteins and to some extent against resident intestinal bacteria (see ch. ). kupffer cells remove any antigen-antibody complexes formed locally in the intestine and prevent them from entering the systemic circulation. up and degraded by reticuloendothelial cells, mainly kupffer cells in the liver. continuous exposure to endotoxin probably has profound effects on the immune system and on the histology of the intestinal mucosa, stimulating development of the immune system in the immature individual, but there are no obvious pathogenic consequences. normal people have low levels of antibody to endotoxin as a result of this continuous exposure. the sick individual may be much more susceptible to endogenous endotoxin, perhaps because of defects in removal by kupffer cells. after trauma or after genito-urinary instrumentation endotoxin is detectable in peripheral blood by the limulus test,* but this leads to no particular signs or symptoms. when large amounts of endotoxin enter the blood there are profound effects on blood vessels with peripheral vascular pooling, a drastic fall in blood pressure, collapse and sometimes death. thus, if enough endotoxin enters the blood during massive gramnegative bacterial sepsis, the vasomotor action of endotoxin becomes important and shock intervenes.! in experimental animals endotoxin also causes vasodilation and haemorrhage into the intestinal mucosa, and sometimes haemorrhage into the placenta with abortion, but these actions do not appear to be important in all gram-negative bacterial infections. to summarise, endotoxin, although studied so carefully and for so long, has not yet been shown to play a definitive role as a toxin in the pathogenesis of any infectious disease. however, there is a growing body of opinion that seeks to implicate endotoxin in the inflammatory response induced in meningitis caused by gram-negative bacteria. but, in spite of its effects on various host defence systems including polymorphs, lymphocytes, macrophages, complement, and on endothelial cells and platelets, its overall role in infection is still not clear. it can, however, cause shock when gram-negative bacteria invade the blood. it is for this reason that considerable effort in recent years has gone into the development of antilipid a antibodies for use as therapeutic agents to combat shock in such situations; the success rate is only partial and the expense enormous. for that reason several groups are seeking to exploit the wealth of chemical and biophysical information available on lps in attempts to develop synthetic derivatives which would neutralise the biological activity of lipid a. we await the outcome of such research. however, the characteristics of the o-antigen polysaccharide are sometimes important in determining virulence (pp. - ). * a sensitive test for endotoxin based on the ability of endotoxin to induce gelation of a lysate obtained from the blood cells of the horseshoe crab, limulus polyphemus. considerable space has been given to toxins because they are being intensively investigated as possible virulence determinants. the account illustrates the complexity of host-microbe interactions when analysed at the molecular level. most toxins are liberated from the microbial cell and can be studied with greater facility than many of the more elusive determinants of pathogenicity. but remember that microbes that replicate inside host cells are less likely to form powerful toxins because they cannot afford to damage at too early a stage the cell in which they are multiplying. thus, toxins are not prominent products in intracellular infections due to mycobacteria, brucella, rickettsiae, or chlamydia, and viruses do not form toxins. although a single molecule of a toxin like diphtheria toxin is enough to kill a cell, other toxins may do no more than impair cell function when present in sublethal concentrations. this can lead, for instance, to defective function in immune or phagocytic cells. low concentrations of the streptococcal streptolysins* will inhibit leucocyte chemotaxis. the ability to form toxins, whether encoded by plasmids or the microbial genome, is subject to selective forces. if toxin production puts a microorganism at a serious disadvantage it will tend to disappear. if it is advantageous it will be maintained, and will spread through the microbial population, just as the genetic changes that confer resistance to antimicrobial drugs are selected for when these drugs are widely used. it is therefore not unreasonable to ask how many of the well-known toxins are actually useful to the microbe as well as being important in causing disease in the host (table . ). however, microbes that multiply extracellularly must produce a variety of enzymes and other molecules involved in nutrition, adherence to substrate, and so on. in the case of free-living microbes these substances, as well as substances that damage or interfere with competing organisms, are of major importance. probably they cannot all be discarded when the parasitic mode of life is adopted. many will have a toxic action. yet, for the infecting microbe, these substances remain as unfortunate necessities, of no particular advantage and perhaps a disadvantage, in the parasitic way of life. in infectious diseases there is nearly always a certain amount of direct microbial damage to host tissues, as discussed above. host cells are destroyed or blood vessels injured as a direct result of the action of * the streptolysins are responsible for ß-haemolysis. most haemolysins will kill phagocytes. ) . inflammatory materials are liberated from necrotic cells, whatever the cause of the necrosis. also many bacteria themselves liberate inflammatory products and certain viruses cause living infected cells to release inflammatory mediators. therefore it is not always clear how much of the inflammation is directly microbial rather than host in origin.* but inevitably the host (see ch. ) generates inflammatory and other tissue responses, and these responses sometimes account for the greater part of the tissue changes. pathological changes can then be regarded as occurring indirectly as a result of these responses to the infection. inflammation causes redness, swelling, pain and sometimes loss of function of the affected part (see ch. ) and is generally a major cause of the signs and symptoms of disease. indirect damage attributable to the host immune response is discussed separately below. in most diseases direct and indirect types of damage both make a contribution to pathological changes, but in a given disease one or the other may be the most important. in a staphylococcal abscess the bacteria produce inflammatory materials, but they also kill infiltrating polymorphs whose lysosomal enzymes are thereby liberated and induce further inflammation. this type of indirect nonimmunological damage is sometimes important in streptococcal infections. virulent streptococci produce various toxins that damage phagocytes, and also bear on their surfaces substances that impede phagocytosis (see ch. ). nevertheless, with the help of antibody, all streptococci are eventually phagocytosed and killed and the infection terminated. unlike the staphylococci, however, killed group a streptococci pose a digestive problem for phagocytic cells. the peptidoglycan component of the streptococcal cell wall is very resistant to digestion by lysosomal enzymes. when streptococci are injected into the skin of a rabbit, for instance, streptococcal peptidoglycans persist in macrophages for as long as days. hence macrophages laden with indigestible streptococcal cell walls tend to accumulate in sites of infection. lysosomal enzymes, including collagenase, leak from these macrophages, causing local destruction of collagen fibres and the connective tissue matrix. macrophages secrete many other substances some of which may contribute to cell and tissue damage (see also p. ). many macrophages eventually die or form giant cells, sometimes giving rise to granulomatous lesions (see p. ). in this way persistent streptococcal materials sometimes cause chronic inflammatory lesions in the infected host. an additional immunopathological contribution to the lesions is to be expected if the host is sensitised to peptidoglycan components. other pathogenic microorganisms that are digested with difficulty by phagocytes include listeria, shigella, candida albicans and, of course, mycobacteria, but the importance of this in the pathogenesis of disease is not generally clear. the expression of the immune response necessarily involves a certain amount of inflammation, cell infiltration, lymph node swelling, even tissue destruction, as described in ch. . such changes caused by the immune response are classed as immunopathological. sometimes they are very severe, leading to serious disease or death, but at other times they play a minimal part in the pathogenesis of disease. with the possible exception of certain vertically transmitted virus infections and the transmissible 'prion' dementias (see ch. ), there are signs of an immune response in all infections. therefore it is to be expected that there will nearly always be some contribution of the immune response to pathological changes.* often the immunological contribution is small, but sometimes it forms a major part of the disease. for instance, in tuberculosis the pathological picture is dominated by the operation of a strong and persistent cmi response to the invading bacillus. in the classical tubercle a central zone of bacilli with large mononuclear and giant cells, often with some necrosis, is surrounded by fibroblasts and lymphocytes. mononuclear infiltrations, giant cells and granulomatous lesions (see p. ) are characteristic pathological features of tuberculosis. there are no recognised toxins formed by tubercle bacilli, and there seems to be no single antigen or other component that accounts for virulence. bacterial glycolipids (e.g. 'cord factor'), resistance to h (see pp. - ) and ability to utilise host fe (see p. ) have been correlated with pathogenicity, and inhibition of phagosome-lysosome fusion in macrophages (see pp. - ) by release of unidentified bacterial components would also contribute to pathogenicity. however, none of these factors is by itself absolutely necessary for virulence, which in such a complex, ancient parasite is likely to be multifactorial. as a gene library for m. tuberculosis is slowly built up there will be opportunities for clearer definition of virulence determinants. when macrophages are killed by intracellular mycobacteria the lysosomal enzymes and other materials released from the degenerating cell contribute to chronic inflammation as in the case of the streptococcal lesions referred to above. * a number of different microbial antigens are produced during most infections (see ch. ) and the possible immunological reactions are therefore numerous. for instance, at least types of circulating malarial antigen are found in heavily infected individuals. the mere enlargement of lymphoid organs during infectious diseases is a morphological change that can often be regarded as pathological. the lymph node swelling seen in glandular fever, for instance, is an immunopathological feature of the disease, and the same can be said of the striking enlargement of the spleen caused by chronic malaria and other infections in the condition known as tropical splenomegaly. as often as not the relative importance of direct microbial damage as opposed to immune and nonimmune inflammatory reactions have not yet been determined, but the picture is clearer in most of the examples given below. in one important human disease, pathological changes are certainly immunopathological in nature, but not enough is known about it to classify the type of reaction (see table . ). this disease is rheumatic fever, which follows group a streptococcal infections of the throat. it is the commonest form of heart disease in many developing countries. antibodies formed against a streptococcal cell wall or membrane component also react with the patient's heart muscle or valves, and myocarditis develops a few weeks later. many strains of streptococci have antigens that cross-react with the heart, and repeated infections with different streptococci cause recurrent attacks of rheumatic fever. there is genetic predisposition to the disease, based either on a particular antigen present in the heart of the patient or on a particular type of antibody response. chorea, a disease of the central nervous system, is a rare complication of streptococcal infection and antistreptococcal antibodies have been shown to react with neurons in the caudate and subthalamic nuclei of the brain. a number of microorganisms have antigens similar to host tissue components (p. ) so that in the course of responding immunologically to such infections the host is vulnerable to autoimmune damage (see ankylosing spondylitis, p. ). the antibodies to host components such as dna, igg, myofibrils, erythrocytes etc. that are seen in trypanosomiasis, mycoplasma pneumoniae, and eb virus infections appear to result from polyclonal activation of b cells (see p. ). it is not clear how important these autoimmune responses are in pathogenesis, but they reflect fundamental disturbances in immunoregulation. four types of immunopathology can be distinguished according to the classification of allergic reactions by coombs and gell, and microbial immunopathology will be described under these headings (see table . ). these depend on the reactions of antigens with reaginic (ige) antibodies attached to mast cells, resulting in the release of histamine, leukotrienes (see p. ) and heparin from mast cells, and the activation of serotonin and plasma kinins. if the antigen-antibody interaction takes place on a large enough scale in the tissue, the histamine that is released can give rise to anaphylactic shock, the exact features depending on the sensitivity and particular reaction of the species of animal to histamine. guinea-pigs suffer from bronchospasm and asphyxia, and in man there are similar symptoms, sometimes with a fall in blood pressure and shock. this type of immunopathology, although accounting for anaphylactic reactions to horse serum or to penicillin, is not important in infectious diseases. when the antigen-ige antibody interaction takes place at the body surface there are local inflammatory events, giving rise to urticaria in the skin, and hayfever or asthma in the respiratory tract. this local type of anaphylaxis may play a part in the pathogenesis of virus infections of the upper respiratory tract (e.g. common cold, respiratory syncytial virus infections of infants), or in skin rashes in infectious diseases. type reactions are common in helminth infections perhaps because ige antibodies have an important role in protection against these parasites. a dramatic type reaction can follow rupture of a hydatid cyst of echinococcus granulosus (the dog tapeworm). slow leakage of worm antigens means that mast cells are sensitised with specific ige antibody, and the sudden release of antigen can cause life-threatening anaphylaxis. when the larvae of ascaris lumbricoides pass through the lung on their journey from blood to intestine, they can give rise to igemediated respiratory symptoms, with infiltration of eosinophils. reactions of this type occur when antibody combines with antigen on the surface of a tissue cell, activates the complement sequence or triggers cytotoxicity by k cells (nk cells or phagocytes with fc receptors). k (killer) cell cytolysis is referred to as antibody-dependent cellular cytotoxicity (adcc). the antibody-coated cell is destroyed. as discussed in ch. , the same reaction on the surface of a microorganism (e.g. enveloped virus) constitutes an important part of antimicrobial defences, often leading to the destruction of the microorganism. cells infected with viruses and bearing viral antigens on their surface are destroyed in a similar way. clearly the antibody-mediated destruction of infected cells means tissue damage, and it perhaps accounts for some of the liver necrosis in hepatitis b, for instance, and probably in yellow fever. infected cells can also be destroyed by sensitised lymphocytes or nk cells independently of antibody (see below). in certain infections antibodies are formed against host erythrocytes and these cells are particularly sensitive to lysis. the haemolysis in malaria is caused by antibodies to parasite-derived antigens that have attached to red cells, rather than by autoantibodies to red cells themselves. in pneumonia due to mycoplasma pneumoniae (atypical pneumonia), antibodies (cold agglutinins) are formed against normal human group o erythrocytes. haemolytic anaemia is occasionally seen, and there is reticulocytosis (see glossary) in % of patients. the lesions in the lungs are perhaps based on cell-mediated immunopathological reactions. the combination of antibody with antigen is an important event, initiating inflammatory phenomena that are inevitably involved in the expression of the immune response. in the infected host, these inflammatory phenomena are most of the time of great antimicrobial value (see ch. ). but there are nevertheless immunopathological features of the infection, and immune complex reactions sometimes do a great deal of damage in the infected individual. the mechanisms by which antigenantibody reactions cause inflammation and tissue damage are outlined in fig. . . iga immune complexes are less harmful. antigens absorbed from the intestine can combine locally with iga antibody and the complex then enters the blood, to be filtered out in the liver and excreted harmlessly in bile (see p. ). when the antigen-antibody reaction takes place in extravascular tissues, there is inflammation and oedema with infiltration of polymorphs. if soluble antigen is injected intradermally into an individual with large amounts of circulating igg antibody, the antigen-antibody reaction takes place in the walls of skin blood vessels, and causes an inflammatory response. the extravasating poly morphs degenerate and their lysosomal enzymes cause extensive vascular damage. this is the classical arthus response. antigen-antibody reactions in tissues are not usually as serious as this, and milder inflammatory sequelae are more common as in the case of allergic alveolitis (see below), or the red zone seen round the borders of a smallpox vaccination site after seven or eight days. in the latter example circulating antibodies pass through vessel walls, meet vaccinia virus antigen in the dermal tissues and an inflammatory response is generated. a similar mild response can be induced experimentally to cause a reaction known as cutaneous anaphylaxis (see glossary), the test antigen being injected into the skin and reacting with blood-borne antibody. the resulting inflammation is detected by the visible local leakage of plasma proteins from blood vessels, circulating plasma proteins having been coloured by the intravenous injection of evans' blue. when the antigen-antibody reaction takes place in the blood to give circulating immune complexes, the sequelae depend to a large extent on size and on the relative proportions of antigen and antibody. if there is a large excess of antibody, each antigen molecule is covered with antibody and is removed rapidly by reticuloendothelial cells, which have receptors for the fc portion of the antibody molecule (see ch. ). when equal amounts of antigen and antibody combine, lattice structures are produced, and these form large aggregates whose size ensures that they are also rapidly removed by reticuloendothelial cells. if, however, complexes are formed in antigen excess, the poorly coated antigen molecules are not removed by reticuloendothelial cells. they continue to circulate in the blood and have the opportunity to localise in small blood vessels elsewhere in the body. the mechanism is not clear, but complexes are deposited in the glomeruli of the kidneys, the choroid plexuses, joints and ciliary body of the eye. factors may include local high blood pressure and turbulent flow (glomeruli), or filtering function of vessels involved (choroid plexus, ciliary body). in the glomeruli the complexes pass through the endothelial windows (fig. . ) and come to lie beneath the basement membrane. the smallest-sized complexes pass through the basement membrane and seem to enter the urine. this is probably the normal mechanism of disposal of such complexes from the body. immune complexes are formed in many, perhaps most, acute infectious diseases. microbial antigens commonly circulate in the blood in viral, bacterial, fungal, protozoal, rickettsial etc. infections. when the immune response has been generated and the first trickle of specific antibody enters the blood, immune complexes are formed in antigen excess. this is generally a transitional stage soon giving rise to antibody excess, as more and more antibody enters the blood and the infection is terminated. sometimes the localisation of immune complexes and complement in kidney glomeruli is associated with a local inflammatory response.* there is an infiltration of polymorphs, swelling of the glomerular basement membrane, loss of albumin, even red blood cells, in the urine and the patient has acute glomerulonephritis. this is seen following streptococcal infections, mainly in children (see below). as complexes cease to be formed the changes are reversed, and complete recovery is the rule. repeated attacks or persistent deposition of complexes leads to irreversible damage, often with proliferation of epithelial cells following the seepage of fibrin into the urinary space. under certain circumstances complexes continue to be formed in the blood and deposited subendothelially for long periods. this happens in certain persistent microbial infections in which microbial antigens are continuously released into the blood but antibody responses are only minimal or of poor quality (see below). complexes are deposited in glomeruli over the course of weeks, months or even years. the normal mechanisms for removal are inadequate. the deposits, particularly larger complexes containing high molecular weight antigens or antibodies (igm) are held up at the basement membrane and accumulate in the subendothelial space together with the complement components. as deposition continues, they gradually move through to the mesangial space ( fig. . ) where they form larger aggregates. mesangial cells, * see also footnote p. ; cells in kidney glomeruli, in joint synovium and in choroid plexuses bear fc or c b receptors. this would favour localisation in these tissues. of cell and tissue damage one of whose functions is to deal with such materials, enlarge, multiply and extend into the subepithelial space. if these changes are gradual there are no inflammatory changes, but the structure of the basement membrane alters, allowing proteins to leak through into the urine. later the filtering function of the glomerulus becomes progressively impaired. in the first place the glomerular capillary is narrowed by the mesangial cell intrusion. also, the filtering area is itself blocked by the mesangial cell intrusion, by the accumulation of complexes (fig. . ) , and by alterations in the structure of the basement membrane. the foot processes of epithelial cells tend to fuse and further interfere with filtration. the pathological processes continue, some glomeruli ceasing to produce urine, and the individual has chronic glomerulonephritis. circulating immune complex deposition in joints leads to joint swelling and inflammation but in choroid plexuses there are no apparent pathological sequelae. circulating immune complexes are also deposited in the walls of small blood vessels in the skin and elsewhere, where they may induce inflammatory changes.* the prodromal rashes seen in exanthematous virus infections and in hepatitis b are probably caused in this way. if the vascular changes are more marked they give rise to the condition called erythema nodosum, in which there are tender red nodules in the skin, with deposits of antigen, antibody and complement in vessel walls. erythema nodosum is seen following streptococcal infections and during the treatment of patients with leprosy. when small arteries are severely affected, for instance in some patients with hepatitis b, this gives rise to periarteritis nodosa. immune complex glomerulonephritis occurs as an indirect immunopathological sequel to a variety of infections. first there are certain virus infections of animals. the antibodies formed in virus infections generally neutralise any free virus particles, thus terminating the infection (see ch. ), but the infection must persist if antigen is to continue to be released into the blood and immune complexes formed over long periods. non-neutralising antibodies help promote virus persistence because they combine specifically with virus particles, fail to render them noninfectious, and at the same time block the action of any good neutralising antibodies that may be present. immune complexes in antigen excess are formed in the blood when the persistent virus or its antigens circulates in the plasma and reacts with antibody which is present in relatively small amounts. virus infections with these characteristics are * it is not clear how inflammation is caused. complement activation would presumably take place while complexes were circulating in the blood. perhaps the complexes bind more antibody after they have localised, or alternatively it is possible that free antigen circulates in the first place, localises, and later binds antibody to generate mediators of inflammation. included in table . . in each instance complexes are deposited in kidney glomeruli and sometimes in other blood vessels as described above. in some there are few if any pathological changes (ldv and leukaemia viruses in mice) probably because there is a slow rate of immune complex deposition, whereas in others glomerulonephritis (lcm virus in mice, adv in mink) or vasculitis (adv in mink) is severe. a persistent virus infection that induces a feeble immune response forms an ideal background for the development of immune complex glomerulonephritis, but there are no known viral examples in man. there are one or two other microorganisms that occasionally cause this type of glomerulonephritis and it is seen, for instance, in chronic quartan malaria and sometimes in infective endocarditis. in both these examples microbial antigens circulate in the blood for long periods. but immune complex deposition does not necessarily lead to the development of glomerulonephritis, and immune complexes are detectable in the glomeruli of most normal mice and monkeys. even in persistent virus infections the rate of deposition may be too slow to cause pathological changes as with ldv and leukaemia virus infections of mice (see table . ). during the acute stage of hepatitis b in man, when antibodies are first formed against excess circulating viral antigen (hepatitis b surface antigen), immune complexes are formed and deposited in glomeruli. but the deposition is short-lived and there is no glomerulonephritis. persistent carriers of the antigen do not generally develop glomerulonephritis, because their antibody is usually directed against the 'core' antigen (nephrotic syndrome in secondary syphilis) unknown causative agents man + + + + of chronic glomerulonephritis of the virus particle, rather than against the large amounts of circulating hepatitis b surface antigen. kidney failure in man is commonly due to chronic glomerulonephritis, and this is known to be mostly of the immune complex type, but the antigens, if they are microbial, have not yet been identified. immune complex glomerulonephritis occurs in man as an important complication of streptococcal infection, but this is usually acute in nature with inflammation of glomeruli, as referred to above. antibodies formed against an unknown component of the streptococcus react with circulating streptococcal antigen, perhaps also with a circulating host antigen, and immune complexes are deposited in glomeruli. streptococcal antibodies cross-reacting with the glomerular basement membrane may contribute to the picture. deposition of complexes continues after the infection is terminated, and glomerulonephritis develops a week or two later. the streptococcal infection may be of the throat or skin, and streptococcus pyogenes types and are frequently involved. when certain antigens are inhaled by sensitised individuals and the antigen reaches the terminal divisions of the lung, there is a local antigen-antibody reaction with formation of immune complexes. the resulting inflammation and cell infiltration causes wheezing and respiratory distress, and the condition is called allergic alveolitis. persistent inhalation of the specific antigen leads to chronic pathological changes with fibrosis and respiratory disease. exposure to the antigen must be by inhalation; when the same antigen is injected intradermally, there is an arthus type reaction (see p. ). there are a number of microorganisms that cause allergic alveolitis. most of these are fungi. a disease called farmer's lung occurs in farm workers repeatedly exposed to mouldy hay containing the actinomycete micromonospora faeni. cows suffer from the same condition. a fungus contaminating the bark of the maple tree causes a similar disease (maple bark stripper's disease) in workers in the usa employed in the extraction of maple syrup. the mild respiratory symptoms occasionally reported after respiratory exposure of sensitised individuals to tuberculosis doubtless have the same immunopathological basis. in addition to their local effects, antigen-antibody complexes generate systemic reactions. for instance, the fever that occurs at the end of the incubation period of many virus infections is probably attributable to a large-scale interaction of antibodies with viral antigen, although extensive cmi reactions can also cause fever. the febrile response is mediated by endogenous pyrogen (interleukin- ) and tumour necrosis factor (tnf) liberated from polymorphs and macrophages, as described on p. . probably the characteristic subjective sensations of illness and some of the 'toxic' features of virus diseases are also caused by immune reactions and liberation of cytokines. systemic immune complex reactions taking place during infectious diseases very occasionally give rise to a serious condition known as disseminated intravascular coagulation. this is seen sometimes in severe generalised infections such as gram-negative septicaemia, meningococcal septicaemia, plague, yellow fever and other haemorrhagic arthropod-borne virus diseases. immune complex reactions activate the enzymes of the coagulation cascade ( fig. . ), leading to histamine release and increased vascular permeability. fibrin is formed and is deposited in blood vessels in the kidneys, lungs, adrenals and pituitary. this causes multiple thromboses with infarcts, and there are also scattered haemorrhages because of the depletion of platelets, prothrombin, fibrinogen etc. systemic immune complex reactions were once thought to form the basis for dengue haemorrhagic fever. this disease is seen in parts of the world where dengue is endemic, individuals immune to one type of dengue becoming infected with a related strain of virus. they are not protected against the second virus, although it shows immunological cross-reactions with the first one. indeed the dengue-specific antibodies enhance infection of susceptible mononuclear cells, so that larger amounts of viral antigen are produced (see p. ). it was thought that after virus replication, viral antigens in the blood reacted massively with antibody to cause an often lethal disease with haemorrhages, shock and vascular collapse. however, it has proved difficult to demonstrate this pathophysiological sequence, and the role of circulating immune complexes and platelet depletion remains unclear. perhaps in this and in some of the other viral haemorrhagic fevers the virus multiplies in capillary endothelial cells. disease seems due to cytokines liberated from infected mononuclear cells. immune complex immunopathology is probable in various other infectious diseases. for instance, the occurrence of fever, polyarthritis, skin rashes and kidney damage (proteinuria) in meningococcal meningitis and gonococcal septicaemia indicates immune complex deposition. circulating immune complexes are present in these conditions. certain african arthropod-borne viruses with exotic names (chikungunya, o'nyong-nyong) cause illnesses characterised by fever, arthralgia and itchy rashes, and this too sounds as if it is immune complex in origin. immune complexes perhaps play a part in the oedema and vasculitis of trypanosomiasis and in the rashes of secondary syphilis. sensitive immunological techniques are available for the detection of circulating complexes and for the identification of the antigens and antibodies in deposited complexes. the full application of these techniques will perhaps solve the problem of the aetiology of chronic glomerulonephritis in man. the mere expression of a cmi response involves inflammation, lymphocyte infiltration, macrophage accumulation and macrophage activation as described in ch. , and can therefore by itself cause pathological changes. the cmi response to infection dominates the pathological picture in tuberculosis, with mononuclear infiltration, degeneration of parasitised macrophages, and the formation of giant cells as central features. these features of the tissue response result in the formation of granulomas (see glossary) which reflect chronic infection and accompanying inflammation. there is a ding-dong battle as the host attempts to contain and control infection with a microorganism that is hard to eliminate. the granulomas represent chronic cmi responses to antigens released locally. various other chronic microbial and parasitic diseases have granulomas as characteristic pathological features. these include chlamydial (lymphogranuloma inguinale), bacterial (syphilis, leprosy, actinomycosis), and fungal infections (coccidiomycosis). antigens that are disposed of with difficulty in the body are more likely to be important inducers of granulomas. thus, although mannan is the dominant antigen of candida albicans, glucan is more resistant to breakdown in macrophages and is responsible for chronic inflammatory responses. the lymphocytes and macrophages that accumulate in cmi responses also cause pathological changes by destroying host cells. cells infected with viruses and bearing viral antigens on their surface are targets for cmi responses as described in chs and . infected cells, even if they are perfectly healthy, are destroyed by the direct action of sensitised t lymphocytes, which are demonstrable in many viral infections. in spite of the fact that the in vitro test system so clearly displays the immunopathological potential of cytotoxic t cells, this is not easy to evaluate in the infected host. it may contribute to the tissue damage seen, for instance, in hepatitis b infection and in many herpes and pox virus infections. antigens from trypanosoma cruzi are known to be adsorbed to uninfected host cells, raising the possibility of autoimmune damage in chagas' disease, caused by this parasite.* it is also becoming * chagas' disease, common in brazil, affects million people, and is transmitted by blood-sucking bugs. after spreading through the body during the acute infection, the parasitaemia falls to a low level and there is no clinical disease. years later a poorly understood chronic disease appears, involving heart and intestinal tract, which contain only small numbers of the parasite but show a loss of autonomic ganglion cells. an autoimmune mechanism is possible (see p. ), because a monoclonal antibody to t. cruzi has been obtained that cross-reacts with mammalian neurons. clear that cells infected with certain protozoa (e.g. theileria parva in bovine lymphocytes) have parasite antigens on their surface and are susceptible to this type of destruction. little is known about intracellular bacteria. the most clearly worked out example of type (cmi) immunopathology is seen in lcm virus infection of adult mice. when virus is injected intracerebrally into adult mice it grows in the meninges, ependyma and choroid plexus epithelium, but the infected cells do not show the slightest sign of damage or dysfunction. after - days, however, the mouse develops severe meningitis with submeningeal and subependymal oedema, and dies. the illness can be completely prevented by adequate immunosuppression, and the lesions are attributable to the mouse's own vigorous cd + t cell response to infected cells. these cells present processed lcm viral peptides on their surface in conjunction with mhc i proteins, and sensitised cd + t cells, after entering the cerebrospinal fluid and encountering the infected cells, generate the inflammatory response and interference with normal neural function that cause the disease. the same cells destroy infected tissue cells in vitro, but tissue destruction is not a feature of the neurological disease. in this disease the cd + t cells probably act by liberating inflammatory cytokines. it may be noted that the brain is uniquely vulnerable to inflammation and oedema, as pointed out earlier in this chapter. the infected mouse shows the same type of lesions in scattered foci of infection in the liver and elsewhere, but they are not a cause of sickness or death. lcm infection of mice is a classical example of immunopathology in which death itself is entirely due to the cell-mediated immune response of the infected individual. this response, although apparently irrelevant and harmful, is nevertheless an 'attempt' to do the right thing. it has been shown that immune t cells effectively inhibit lcm viral growth in infected organs. however, a response that in most extraneural sites would be useful and appropriate turns out to be self-destructive when it takes place in the central nervous system. another type of t cell-mediated immune pathology is illustrated by influenza virus infection of the mouse. when inoculated intranasally, the virus infects the lungs and causes a fatal pneumonia in which the airspaces fill up with fluid and cells. the reaction is massive and the lungs almost double in weight. effectively the animal drowns. the cause is an influx of virus-specific cd + t cells. normally when an appropriate number of t cells had entered the lungs, the t cells would issue a feedback response to prevent such over-accumulation, but it is thought that influenza virus infects the t cells and inhibits this control process, so that the lungs are eventually overwhelmed. the action of the virus is subtle as it does not multiply in or kill the infected t cells, and it is presumed that it undergoes limited gene expression. one human virus infection in which a strong cmi contribution to pathology seems probable is measles. children with thymic aplasia show a general failure to develop t lymphocytes and cell-mediated immunity, but have normal antibody responses to most antigens. they suffer a fatal disease if they are infected with measles virus. instead of the limited extent of virus growth and disease seen in the respiratory tract in normal children, there is inexorable multiplication of virus in the lung, in spite of antibody formation, giving rise to giant cell pneumonia. this indicates that the cmi response is essential for the control of virus growth. in addition there is a total absence of the typical measles rash, and this further indicates that the cmi response is also essential for the production of the skin lesions. there is evidence that cell-mediated immune responses also make a contribution to the rashes in poxvirus infections. sometimes in infectious diseases there are prominent pathological changes which are not attributable to the direct action of microbes or their toxins, nor to inflammation or immunopathology. the stress changes mediated by adrenal cortical hormones come into this category. stress is a general term used to describe various noxious influences, and includes cold, heat, starvation, injury, psychological stress and infection. an infectious disease is an important stress, and corticosteroids are secreted in large amounts in severe infections (see also ch. ). they generally tend to inhibit the development of pathological changes, but also have pronounced effects on lymphoid tissues, causing thymic involution and lymphocyte destruction. these can be regarded as pathological changes caused by stress. it was the very small size of the thymus gland as seen in children dying with various diseases, especially infectious diseases, that for many years contributed to the neglect of this important organ, and delayed appreciation of its vital role in the development of the immune system. appreciation of the effects of stress on infectious diseases and the immune response in particular has led to the establishment of the science of neuroimmunology. properly controlled experiments are difficult to mount but there is a persuasive and growing body of evidence which shows that the nervous system affects the functioning of the immune system. the pathways of this communication are still poorly understood. work on mycobacterium bovis grew out of observations from the turn of the century that stress appears to increase the death rate in children with tb. in one type of experiment mice were stressed by being kept in a restraining device where movement was virtually impossible. this resulted in the reduction of expression of mhc class ii antigens on macrophages, which correlated with increased susceptibility to infection. similarly stressing mice infected with influenza virus caused several immunosuppressive events including reduction of inflammatory cells in the lung, and decreased production of interleukin- . suppression of antibody responses is found in people suffering a type of stress familiar to students-examinations! the best responses to hepatitis b vaccine in students immunised on the third day of their examinations were found in those who reported the least stress. finally, in a double blind trial at the common cold research unit in england with five different respiratory viruses, it was ascertained in human volunteers that stress gave a small but statistically significant increased likelihood of an individual developing clinical disease. pathological changes are sometimes caused in an even more indirect way as in the following example. yellow fever is a virus infection transmitted by mosquitoes and in its severest form is characterised by devastating liver lesions. there is massive mid-zonal liver necrosis following the extensive growth of virus in liver cells, resulting in the jaundice that gives the disease its name. destruction of the liver also leads to a decrease in the rate of formation of the blood coagulation factor, prothrombin, and infected human beings or monkeys show prolonged coagulation and bleeding times. haemorrhagic phenomena are therefore characteristic of severe yellow fever, including haemorrhage into the stomach and intestine. in the stomach the appearance of blood is altered by acid, and the vomiting of altered blood gave yellow fever another of its names, 'black vomit disease'. haemorrhagic phenomena in infectious diseases can be due to direct microbial damage to blood vessels, as in certain rickettsial infections (see p. ) or in the virus infection responsible for haemorrhagic disease of deer. they may also be due to immunological damage to vessels as in the arthus response or immune complex vasculitis, to any type of severe inflammation, and to the indirect mechanism illustrated above. finally there are a few infectious diseases in which platelets are depleted, sometimes as a result of their combination with immune complexes plus complement, giving thrombocytopenia and a haemorrhagic tendency (see also disseminated intravascular coagulation, p. ). thrombocytopenic purpura is occasionally seen in congenital rubella and in certain other severe generalised infections. infection during pregnancy can lead to foetal damage or death not just because the foetus is infected (pp. - ), but also because of infection and damage to the placenta. this is another type of indirect pathological action. placental damage may contribute to foetal death during rubella and cytomegalovirus infections in pregnant women. certain viruses undoubtedly cause tumours (leukaemia viruses, human papillomaviruses, several herpes viruses in animals) and this is to be regarded as a late pathological consequence of infection. as was discussed in ch. the tumour virus genome can be integrated into the host cell genome whether a tumour is produced or not, so that the virus becomes a part of the genetic constitution of the host. sometimes the host cell is transformed by the virus and converted into a tumour cell, the virus either introducing a transforming gene into the cell, activating expression of a pre-existing cellular gene, or inactivating the cell's own fail-safe tumour suppressor gene. the transforming genes of dna tumour viruses generally code for t antigens which are necessary for transformation, and the transforming genes of rna tumour viruses are known as one genes.* transformation has been extensively studied in vitro, and the features of the transformed cell described (changed surface and social activity, freedom from the usual growth restraints). simultaneous infection with two different microorganisms would be expected to occur at times, merely by chance, especially in children. on the other hand, a given infection generates antimicrobial responses such as as interferon production and macrophage activation which would make a second infection less likely. dual infections are commonest when local defences have been damaged by the first invader. the pathological results are made much more severe because there is a second infectious agent present. this can be considered as another mechanism of pathogenicity. classical instances involve the respiratory tract. the destruction of ciliated epithelium in the lung by viruses such as influenza or measles allows normally nonpathogenic resident bacteria of the nose and throat, such as the pneumococcus or haemophilus influenzae, to invade the lung and cause secondary pneumonia. if these bacteria enter the lung under normal circumstances, they are destroyed by alveolar macrophages or removed by the mucociliary escalator. in at least one instance the initial virus infection appears to act by interfering with the function of alveolar macrophages. mice infected with parainfluenza (sendai) virus show greatly increased susceptibility to infection with haemophilus influenzae, and this is largely due to the fact that alveolar macrophages infected with virus show a poor ability to phagocytose and kill the bacteria. specialised respiratory pathogens such as influenza, measles, parainfluenza or rhinoviruses damage the naso-* one genes (oncogenes) are also present in host cells, where they play a role in normal growth and differentiation, often coding for recognised growth factors (e.g. human platelet-derived growth factor). they can be activated and the cell transformed when tumour viruses with the necessary 'promoters' are brought into the cell. the one genes of the rna tumour viruses themselves originate from cellular oncogenes which were taken up into the genome of infecting viruses during their evolutionary history. pharyngeal mucosa and can lead in the same way to secondary bacterial infection, with nasal catarrh, sinusitis, otitis media or mastoiditis. the normal microbial flora of the mouth, nasopharynx or intestine are always ready to cause trouble if host resistance is lowered, but under normal circumstances they hinder rather than help other infecting microorganisms (see ch. ). one interesting example of exacerbation of infection occurs in mice dually infected with influenza virus and microorganisms such as streptococcus aureus or serratia marcescens. under these conditions animals suffer a more severe viral infection. this results from the need to proteolytically cleave the viral haemagglutinin protein which is done by a cellular enzyme. if the appropriate protease is in short supply or lacking completely, virions are formed but they are not infectious. under these circumstances the haemagglutinin can be cleaved extracellularly by microbial proteases with resulting increased amounts of infectious virus and disease. as a final example of dual infections, microorganisms that cause immunosuppression can activate certain pre-existing chronic infections. in measles, for instance, there is a temporary general depression of cmi; tuberculin-positive individuals become tuberculin negative, and in patients with tuberculosis the disease is exacerbated. in aids (see p. ) immunosuppression by hiv activates a variety of pre-existing persistent infections. diarrhoea deserves a separate section, since it is one of the commonest types of illness in developing countries and a major cause of death in childhood. particularly in infants, who have a very high turnover of water relative to their size, the loss of fluid and salt soon leads to life-threatening illness. it is estimated that on a global scale diarrhoea is responsible for - million deaths per year in children under five years old. in villages in west africa and guatemala the average - -year-old child has diarrhoea for about two months in each year.* diarrhoea also interacts with malnutrition and can cause stunted growth, defective immune responses and susceptibility to other infections (pp. - ). diarrhoea is also a common affliction of travellers from developed countries, * diarrhoea on a massive scale is not always confined to developing countries. there was a major outbreak of cryptosporidium infection in milwaukee, usa, in with more than cases; of these were diagnosed in the laboratory and they suffered watery diarrhoea (mean stools a day) for a mean of nine days. the small ( - μιη) oocysts, probably from cattle, had entered lake michigan, and then reached the community water supply because of inadequate filtration and coagulation treatment. and business deals, athletic successes and holiday pleasures can be forfeited on the toilet seats of foreign lands. the most reliable prophylaxis is to 'cook it, peel it, or forget it'. most attacks of diarrhoea are self-limiting. fluid and electrolyte replacement is a simple, highly effective, life-saving treatment that can be used without determining the cause of the diarrhoea. oral rehydration therapy (orf) means giving a suitable amount of salt and sugar in clean water and this is something that can be done by the mother. diarrhoea means the passage of liquid faeces,* or faeces that take the shape of the receptacle rather than have their own shape. this could arise because of increased rate of propulsion by intestinal muscles, giving less time for reabsorption of water in the large bowel, or because there was an increase in the amount of fluid held or produced in the intestine. in many types of infectious diarrhoea the exact mechanism is not known. diarrhoea, on the one hand, can be regarded as a microbial device for promoting the shedding and spreading of the infection in the community, or on the other hand as a host device to hasten expulsion of the infectious agent. diarrhoea is a superb mechanism for the dissemination of infected faeces (see p. ) and there is no doubt that strains of microbes are selected for their diarrhoea-producing powers. the advantages to the host of prompt expulsion of the infectious agent was illustrated when volunteers infected with shigella flexneri were given lomotil, a drug that inhibits peristalsis. they were more likely to develop fever and had more difficulty in eliminating the pathogen. in recent years significant strides have been made in our understanding of the pathophysiology of diarrhoeal disease. first, a quick resume of the normal structure and function of gut before attempting to understand the processes whereby it may be perturbed. the main function of the gut is the active inward transport of ions and nutrient solutes which is followed by the passive movement of water ( fig. . ). the driving force is the na + /k + atpase situated in the basolateral membrane of enterocytes on the villus (fig. . ) which maintains a low intracellular [na" "] thus creating the electrochemical gradient favourable for na + entry and, a high regional [na + ] in the intercellular spaces; cl~ follows na + . a similar situation exists in crypt cells: na + /k + atpase drives secretion. the key difference is the location of the carrier systems responsible for the facilitated entry of the actively transported species. in villus cells the carriers are present in the brush border, whereas in crypt cells they are located in the basal membrane: this is responsible for the vectorial aspects of ion/fluid traffic in villus/crypt assemblies. however, it is clear that several factors in addition to enterocytes are involved in * liquid faeces are not abnormal in all species. the domestic cow experiences life-long diarrhoea, but presumably does not suffer from it. (a) two methods of n a + cotransport are shown involving a glucose-linked symport and two coupled antiports; the latter results in the cotransport of cl~. the coupled antiports are functionally linked via h + and hc ~, the relative concentrations of which are a reflection of metabolic activity. these processes occur within the same cells but are shown separately for clarity. the driving force for n a + uptake is the low na + concentration maintained by the na + /k + pump (atpase) which creates the electrochemical gradient which promotes the inward movement of na + ; cl~ follows n a + by diffusion. water is drawn osmotically across the epithelium paracellularly (i.e. across tight junctions) and/or transcellularly, the former pathway accounting for approximately % of fluid movement. (b) secretion is the result of the coupled entry of n a + and cl~ across the basolateral membrane. n a + is recycled by the na + /k + pump and cl~ exits by diffusing down an electrochemical gradient and across the undifferentiated crypt cell apical membrane; n a + follows cl~ and water follows passively. if by whatever means, the level of nacl were to increase in such cells (as for example in rotavirus infection of neonatal mice, see below) then one could perceive how this could give rise to a cl~ driven hypersecretion of water. note, (i) the driving force results from the same mechanism that powers absorption i.e. the n a + / k + pump located in the basolateral membrane; it is the location of the 'port' 'diffusion' systems that determines the vectorial aspects of ion movement, (ii) the tight junctions are less tight in the crypts than villi. (iii) the apical membrane of the crypt cell is undifferentiated and only acquires micro villi during ascent into villous regions, φ : na + /k + pump; o: sym-, anti-port or diffusion channel. r e g u l a t i n g fluid t r a n s p o r t in t h e gut; t h e s e include t h e enteric n e r v o u s s y s t e m a n d t h e a n a t o m y of t h e microcirculation. t h e l a t t e r plays a profoundly i m p o r t a n t role in t h e u p t a k e of fluid. this is i l l u s t r a t e d in fig. . , which shows t h e existence of zones of g r a d e d osmotic fig. . schematic representation of a villus. note the central arterial vessel (av) which arborizes at the tip into a capillary bed drained by a subepithelial venous return (vr). movement of sodium into vr creates a concentration gradient between vr and av causing absorption of water from av and surrounding tissue. this results in a progressive increase in the osmolarity of incoming blood moving into the tip region through to vr. tip osmolarity is about three times higher than normal. this counter current system has been demonstrated in man and can be inferred in mice from the morphology of red blood cells which changes during ascent of the same vessel from base to tip regions of villi. the shaded areas indicate a vertical increase in osmolarity. left crypt: represents normal physiological secretion. right crypt: represents hyper secretion. ens, enteric nervous system, depicted schematically and not anatomically. potential. at the tips of villi in adult human gut, osmolalities range from to mosmkg _ h , which would generate huge osmotic forces. thus, current perceptions are that enterocytes are responsible for generating this gradient and the blood supply acts as a countercurrent multiplier which amplifies the gradient in a manner analogous to the loops of henle in the kidney. the hypertonic zone has been demonstrated directly in whole villi of infant mice in terms of the changing morphology of erythrocytes: in the lower regions of villi they show characteristic discoid morphology, whereas in the upper region they are crenated, indicating a hyperosmotic environment. the hypertonicity is dissipated if the blood flow is too slow and washed out if too fast. it is the villus unit rather than enterocytes by themselves which is responsible for fluid uptake. another consequence of the microcirculatory anatomy is that villus tip regions are relatively hypoxic. in addition, neonatal brush borders contain disaccharidases (principally lactase) which break down nonabsorbable disaccharides (e.g. lactose) into constituent absorbable monosaccharides. villus tips and crypts are regarded as the anatomical sites of physiological absorption and secretion respectively. fluid transport is a bidirectional process in the healthy animal with net absorption in health and net secretion in disease. the balance between absorption and secretion is poised at different points throughout the intestinal tract reflecting differences in both structure and function. proximal small intestine is relatively leaky; in contrast the colon is a powerfully absorptive organ. finally, crypts are the principal sites of cell regeneration, replacing cells which migrate up the epithelial escalator. the epithelium is renewed in approximately - days. at villus tips senescent cells are shed. diarrhoeal disease can result from interference with almost any one, or combination of these systems. diarrhoea-producing microbes are listed in table . . some examples are considered in detail below. rotaviruses are known to invade intestinal epithelial cells and cause diarrhoea in man, foals, dogs, pigs, mice etc. extensive multiplication takes place and very large amounts of virus ( particles g _ ) are shed in faeces. the conventional wisdom is that tips of villi especially are affected, leading to reduced absorption of fluid from the lumen. in addition destruction of enterocytes leads to a loss in lactase resulting in an accumulation of lactose in the gut causing an osmotic flux of fluid into the intestine. a major study of rota virus-induced diarrhoea in neonatal mice provides a different model of this important disease of children. oral infection of baby mice with a murine strain of rotavirus resulted in virus replication in enterocytes of the small intestine. before there had been detectable virus replication, villi became ischaemic, enterocytes severely damaged and villi shortened. in gut enterocytes this virus was demonstrably not cytopathic; the cell damage and villus responses were almost identical to that seen in experimentally induced ischaemia in physiological experiments. at the peak of virus replication, which was coincident with maximum shortening of villi, the gut was still absorptive, glucose transport intact and sufficient lactase activity remained to deal with the normal lactose load delivered to the stomach. after this the gut became secretory and diarrhoea clinically evident. villi were rapidly regenerated, but hyperaemia and diarrhoea persisted until the regeneration of the hypertonic zones as judged by the morphology of the red blood cells. peak diarrhoea coincided with the resynthesis of new villi (as judged by thymidine kinase levels, increased mitotic activity and morphometric analyses), and increased levels of intracellular nacl in the zone of cells where cell division was most active. transiently high levels of nacl have also been observed in dividing cells in culture. since ischaemic and hyperaemic villi were occasionally seen in control villi it may well be the case that the pathological changes reflect an exaggerated synchronised response of basic circulatory control mechanisms, which are part of the normal homeostatic mechanisms of villus physiology. thus, the pathophysiology of rotavirus-induced diarrhoea in neonatal mice may be summarised as follows: the reduction of red blood cells flowing through villi in the early stages of infection instigates hypoxia and hence atrophy of villi. the degree of atrophy in this infection is not associated with lack of absorption. the ensuing villus repair processes induce hypersecretion. the increase in blood flow throughout the remaining course of the infection reduces the hypertonicity of villi which impairs water absorption and thus prolongs diarrhoea. the preceding description of the self-limiting diarrhoea induced by rotavirus in neonatal mice is probably applicable to many diarrhoeas. however, the observed pathology may be different according to age, species, or the inducing pathogen. for example, in rotavirus-infected lambs, villus atrophy and crypt hypertrophy occur (the latter indicative of crypt cell division) but as in mice, infected lambs are not lactose intolerant. in rotavirus-infected piglets, crypt hypertrophy occurs but villus atrophy is severe, the animals are lactose intolerant and mortality high; a similar situation exists for the coronavirus, transmissible gastroenteritis (tge) virus. the latter has often been used as the model for infantile diarrhoea but the question is whether human infants are more like piglets or lambs. clinical studies have shown that recovery from mild, acute gastroenteritis of rota virus origin occurs within two weeks irrespective of the carbohydrate ingested. clearly, the severity of disease and the clinical outcome will depend on the extent of vertical' villus/ crypt involvement and the regions of intestine infected. when villus erosion is severe, then lactose may cause an 'osmotic' purge or be fermented by intestinal bacteria to short chain fatty acids which stimulate secretion in the colon. astroviruses, norwalk virus, certain coronaviruses, certain adenoviruses and probably toroviruses all cause gastroenteritic disease by infecting enterocytes. however, parvoviruses cause severe intestinal disease in dogs by virtue of their predilection for the mitotically active crypt cells; this causes the near complete erosion of villi similar to that seen after exposure to sublethal doses of irradiation. the classic paradigm for bacterial watery diarrhoea is cholera caused by vibrio cholerae in the small intestine. v. cholerae attaches to enterocytes of the proximal small bowel and is capable of producing at least three toxins: classical cholera toxin (ct); a toxin which disrupts the zonula occludens tight junction (designated zot); and another less well defined, auxilliary cholera enterotoxin (designated ace). as a result of toxic action, water and electrolytes are lost through the intact epithelial cells into the small intestine. as the multiplying bacteria increase in numbers and more and more epithelial cells are affected, the absorptive capacity of the colon is overwhelmed and there is profuse watery diarrhoea, as much as l l h - in severe cases.* the massive loss of isotonic fluid with excess of sodium bicarbonate and potassium leads to hypovolaemic shock, acidosis and haemoconcentration. anuria develops, and the collapsed, lethargic patient may die in - h. lives are saved by replacing the lost water and salts. the effect of toxin on an intestinal epithelial cell is long lasting, but the patient recovers as affected cells are shed and replaced in the normal fashion. the infection is particularly severe in children who easily develop low levels of plasma potassium. however, on a global scale this greatly feared disease, cholera, is only responsible for less than % of the total deaths due to diarrhoea. administration of as little as μg of purified ct alone to a healthy volunteer will reproduce the hugely dramatic, potentially lethal, purge of near isotonic fluid emphasising the central importance of ct in the causation of disease. the effect of zot has been demonstrated by electron microscopy in human biopsies, but the relative importance of zot and ace in human disease has not been quantified. how does ct work? as already outlined (pp. - ) the a subunit of this toxin is an adpribosyl transferase whose target is the a s subunit of the regulatory g protein which governs the expression of adenylate cyclase and hence the production of camp. this in turn results in the perturbation of the transport systems leading to a net efflux of ions and hence water. things are slightly more complicated, because elevation of camp occurs in villus tip cells (hence affecting absorption) but not in crypt cells. probably a signal is generated in intoxicated tip enterocytes which is transmitted to the crypt region by the ens causing release of neurotransmitters (e.g. serotonin, acetyl choline, and vasoactive peptide) which act on enterochromaffin (secretory) cells and on intestinal smooth muscle thereby augmenting crypt secretion and increasing peristalsis. studies have been made of sequential histological changes in human biopsies, taken from a series of patients from the onset of symptoms to recovery. it was clear that fluid secretion was not the result of massive desquamation of the epithelium. histological changes included 'engorgement of the capillaries' (i.e. interference with the blood supply), 'dilation of lacteals, vacuolation of enterocytes, exhaustion atrophy of enterocytes' (remember this is a noninvasive pathogen), 'accelerated shedding of cells, and increased mitotic activity'. most of these changes are similar to those described above for experimental murine rotavirus at post-peak diarrhoea and it seems that the range of histological reactions that can occur in the small intestine are limited so that qualitatively the picture is similar in different diarrhoeas. despite the undoubted importance of ct in the causation of the disease, and the potent antigenicity of ct, it is now recognised that protective immunity is very largely antibacterial. it is stopping effective colonisation which is important rather than neutralisation of the toxin. this has been partially achieved by using killed whole cell vaccines. several attempts have been made in the laboratory to genetically manipulate virulent strains (in practice this means deleting or inactivating the known toxin genes) such that the attenuated strain will colonise the gut and stimulate local immune responses and thereby prevent colonisation of the gut by virulent strains. to date, attenuated strains have been developed which fulfil these criteria but induce a mild transient diarrhoea which has prevented their adoption into vaccination programmes. however the recent emergence of v. cholerae strain (a new, third type) in india and bangladesh is a reminder that today's solution may not be adequate for tomorrow's problems. current vaccines are ineffective against this new strain. the picture withi£. coli is complicated since there are several biotypes of this pathogen which include: etec (enterotoxigenic e. coli, the principal cause of travellers' diarrhoea), epec (enteropathogenic e. coli), eiec (enteroinvasive e. coli), and ehec (enterohaemorrhagic e. coli) variants (see ch. ). the situation with etec is very similar to v. cholerae as far as heat labile toxin (lt, which is very closely related to ct) is concerned. it is additionally complicated by the fact that some strains of etec also make heat stable toxins (sts), which are nonantigenic low molecular weight peptides which activate membrane-bound guanylate cyclase which causes the production of cgmp which is the functional equivalent of camp. the 'classical' epec lesion is the formation of a characteristic pedestal-like lesion in the brush border of enterocytes with only limited invasion of the mucosa. just how it causes diarrhoea is not wholly clear. eiec is almost indistinguishable from shigella, and causes similar disease. ehec strains, which belong mainly (but not exclusively) to serogroup :h , cause a spectrum of intestinal disease ranging from asymptomatic carriage through mild diarrhoea to severe haemorrhagic colitis. they also cause extraintestinal infections such as haemolytic uraemic syndrome (hus). the initial lesion seen in the gut is similar to that caused by epec but ehec do not remain localised there: they multiply in the lamina propria and glandular crypts. while there is no conclusive proof of the involvement of slt in the pathogenesis of ehec diarrhoea there is a correlation between slt production and the severity of diarrhoeal disease or hus caused by this organism. salmonella spp. cause acute gastroenteritis and systemic typhoid disease in man, as well as other important infections in domestic animals. virulence in salmonella, especially in their natural hosts (see table . , footnote) isxomplex and mediated by numerous genes, some of which are present in otherwise nonpathogenic organisms such as e. coli. presumably all these bacteria share nutritional and environmental problems, whether inside or outside a host, that call for such genes. those restricted to salmonella are more likely to be critical in vivo. the clinical features of salmonella-induced diarrhoea (gastroenteritis) in humans and systemic typhoid infections are quite different. for example, gastroenteritis may follow - h after ingestion of contaminated food, whereas typhoid follows an incubation period of - days. diarrhoea, (which is usually watery, but may be severe, and sometimes bloody) is the predominating feature of gastroenteritis, whereas in adults, constipation is seen in the early clinical stages of typhoid; diarrhoea may occur much later. although fever may occur in gastroenteritis, in typhoid this may be so severe as to cause delirium. current perceptions are that gastroenteritis results from the initial interactions of salmonella (any one of many serotypes but frequently typhimurium or enteriditis, and/or their products) with the gut mucosa, whereas typhoid fever is produced by s. typhi organisms which translocate the mucosa, survive within macrophages, multiply and release endotoxin which triggers the highly complex endotoxin cascade. gastroenteritis is usually self-limiting, whereas in untreated typhoid mortality can be as high as %. the pathophysiology of fluid secretion caused by salmonellae is highly complex and as yet incompletely understood. the only two biological parameters which have been shown to correlate with induction of fluid secretion in a rabbit model (the best available small laboratory animal for human gastroenteritis) is the ability to invade the gut mucosa, and the induction of a leucocyte (mainly polymorphonuclear cell) influx into the mucosa and lumen of the gut. fluid secretion does not correlate per se with the ability to make an enterotoxin: avirulent as well as virulent strains make a cholera-like toxin though neither appear to release the toxin readily. the latter is therefore either not primarily involved, or is involved only after release from bacteria by means other than normal protein secretion mechanisms. these questions are as yet unresolved and the reader is referred to the reference list for a fuller discussion of these complex matters. currently, the major diarrhoeagenic pathogen of the small intestine in the developed world is campylobacter jejuni. the latter are bacteria present in wild birds, chickens and in the faeces of up to % of healthy cows in the uk. milk becomes contaminated and those who drink raw (unpasteurised) infected milk develop diarrhoea, sometimes with dysentery (blood and pus in the stools) and fever. the incubation period is - days and diarrhoea occurs after bacterial replication in the upper small intestine (jejunum). however, we are only at the beginning of understanding the detailed mechanisms and determinants responsible for disease causation by this pathogen. giardia lamblia is a noninvasive protozoan pathogen of the human small bowel which causes a spectrum of infection ranging from asymptomatic carriage through acute watery diarrhoea to chronic diarrhoea and malabsorption. giardia has a simple life cycle, existing in two forms: the multiplying trophozoite which infects mammalian hosts to cause disease and the environmentally resistant cyst. after ingestion, excystation is triggered by the ph in the stomach, and the trophozoite colonises the small bowel; the putative adherence factors have been described in ch. . the precise mechanisms involved in subsequent stages responsible for diarrhoea, malabsorption and cystation are less well understood. entamoeba histolytica causes lysis of target cells apparently by direct contact with the cell membrane. this pathogen produces under in vitro conditions a spectacular array of potential (but as yet unproven) virulence determinants including: proteases that round up cells, poreforming proteins, collagenases and oligosaccharidases and neurotransmitter-like compounds; the latter can induce intestinal fluid secretion. some of these factors have been implicated as the determinants responsible for liver abscess formation. not all diarrhoeal disease is the result of small bowel dysfunction. two important pathogens of the colon will be considered-clostridium difficile and shigella dysenteriae. c. difficile causes a spectrum of disease varied hepatitis a there are more than serotypes of salmonella, distinct from salmonella typhi and salmonella paratyphi. they are primarily parasites of animals, ranging from pythons to elephants, and their importance for man is their great tendency to colonise domestic animals. pigs and poultry are commonly affected, and human disease follows the consumption of contaminated meat or eggs. b other campylobacters cause sepsis, abortion and enteritis in animals. ranging from asymptomatic carriage through mild antibiotic-associated diarrhoea (aad) to fatal pseudomembranous colitis (pmc). normal flora play a major part-by means which are still not completely understood-in suppressing outgrowth of resident c. difficile spores. disruption of the normal flora, by antibiotic treatment for example, results in vegetative growth and production of several toxins of which c. difficile toxin a seems to be the most important in disease. this toxin is responsible for the secretory and inflammatory response in the intestine, and epithelial disruption. it probably acts by disrupting tight junctions between cells, and by causing chemotactic and other responses in polymorphs. diarrhoea could be due to the destruction of the 'absorptive epithelium' and failure to cope with the normal fluid load delivered by the small intestine, or an active type of secretion caused by replacement of the damaged epithelium, or both. the second example is that of dysentery caused by shigellae spp., in particular s. dysenteriae and s. flexneri. we have already dealt with the capacity of shigellae to invade (ch. ). invasiveness is a vital part of the virulence armoury of shigellae spp.; non-invasive mutants are a virulent. the pathogen invades the gut via m cells in peyer's patches and thence adjacent microvillus-bearing colonocytes. what is the role played by shiga toxin? mutants of s. dysenteriae devoid of the ability to make shiga toxin still retained the ability to cause lethal fulminant dysentery (low volume fluid production, pus cells, mucus) in macaque monkeys, and they invaded, multiplied within and rapidly killed hela cells. the major difference between the mutants and the toxin-positive wild type was that the latter caused a more severe disease with more haemorrhage, giving rise to blood in the stools, and, in some instances greater destruction of mucosal tissues. evidently the production of shiga toxin is not a sine qua non for causing bacillary dysentery: it exacerbates the disease. although much research has been focused on toxins, their mode of action, and their role in disease, it is useful to compare different types of intestinal infection and to refer to the concept of food poisoning. types of intestinal infection are set out in table . . food poisoning is a loosely used term, and usually refers to illnesses caused by preformed toxins in food, or sometimes to illnesses that come on within a day or so after eating contaminated food. food may be contaminated with plant poisons, fungal poisons (e.g. poisoning due to amanita phalloides), fish poisons,* heavy metals, as well as with bacterial toxins or bacteria. sourcebook of bacterial toxins the virology and immunology of lymphocytic choriomeningitis virus infection * ingestion of scombroid fish (mackerel etc.) containing large amounts of histamine or similar substances leads to headache, flushing, nausea and vomiting within an hour response of man to infection with vibrio cholerae. . clinical, serologic and bacteriologic responses to a known inoculum epitopes of streptococcal m proteins shared with cardiac myosin bacterial infections of respiratory and gastrointestinal mucosae pathogenesis and immunology of treponema pallidum t-lymphocyte stimulation by microbial antigens lessons from diarrhoeal diseases, demography to molecular pharmacology clostridium botulinum toxins: a general review of involvement in disease, structure, mode of action and preparation for clinical use disseminated intravascular coagulation: a review campylobacterpyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium typhoid fever: pathogenesis and immunologie control comparison of the alpha-toxin genes of clostridium perfringens type a and c strains: evidence for extragenic regulation of transcription pathogenic mechanism ofneisseriagonorrhoeae: observations on damage to human fallopian tubes in organ cultures by gonococci of colony type i or type rift valley fever virus in mice vi: histological changes in the liver in relation to virus multiplication viral aetiology of diseases of obscure origin campylobacter jejuni. current status and future trends cytolytic pore-forming proteins and peptides: is there a common structural motif? treatment of spasmodic torticollis with local injections of botulinum toxin pathogenesis of diseases caused by entamoeba histolytica: studies of adherence, secreted toxins and contactdependent cytolysis pituitary dwarfism in mice persistently infected with lymphocytic choriomeningitis virus tetanus and botulinum-b neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc experimental salmonella typhimurium induced gastroenteritis bacterial phospholipases c the development of respiratory syncytial virus-specific ige and the release of histamine in naso-pharyngeal secretions after infection immune complexes in human diseases key: cord- -b sbvy g authors: marques neto, lázaro moreira; kipnis, andré; junqueira-kipnis, ana paula title: role of metallic nanoparticles in vaccinology: implications for infectious disease vaccine development date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: b sbvy g subunit vaccines are safer but less immunogenic than live-attenuated vaccines or whole cell inactivated vaccines. adjuvants are used to enhance and modulate antigen (ag) immunogenicity, aiming to induce a protective and long-lasting immune response. several molecules and formulations have been studied for their adjuvanticity, but only seven have been approved to formulate human vaccines. metallic nanoparticles (menps), particularly those containing gold and iron oxides, are widely used in medicine for diagnosis and therapy and have been used as carriers for drugs and vaccines. however, little is known about the immune response elicited by menps or about their importance in the development of new vaccines. there is evidence that these particles display adjuvant characteristics, promoting cell recruitment, antigen-presenting cell activation, cytokine production, and inducing a humoral immune response. this review focuses on the characteristics of menps that could facilitate the induction of a cellular immune response, particularly t-helper and t-helper , and their potential functions as adjuvants for subunit vaccines. (tlr agonists) are immunomodulatory molecules, capable of generating a th response ( ) . there is a demand for safe adjuvants capable of inducing efficient cellular immunity, especially th and th , to be used against tuberculosis, leishmaniasis, malaria, and other diseases caused by intracellular microorganisms ( , ) . the majority of molecules with this type of adjuvanticity (th driven) are related toward the response of danger receptors to trigger inflammation, thus safety and tolerance could be major barriers that prevent their use in human vaccines ( ) . however, comparing alum and cpg/dna adjuvants in human trials, only common adverse effects, including local site reaction, flu-like symptoms and headache were observed when cpg/dna was used ( ) . also, verstraeten et al. ( ) , analyzing more than , individuals, who received vaccine-containing as , observed that only common side effects occurred. nanoparticles (nps) are classically described as structures smaller than nm and can be classified, based on their composition, as polymeric, inorganic, liposomes, immunostimulating complexes, virus-like particles, emulsions, or self-assembled proteins ( ) . they are made of different materials and differ in size, shape, and surface properties; interactions with biological systems, therefore, are varied, with several applications in modern medicine. in vaccinology, they are classically thought to have delivery and deposit properties. however, many nps have been shown to stimulate immune responses, including cell recruitment, activation of antigen (ag)-presenting cells (apcs), and induction of cytokine and chemokine release. the development of nanostructures and nanoadjuvants may therefore offer alternatives to currently used adjuvants once studies establish ways for them to elicit innate immune response and support the development of adaptive immune response in the context of vaccine formulations ( ) . metallic nanoparticles (menps) are relatively non-biodegradable, have rigid structures, and possess simple synthesis methodology. many have been studied for their immunological properties ( ) . however, there are still gaps in understanding the immune response generated by nps, especially menps. few studies have compared nps of different types and there is no standardization among published methodologies, which hampers comparisons of immunostimulatory characteristics. several important characteristics, therefore, have not been well studied. for example, how chemical and physical properties (including material composition, size, shape, surface charge, and hydrophobicity) impact vaccine immune response ( ) . this review focuses on the use of menps in formulations against infectious diseases, aiming to assess progress of their use in vaccinology and their possible applications as adjuvant. the immune response generated by menp-formulated vaccines table summarizes the articles that report the use of menps as part of vaccine formulations against infectious diseases and the immune responses they elicited. a range of immune responses is required to fight a diverse group of microorganisms. the type of protective immune response can be simplistically divided based on the type of microorganism: extracellular bacteria and toxin, intracellular bacteria, viruses, fungi, and protozoa. among the vaccines targeting extracellular bacteria and toxin, two were formulated with lipopolysaccharide (lps) in glycopeptide ag. the use of glycoantigen and lps can trigger an intense response through tlr and b cell receptor activation; the presence of gold nps (aunps) may have minimal influence on this response. however, in the work of gregory et al. ( ) and torres et al. ( ) , the use of aunps in the formulation generated a different response, improving anti-lps immunoglobulin g (igg) response, decreasing bacterial burden, generating a more efficient humoral response, and improving animal survival, showing that aunps may influence immune response and protection. using protein ag, barhate et al. ( ) formulated a vaccine using aunps and toxoid ag and demonstrated that their formulation could induce a mucosal and systemic igg and iga response. when co-administered with asparagus racemosus extract, a botanically derived adjuvant, the response was further enhanced ( ) . dakterzada et al. ( ) developed a vaccine against pseudomonas aeruginosa using the flagellin subunit and aunps that elicited an igg response comparable to that induced by freund adjuvant. flagellin is a tlr agonist but the recognition and signaling is structure dependent. this study, however, used only the - aa from flagellin and its ability to activated tlr could not be maintained ( ) . gregory et al. ( ) used an f yersinia pestis ag conjugated to aunps that induced an ab response with higher igg a associated with higher levels of interferon gamma (ifnγ), suggesting activation of th cells. among the studies that used menps in vaccine formulation, only one targeted intracellular bacteria (listeria monocytogenes). the protective immune response against intracellular bacterial infections requires th activation and, therefore, apcs activation and ag presentation through major histocompatibility complex ii (mhc ii). to generate a th response, an aunp and listeria ag formulation were used in different strategies. although the authors tested direct vaccination, when dendritic cells (dc), in vitro loaded with aunp plus listeria ag, were adoptively transferred to a naïve animal, they induced th , cd +, and natural killer (nk) cells that provided better protection against l. monocytogenes than the traditional vaccine approach ( ) . in evaluating vaccines developed with menps against viral infections, niikura et al. ( ) ( ) also evaluated the addition of cytosine and guanine linked by phosphodiester unmethylated (cpg/ dna) and found that it improved ab levels and animals' survival rates. another important feature of studies by niikura et al. ( ) and chen et al. ( ) was the use of various np sizes and the demonstration that all different np shapes were capable of inducing a humoral response. the levels of ab were size dependent, but the results were inconsistent: the first study found that a nm sphere was the most efficient ab inducer and the second study found that the nm and nm spheres performed best. a special case of the use of menps was the use of nickelfunctionalized nanolipoprotein particles (ninlps) by yan et al. ( ) and wadhwa et al. ( ) in combination with hiv ag. ninlps are nanometer-sized nanolipoprotein particles with nickel incorporation into their surface in order to induce polyhistidine tagged proteins adsorption ( ) . they demonstrated that specific igg (igg and igg a) levels were greater than those obtained when alum was used in the formulation. fischer et al. ( ) used truncated wnv envelope protein ag and found that a single dose vaccination induced a superior anti-wnv igg response and improved protection against a wnv challenge ( ) . these responses were associated with nickel functionalization, described as a hapten, and triggered responses through activation of human tlr and intracellular transduction signals through myeloid differentiation primary response (myd- ), nuclear factor-κb (nf-κb), and mitogenactivated protein kinase (mapk), inducing pro-inflammatory responses [tumor necrosis factor (tnf)-α and interleukin (il)- ] ( , ) . for protozoan infections, parween et al. ( ) , using plasmodium falciparum merozoite surface protein subunit and aunps, evaluated the humoral immune response (igg , igg a, igg b, and igg ) and found an intense igg response compared with the alum formulation ( ). kaba et al. ( ) , using p. berghei circumsporozoite protein and aunps, generated long-lasting protective immunity with th that produced il- and mixed high avidity igg /igg a (th /th ) ( ) . in other studies, these authors replaced ag with p. falciparum circumsporozoite protein; vaccination was shown to induce protective cytotoxic (cd +) cells, high avidity ab titers, and specific effector memory, central memory, and long-term central memory cd + t cells in draining lymph nodes, spleen, and liver ( ) . this response was shown to be generated by dc cross-presentation, which had delayed fusion and interaction of endosomes with lysosomes caused by the aunp formulation ( ) . finally, pfmsp was used with dextrancoated iron oxide nps (ionps) and was capable of inducing a humoral response in two animal models (mouse and monkey). this response was also shown to inhibit parasite growth by - % ( ). most studies evaluated immunogenicity through measurement of the humoral immune response. according to their findings, the use of nps was efficient in inducing an ab-based response. based on heavy chain structure, there are five types of ab, each with a different role: igg, igm, iga, igd, and ige. igg and iga can be subdivided as igg , igg , igg , igg , iga , and iga based on additional small differences in their heavy chain. with regard to vaccination, humoral immunity is especially important in responding to infection by extracellular pathogens, toxins, protozoa, and viruses. its importance is associated with the biological activities of immunoglobulins, including microorganism opsonization and phagocytosis; complement activation ( ); toxins and microorganism neutralization ( ) ; and mast cells and basophil activation ( , ) . in addition, immunoglobulins can help target cytotoxicity against infected cells (ab-dependent cell cytotoxicity of cd t cells and nk). in some cases, however, the pathogens have the ability to evade the humoral system or can even use immunoglobulins as a way to facilitate cell invasion, as in the cases of mycobacterium tuberculosis and leishmania spp. ( , ) . the studies described above clearly show that menps (gold, iron, and nickel) can be used for vaccine development. different menps were used in conjunction with several ag for distinct microorganisms and showed the ability to generate humoral and cytotoxic responses. although the generation of igg a and ifn-γ shown in some studies are indicators of th responses using menps as adjuvant, further research is needed to specifically assess the role of different menp vaccines in th induction. to understand the possible uses of menps as platforms for vaccines against infectious diseases, analysis is needed of the impact of different physicochemical characteristics of nps on the innate immune response (figure ) . several strategies have included menps as vaccine platforms, involving menps of different materials (including gold, iron oxide, and nickel); shapes (including spheres, cubes, rods, and disks); sizes (from nm to over nm); and types of coating [e.g., citrate, chitosan, dextran, or cetyltrimethylammonium bromide/ -styrenesulfonic acid-comaleic acid (ctab/pss-ma)]. the material from which an np is made has a direct influence on the functions of apcs; gold nps (aunps) have been most commonly used in vaccinology ( table ) . the most recent studies involving aunps demonstrate the effects of gold sodium thiomalate on macrophage function, showing lysosomal enzyme inhibition and reducing phagocytosis ( ) . similar effects were seen in macrophages of several origins, which, when stimulated with aunps, showed diminished bactericidal activity against staphylococcus aureus ( ) and low or absent cytokine production il- , il- , and tnf-α ( , ) . moreover, when splenocytes were stimulated with lps, the addition of aunp reduced il- and tnf-α release ( ) . some of these results raise the concern on the use of aunps as adjuvants, since these immunomodulatory properties can act inhibiting the generation of th . however, the response to aunps is also correlated with other physicochemical characteristics that will be discussed below, which may be tailored to improve immunostimulatory or immunomodulatory capacity. iron oxide nanoparticles have also been used as adjuvants. iron is an important ion in the homeostasis of all cells and in generating immune responses to several microorganisms. the effect of ionps phagocytosis have been explored in several studies, for example, m macrophages after exposure to ionps induced reactive oxygen species (ros), but after h induced il- production ( ) . the use of ionps in balb/c mice demonstrated the immunomodulatory capacity of this np by diminishing splenocyte cytokine production (il- and ifn-γ) ( ) as well as suppressing the response to pancreatic ag in diabetic mice ( ) . sindrilaru et al. ( ) , however, showed that macrophages, under iron overloaded conditions, became unrestrained m (with an incomplete switch to m macrophages) and produced more tnf-α, which impaired wound healing and had an important role in the immunopathology of chronic venous leg ulcers. consequently, ionp response seems to have direct correlation with time and dose, once iron overload seems to be a requisite to developed pro-inflammatory response and this aspect must be evaluated to avoid the inhibition of the desired immune response. other critical characteristics are the shape and size of nps, which have a direct impact on vaccine efficiency, ag load capacity, and interaction with cells (phagocytes and apcs). these characteristics have been studied in different nps; shah et al. ( ) published a review focusing on the impact of size for alum, oil-in-water, emulsion, polymeric particles, and liposome adjuvanticity, but did not evaluated menps. in the studies reviewed here, np sizes range from nm nanospheres to nm nanocapsules. two authors have evaluated the impact of size and shape for menps ( table ) : chen et al. ( ) evaluated differences in immune response based on aunp sizes (ranging from to nm nanospheres) and found that and nm were the most drained np ( ); niikura et al. ( ) went further and, using four different shapes of np ( nm sphere, nm sphere, cube, and rod), showed that ab responses and tnf-α were directly correlated with the specific np surface area (the ratio of the total surface area per single np volume). furthermore, nm spheres appear to be the most efficient in generating immune responses (il- and il- ) and granulocyte macrophage colony-stimulating factor production. surface charge and hydrophobicity are additional important np characteristics for immune response induction and are directly influenced by np functionalization (chemical modification of nps surface by adding or replacing functional groups) and coating (ag) ( ) . most studies used citrate-coated nps, but dextran and ctab/pss-ma have also been used; all three result in negatively charged (anionic) particles. only one np, revised here, used positive charged (cationic) functionalization [( ); table ]. the higher hydrophobicity of aunp was shown to activate the innate immune system (tnf-α secretion) ( ) . although the surface charge of other non-metallic nps has been studied ( ) , to our knowledge the studies using menps did not address the other characteristics associated with immune response induction. for non-metallic nps, it appears that a positive charge signified a greater ability to induce immune responses than a negative charge. interestingly, negatively charged non-metallic nps were associated with ag-specific tolerance ( ) . further studies are needed to investigate whether or not the charge imputed by np coating influences the immune response. though the size and shape of menps had little to no impact on the innate response elicited, coating modifications may improve the capacity of these molecules to influence immune responses. finally, it is important to note that the majority of adjuvant characteristics were evaluated using non-metallic nps. t-helper cells are associated with immunity against intracellular pathogens and the secretion of ifn-γ, which, in turn, is essential for the activation of mononuclear phagocytes, including macrophages, resulting in enhanced phagocytic activity ( ). th cells (il- a and il- f producer cells) are associated mainly with stimulation and chemotaxis of neutrophils to the site of inflammation. however, their function goes beyond this and includes the targeting of various cells types, including nonlymphoid cells and the stimulation of cytokine, chemokine, and prostaglandin production. another characteristic of these cells is their memory effector subset, which is maintained in mucosal tissues for extended periods. this subset has high plasticity and is able to transform into th or th phenotypes depending on the cytokine milieu at mucosal sites. this diversity of function and actuation make th cells very important in defense against several microorganisms, mainly those acquired through mucosal routes ( , ) . t-helper and th cells have their own distinct sets of functions and differentiation factors. both cell types require t cell receptor downstream activation by ag presentation cells through mhc ii and co-stimulatory molecules ( ) . consequently, cytokine release during ag presentation is correlated with the type of adaptive immune response generated. while th differentiation requires stimulation by il- , th generation requires transforming growth factor-β and il- . however, this generation is influenced by other factors and how menp are involved in the possible induction of th or th will be discussed below. in this review, only one study investigated the development of the direct th (type t helper cell) and th response. using a listeria ag, rodriguez-del rio et al. ( ) showed that in contrast to advax™ adjuvant alone, a combination of nm aunps and advax™ was capable of inducing the highest th response. pusic et al. ( ) immunized mice with ionps covered with rmsp , a p. falciparum merozoite ag, and showed that after immunization (intramuscular, subcutaneous, or intraperitoneal), production of il- was greater than that of ifn-γ, suggesting a predominant th response (although the cellular immune response was not directly evaluated). the first major determinant in generating th and th populations is the route of vaccine administration, which dictates the cell dynamic and initial response to the vaccine. for example, mohanan et al. ( ) , in a cross-sectional study using a liposome plus ag (ova) vaccine formulation, compared intradermal (high igg ; intermediate igg ; and ifn-γ), intralymphatic (high igg , igg , and ifn-γ), intramuscular (high igg ; intermediate igg and ifn-γ), and subcutaneous (high igg ; low igg and ifn-γ) routes of administration ( ) . the predominant th response to administration through the intradermal route was most likely due to the cooperation between langerhans cells, the primary innate immune response cells and keratinocytes that may also be stimulated by the formulation. these elicited the production of cytokines and chemokines that helped in the activation of other apcs ( ) . the early phase of vaccination is characterized by recruitment of neutrophils and monocytes to the site of inoculation. both cell types can also act as apcs, delivering ag-specific and co-stimulatory signals to t cells. their collaborative endeavors have been found to modulate (positively or negatively) the activity of different effector t cell subsets ( , ) . neutrophils are the first cell lineage to migrate to inflammation sites and, when stimulated, they produce cytokines and chemokines that will attract and activate other cell types. for example, neutrophils were shown to be an important inducer of th and th cells ( ) , but their role in cytokine secretion is much broader ( ) . moreover, signals may elicit different function in neutrophils and therefore, influence the quality of t cell responses. for example, aunps have been described as capable of inducing neutrophil extracellular traps, which act as damage-associated molecular patterns and stimulate immune system through dna receptors such as tlr ( ) . upon stimulation by nps (tio -titanium dioxide-and alum), duffin et al. ( ) demonstrated neutrophil influx to the lungs and also induced production of il- . silver nps were also shown to be capable of interacting with neutrophils, inducing apoptosis of these cells, and inducing caspase- \ caspase- partially dependent il- β secretion ( ) . in another study, cobalt and nickel nps were shown to induce higher nitric oxide, tnf-α, and cxcl chemokine production, by human peripheral blood neutrophils, than titanium nps (tio np) ( ) . nonetheless, tio nps also induced polymorphonuclear cell activation through phosphorylation of several proteins, including p mapk and extracellular signal-regulated kinases- / (erk- / ), which were associated with increased neutrophil life-span and production of several cytokines and chemokines ( ) . classically, apcs, macrophages, and dcs act at the site of vaccine inoculation by sensing foreign agents, through tlrs and other receptors, and triggering inflammation. apcs play a key role in the initiation, maintenance, and selectivity of inflammation, through their three major functions: endocytosis, ag presentation, and production of various cytokines and growth factors ( ) . the main family of pattern recognition receptors in microbial recognition is the tlrs, part of the family of transmembrane proteins, which affect the transcription of genes involved in inflammatory and immune response-enhancing cellular activities such as phagocytosis, endocytosis, cytotoxic functions, and cytokine production ( , ) . the adjuvants most frequently used for the induction of th and th responses are tlr agonists, such as as , cpg/ dna, and others. menps seems to have capacity to induce the expression of toll-like receptors, such as tio nps and zirconium oxide nps that have been described to enhance tlr , tlr , and tlr expression in macrophages ( ) and tlr and tlr in mouse liver cells ( ) . zinc oxide nps (plus ova) generated an inflammatory response in balb/c mice and also improve tlr- , - and - expression, followed by activation of src family kinases ( ) . consequently, tio nps and ionps were shown to induce dc upregulation of co-stimulatory molecules (mhc ii, cd ) ( , , ) , which can also be related to tlr stimuli pathways. however, none of these works demonstrate the direct interaction of nps with tlr (using knock-out mice, agonists, or antagonist molecules) thus, this interaction must be further studied. the next step in the generation of adaptive responses is the tailoring of cytokine secretion by apcs at immunological synapses, which will guide the development of the response. several nps have been reported to trigger cytokine and chemokine production, which may be used as biomarkers for immunotoxicity ( ). among those described, tio nps were used in mimetic systems composed of blood vein endothelial component (including pbmc) and was reported to trigger pro-inflammatory cytokines (il- , ifn-γ, and tnfα) ( ); zinc oxide nps were shown to be preferentially associated with monocytes and, when used in pbmc, induced ifn-γ, tnf-α, and il- cytokine production ( ); aunp-stimulated bone marrow-derived dc produced il- , tnf-α, and ifn-γ ( ) ; and ionps were shown to induce the activation of apcs with an increase of il- , tnf-α, ifn-γ, and il- , as well as chemokines. the response generated by ionps, however, was weaker than that generated by the positive control lps which may be beneficial in controlling possible side effects ( ) . the generation of a cellular response associated with protection against intracellular pathogens is the ultimate goal of vaccination. however, the direct effects of nps on cellular responses have been evaluated in only a few studies. tio nps were shown to activate and induce proliferation of naïve cd + t cells and to generate a pronounced th response with ifn-γ and tnf-α production, associated with pro-inflammatory cytokine production (il- , il- a, il- b) and dc maturation (cd + and cd + expressions increase). schanen et al. ( ) hypothesized that the oxidative capacity of an np could impact the response and trigger pro-inflammatory (oxidant capacity) or anti-inflammatory (antioxidant capacity) responses. this oxidant effect could control ros generation and thus control downstream pro-inflammatory effects while antioxidants prevent the initiation of the innate immunity in lps-stimulated macrophages ( ) . this study was, however, conducted with mitogens (non-specific stimuli) and not with vaccine stimuli, but nevertheless serves as a warning about the direct action of nps, not only on the innate immune system but specifically on t cells. there is enough evidence to suggest that menps are not only particulate formulations but also immunostimulatory molecules with several studies demonstrating their capacity to generate humoral and cytotoxic responses. menps clearly have figure | metallic nanoparticles adjuvanticity and its prediction capacity to generate t-helper (th ) and th responses. to generate a cellular immune response, the np must be able to be recognized by the host innate immune response and stimulate a sequence of events that will lead to the release of a specific milieu of cytokines and better antigen presentation (bottom arrow). in the top arrow is the immune response elicited by metallic nanoparticles to aid th and th generation. nf-κb, nuclear factor kappa b; ccl, chemokine ligand; cxcl, chemokine (c-x-c motif) ligand; gm-csf, granulocyte macrophage colonystimulating factor; ifn, interferon; il, interleukin; m-csf, macrophage colony-stimulating factor; myd, myeloid differentiation factor; tcr, t cell receptor; th, t-helper cell; tlr, toll-like receptor; tnf, tumor necrosis factor. immunostimulatory capacity and can induce several reactions in all phases of vaccine development. these capabilities correlated with np physicochemical characteristics such as size, charge, and hydrophobicity, but there are several gaps in our understanding of their mechanism of actions and how they may lead to adjuvanticity, immunomodulation, or tolerance to the ag formulated with nps. there are also evidence of menp being capable of help in the generation of th and th ; figure presents an overview of the generation of these cells subsets and the possible role of menp in this induction. ln designed the review and wrote the first draft. aj-k edited the first draft and critically reviewed the manuscript. ak edited the first draft and critically reviewed the manuscript. all authors read and approved the final version of the manuscript and agreed to submission. this work is part of ln phd thesis at biotechnology and biodiversity graduate program from capes. this work was funded by fapeg (grant number - ) and cnpq (grant number: / - ). ln received a phd fellow from capes, and apjk (# / - ) and ak (# - - ) received a productivity research fellow from cnpq. key roles of adjuvants in modern vaccines novel adjuvant formulations for delivery of anti-tuberculosis vaccine candidates landmark 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tio( ) nanoparticles the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -wjx xki authors: da silveira, matheus pelinski; da silva fagundes, kimberly kamila; bizuti, matheus ribeiro; starck, Édina; rossi, renata calciolari; de resende e silva, débora tavares title: physical exercise as a tool to help the immune system against covid- : an integrative review of the current literature date: - - journal: clin exp med doi: . /s - - - sha: doc_id: cord_uid: wjx xki acute viral respiratory infections are the main infectious disease in the world. in , a new disease caused by severe acute respiratory syndrome coronavirus (sars-cov- ), coronavirus disease (covid- ), became a global pandemic. the immune response to the virus depends on factors such as genetics, age and physical state, and its main input receptor is the angiotensin-converting enzyme . the practice of physical exercises acts as a modulator of the immune system. during and after physical exercise, pro- and anti-inflammatory cytokines are released, lymphocyte circulation increases, as well as cell recruitment. such practice has an effect on the lower incidence, intensity of symptoms and mortality in viral infections observed in people who practice physical activity regularly, and its correct execution must be considered to avoid damage. the initial response is given mainly by type i interferons (ifn-i), which drive the action macrophages and lymphocytes, followed by lymphocyte action. a suppression of the ifn-i response has been noted in covid- . severe conditions have been associated with storms of pro-inflammatory cytokines and lymphopenia, as well as circulatory changes and virus dispersion to other organs. the practice of physical activities strengthens the immune system, suggesting a benefit in the response to viral communicable diseases. thus, regular practice of adequate intensity is suggested as an auxiliary tool in strengthening and preparing the immune system for covid- . further studies are needed to associate physical exercise with sars-cov- infection. acute respiratory infections (aris) are caused by respiratory viruses and bacteria, being the most infectious disease in humans [ , ] . these can be caused by more than different viruses, with rhinovirus being the most common etiological agent [ ] [ ] [ ] . in december , a new coronavirus outbreak was reported in china, being called the severe acute respiratory syndrome coronavirus (sars-cov- ), spreading rapidly and infecting more than million people, being declared a health emergency international public service on january , [ , ] . the main mode of transmission is contact with droplets containing viral particles eliminated through the cough or sneeze of an infected person, and the incubation period usually varies from to days. approximately % of the cases are asymptomatic or with mild symptoms, and the others can be severe or critical and can lead to death [ ] . the development of coronavirus disease (covid- ) is dependent on the interaction between sars-cov- and the host's immune system, the immune response being influenced by genetics (hla genes), age, sex, nutritional status and status physical [ ] . the immune response includes two stages, innate immunity and adaptive immunity. the first one comprises physical and chemical barriers and the action of cells such as macrophages, dendritic cells (dcs), natural killer cells (nks), neutrophils and molecules such as cytokines, interleukins (ils), nitric oxide (no) and superoxide anion (o -). the second one has as mechanism of action the t lymphocytes (tcd + and tcd +) and b lymphocytes and their products, such as antibodies and cytokines. furthermore, the adaptive immune response can be subdivided into cellular immunity (mediate by cells as macrophages and lymphocytes) and humoral immunity (mediates by cells as macrophages and lymphocytes) and humoral immunity (mediated by antibodies) [ , ] . the regular practice of physical exercises promotes improvements in quality of life and can act in the immune response, reducing the risk of developing systemic inflammatory processes and stimulating cellular immunity [ ] . therefore, the present article aims to perform an integrative review of the literature relating the role of physical exercise on the immune system in the fight against covid- . for this purpose, the bibliographic study included knowledge about respiratory infections, influences of physical exercise on the immune system and proposed the comprehension of the most recent information about the immunopathogenesis of sars-cov- infection, also comprising its relationship with the host's physical and health conditions. physical activity is considered one of the main components of healthy living. in addition to the functions related to the prevention of excess body weight, systemic inflammation and chronic non-communicable diseases, a potential benefit of physical exercise in reducing communicable diseases, including viral pathologies, is suggested [ ] . the practice of physical exercise, both in its acute form and in its chronic form, significantly alters the immune system [ , ] . studies indicate that the modulation of the immune response related to exercise depends on factors such as regularity, intensity, duration and type of effort applied [ , ] . moderate-intensity physical exercises stimulate cellular immunity, while prolonged or high-intensity practices without appropriate rest can trigger decreased cellular immunity, increasing the propensity for infectious diseases [ , ] . according to the international society for exercise and immunology (isei), the immunological decrease occurs after the practice of prolonged physical exercise, that is, after min of moderate-to high-intensity physical activity [ ] . cellular changes due to physical activity are illustrated in fig. . cytokines are classified as anti-inflammatory and proinflammatory according to their functions. among the antiinflammatory cytokines, we highlight il- and transforming growth factor-beta (tgf-β), responsible for inhibiting the production of pro-inflammatory cytokines [ ] . among the pro-inflammatory cytokines, we highlight il- , il- , il- , il- , interferon-gamma (ifn-γ) and tumor necrosis factor-alpha (tnf-α) [ ] . cytokine production can be modified due to hormonal or oxidative stress and physical exercise. the muscle contraction has the effect of increasing the release of antinflammatory and pro-inflammatory cytokines at levels that vary according to the volume of contractile mass involved, duration and intensity of exercise [ ] . during the practice of physical exercise, the activation of the muscle fiber is responsible for increasing the release of calcium (ca +) and, therefore, promoting the synthesis of proinflammatory cytokines, namely tnf-α and il- β, which act in the regulation of selectins, which, in turn, attract neutrophils to the site [ ] . the neutrophilia induced by physical activity is due to the release of neutrophils from the bone marrow due to the influence of cortisol [ ] . after aerobic physical exercise (approximately h), there is a significant reduction in neutrophil chemotaxis, however, without compromising bactericidal activity. the reduction in neutrophil chemotaxis is reversed within h after physical activity, during which the opportunistic activity of infectious microorganisms can occur [ ] . physical activity is also responsible for increasing the concentration of circulating leukocytes [ ] . this is due to shearing of immune cells in blood vessels, especially secondary lymphoid tissues such as liver, spleen and lung [ ] . the leukocyte concentration remains high with a peak of - min after constant physical activity, which may persist for up to h after [ ] . the practice of aerobic physical exercise in an exacerbated manner is responsible for decreasing the expression of tolllike receptors (tlrs) in macrophages, considerably reducing the presentation of antigens to t lymphocytes, thus causing the suppression of the inflammatory t helper type (th ) response. thus, the failure to develop an inflammatory activity precludes possible tissue damage resulting from inflammatory mediators and, consequently, the risk of chronic inflammatory processes. however, the susceptibility to infections due to intracellular microorganisms increases [ ] . during physical activity, blood flow increases in order to supply the metabolic demands of the human body. the recruitment of nk cells occurs through cellular stress promoted by exercise and a consequent decrease in adhesion molecules induced by catecholamines [ ] . however, physical activity lasting more than three hours causes the concentration of nk cells to return to the pre-exercise state or even lower than this. this is because the nk cells migrate to the muscle injury site [ ] . during moderate physical exercise, the concentration of lymphocytes increases in the vascular bed and, after strenuous exercise, decreases to levels below the pre-exercise period [ , ] . the cd +:cd + ratio decreases as tcd + cells increase [ ] . tcd + cells decrease due to the increase in nk cells [ , ] . after physical activity, the lymphocyte concentration decreases due to the apoptosis mechanism [ ] . thus, the increase in lymphocyte concentrations favors the th mediated immune response, preventing infections by intracellular microorganisms [ ] (fig. ). sars-cov- is constituted by single-strand positivesense rna and belongs to the genus betacoronavirus, lineage b and subgenus sarbecovirus. viral genome studies have identified similarity of sars-cov- with bat coronaviruses, as well as coronaviruses responsible for two previous pandemics: the severe acute respiratory syndrome-related coronavirus (sars-cov) and the middle eastern respiratory syndrome coronavirus (mers-cov) [ , ] . similar to sars-cov, the novel coronavirus uses a structural glycoprotein to infect cells: the envelope spike protein (s), using the angiotensin-converting enzyme- (ace ) as entry receptor [ , ] . ace consists of a cell membrane protein abundantly expressed in the organism, which is present in cardiac, pulmonary, renal, intestinal and vascular cells, and the binding of sars-cov- with this enzyme has a strong affinity, which may explain the high transmissibility of the virus [ , , ] . due to mainly respiratory symptoms, it is believed that the target cells of sars-cov- are in lower airways [ ] , as is the case of type pneumocytes or alveolar cells, the main site of expression of ace receptors [ ] . the result of the interaction between the s receptor binding domain (rbd) and ace is the fusion of the viral and host membranes, which proceeds for viral replication and dissemination and may reach the other cells with ace expression in the organism [ ] . with the purpose of containing the infection, the innate and adaptive immune system is activated by mechanisms that are not yet completely elucidated. in spite of this, it is known that effective immunological actions are essential to control viral replication and dissemination, cellular inflammation and tissue injury, and many studies have reported that the immune response of the host influences the severity of covid- [ , [ ] [ ] [ ] . to initiate the antiviral response, cells of the innate immune system need to recognize the infection, a process performed through pattern-recognition receptors (prrs) such as tlr, nod-like receptor (nlr), c-type lectinlike receptor (clr), rig-i-like receptor (rlr) and freemolecule receptors in the cytoplasm, which detect pathogen-associated molecular patterns (pamps). once viral nucleic acids are recognized as pamps, prrs activate molecular pathways of inflammatory response, stimulating chemotaxis, maturation of immune cells, phagocytosis and expression of inflammatory factors [ ] . viral recognition by tlr , tlr and rig-i receptors leads to activation of the nuclear factor-κb (nf-κb) and irf signaling cascade, with nuclear transcription and expression of type i interferons (ifns-i) and pro-inflammatory cytokines, creating the first line of defense against viral infections. the ifns-i (ifn-α and ifn-β) are the most important antiviral cytokines, and they act as immunomodulators influencing the activities of macrophages and lymphocytes, performing actions such as protection of non-infected cells, containment of viral replication and effective activation of the adaptive immune system [ , , ] . a suppression or delay of ifns-i response-due to viral evasion mechanisms-results in impairment of early infection control, hyperinflammatory infiltrate of neutrophils, macrophages and monocytes into the lungs, production of cytokines by these cells and lung tissue damage. this process, described in sars-cov and mers-cov, has been suggested as a possible strategy to trigger or collaborate to the pathology of covid- [ , ] . macrophages and dcs act as apcs for lymphocytes via mhc and produce a microenvironment of signaling cytokines, activating the adaptive immune system. t lymphocytes perform important functions against viral microorganisms, since tcd + can cause direct cytotoxicity against infected cells and tcd + stimulate b lymphocytes to produce neutralizing antibodies. in turn, t helper lymphocytes (th)-predominantly th -contribute to the organization of the adaptive response and release cytokines that are able to recruit monocytes and neutrophils and to promote other cascades of pro-inflammatory molecules, amplifying the immune response [ , ] (fig. ). the complement system can also be activated and has an important role in coronavirus infections, because it helps the innate immune system to identify antigens. however, its activation can contribute to the disease due to its potent capacity to stimulate neutrophils and to recruit inflammatory cells, which can trigger tissue damage [ ] . studies suggest that in mild cases, pulmonary tissue macrophages are capable of containing sars-cov- and innate and adaptive immune responses are efficiently activated against viral replication. however, severe cases of covid- are associated with an imbalance in antiviral immunity, characterized by two main situations: a pro-inflammatory cytokine storm and a lymphopenia state [ , ] . the degree of lymphopenia and cytokine storm was related to the severity of covid- [ ] , and similar situations had already been reported in other respiratory viral infections, including influenza, sars-cov and mers-cov. activation of the complement system and abnormalities in coagulation were also observed in severe patients with markers such as c-reactive protein (crp), dimero-d and fibrin degradation products, which are usually elevated in advanced stages of the disease [ , ] . the mechanism of cytokine storm and lymphopenia associated with circulatory alterations and viral dissemination to several organs was proposed as being responsible for viral sepsis. among the complications of this condition are acute respiratory distress syndrome (ards), septic shock, multiple organ failure (mof) and death [ , ] . in patients with severe covid- , increased levels of cytokines were observed, including il- β, il- , il- , il- , il- , il- , interferon-gamma (ifn-γ), tumor necrosis factor alpha (tnf-α), granulocyte-colony stimulating factor (g-csf), gamma-induced protein (ip ), monocyte chemoattractant protein (mcp ), macrophage inflammatory protein alpha (mip -α) and other molecules, characterizing the cytokine storm [ , ] . additionally, elevations of il- β, ifn-γ, ip and mcp in infections by the novel coronavirus were associated with the th response; however, an increase in interleukins of the t helper type (th ) profile, such as il- , il- , il , which suppress the inflammation, was also associated with a greater severity of covid- , which may demonstrate an imbalance in immune regulation and an attempt to minimize tissue inflammatory damage [ , ] . the cytokine storm generates an immunological system attack against the organism, which can cause substantial lesions in organs such as the lung, heart, brain, kidneys, spleen, liver and lymph nodes. the increase of neutrophils, macrophages and monocytes in association with a dysfunction of the ifn-i response has been reported as the main cause of lethality in sars-cov and mers-cov pneumonia, and a similar conclusion has been suggested for sars-cov- [ , , ] . in the lungs, alveolar macrophages and epithelial cells are the most responsible for the production of cytokines and chemokines. during infection by the novel coronavirus, excessive secretion of these molecules by the immune cells mediates a massive pulmonary infiltrate of neutrophils, monocytes and macrophages, which results in alveolar damage due to wall thickening and formation of hyaline membranes. in addition, the accumulation of neutrophils in the lungs increases the production of reactive oxygen species (ros) and pro-inflammatory molecules, predisposing to injury [ ] . therefore, high levels of pro-inflammatory cytokines are associated with respiratory insufficiency, ards and may lead to shock, mof and death in the covid- [ , ] . in comparison, pulmonary disease caused by sars-cov also presents with formation of hyaline membranes, desquamation of alveolar space pneumocytes and interstitial infiltration with lymphocytes and mononuclear cells. in the serum of patients who develop sars, there are high levels of cytokines and pro-inflammatory chemokines [ ] . additionally, it has been observed that during viral infections, pro-inflammatory cytokines can stimulate an increase in the levels of ace protein, the receptor for sars-cov- . a larger quantity of this protein may accelerate the entry of the novel coronavirus into the host cells and contribute to its dissemination in the organism, negatively influencing the antiviral response [ ] . in critically ill patients, a state of immune suppression is also described, with a significant and sustained decrease in the absolute number of tcd + and tcd + lymphocytes, possible reduction of b lymphocytes, nk cells, monocytes, eosinophils and basophils [ , ] . the study conducted by liu et al. [ ] did not observe significant changes in the total counts of b lymphocytes, nk cells and monocytes; however, there was a considerable reduction in the lymphocyte count in peripheral blood of patients with severe covid- at the beginning of the disease, especially cytotoxic tcd + cells [ ] . activation markers for tcd + and tcd + lymphocytes showed excessive stimulation and exhaustion markers for tcd + lymphocytes were elevated in the disease, suggesting lymphocyte dysfunction [ , ] . additionally, an increase in the number of neutrophils and a greater neutrophil-to-cd + t cell ratio (n r) was associated with severe covid- and proposed as the most significant predictor of poor prognosis [ , ] . cell death induced by interaction between fas and fas ligand by activation of the tnf-related apoptosis inducing ligand axis and by direct infection of t lymphocytes by sars-cov- may be responsible for the origin of lymphopenia in covid- [ ] . the cytokine storm can also influence the lymphopenia, since in the study of liu et al. [ ] , the peaks in cytokine levels il- , il- , il- , tnf-α and ifn-γ coincided with the lowest t lymphocyte counts, about - days after the onset of severe covid- ; therefore, the restoration of t cell numbers was associated with reductions in circulating cytokines [ ] . the fact that t cells are important regulators of the activation of the immune system during a viral infection may explain how lymphopenia is related to the worsening of inflammatory responses [ ] . in consequence of lymphopenia and lymphocyte dysfunction, the adaptive immune response is ineffective and the infection is not adequately controlled, further increasing the stimulation of cytokines and cellular infiltrations [ ] . it was observed that patients with sars-cov- infection present increased risk of venous thromboembolism (vte) and disseminated intravascular coagulation (dic). coagulopathy associated with covid- , as it may be named, is characterized by hypercoagulability and thrombosis and is associated with worse prognosis in infection. among the altered coagulation parameters in patients with severe covid- are exacerbated coagulation activation, coagulation factor consumption, prolongation of prothrombin time (pt) and activated partial thromboplastin time (appt), moderate to severe thrombocytopenia, increased d-dimer and reduction of fibrinogen [ ] . through a retrospective analysis of patients with coronavirus pneumonia, high levels of the d-dimer and fibrin degradation product have been identified, in addition to prolonged pt, as well as appt in patients who had deceased. tang et al. [ ] considered the d-dimer as an important coagulopathy marker in cases of sars-cov- infection. the same findings were found in the studies by han et al. [ ] who see the use of hemostasis tests as tools to be used in early diagnosis and in monitoring disease progression [ ] . blood coagulation is the fastest mechanism in the confinement and inactivation of infections, being the first and the last defense line of the innate immune system to take place in tissues and blood circulation. the sore promotes the activation of the endothelial cells and the dysfunction of the endothelium, thus generating a pro-thrombotic state [ ] . in cases hyperactivation of the immune system, the coagulation may become intravascular and disseminated, therefore causing multiple organ failure. after all, the amount of vascular endothelial lesions of organs and tissues is, due to their activation intent, inversely proportional to the amount of existing coagulation factors [ ] . thereupon, the first clinical studies carried out in patients with pneumonia caused by the coronavirus confirmed the occurrence of organ dysfunction and coagulopathy as possible causes of the negative outcomes of the disease [ ] . the activation of the immune system, in response to the infection, leads the production of cytokines and tissue factor expression. the cytokines, in large amounts, harm the gas exchange and lead not only to inflammation but fibrinolysis, thus increasing d-dimer concentration [ ] . the tissue factor is related to an increase in thrombin generation and fibrin deposition, leading to hypercoagulability and civd and, thus, a worse prognosis [ , ] . as a contributor to the coagulation process, there is the presence of polymorphonuclear leukocytes (pmn) that are activated during the inflammatory process, releasing extracellular neutrophil traps (nets) which contain proteases that generate the inactivation of endogenous anticoagulants and the propagation of a procoagulant state. the interaction of activated platelets with pmn can form vaso-occlusive thrombotic complexes [ ] . some authors also elucidate the virus's relationship with the eca functional receptor, present in the arterial and venous endothelial cells of most human organs and part of both axes of the renin-angiotensin system, the vasoconstrictor eca/ang/at r and the vasodilator eca /ang-( - )/mas [ , ] . dalan et al. [ ] cite that both aging and metabolic disorders positively regulate the eca/ang/at r axis, leading to inflammatory, oxidative, vasoconstrictor and fibrotic effects. therefore, the eca / ang-( - )/mas axis is negatively regulated, resulting in a decrease in the anti-inflammatory and anti-fibrotic effects [ ] . the connection of sars-cov- to the eca receiver affects the balance between eca/ang/at r and eca /ang-( - )/mas, making the effects of the eca/ ang/at r axis even more prominent [ ] . the different immune responses of the host to the sars-cov- infection may explain the reason why men and women, young and old, infected by the virus can suffer a different severity of the disease [ ] . therefore, a considerably higher mortality rate was observed in patients with advanced chronological age [ ] . immune aging is related to an increase in individuals' susceptibility to infections, due to the decline in immune function, which can occur at any stage of the immune response. such changes can be seen, especially when associated with emotional stress [ ] . immune senescence is associated with the suppression of the activation and presentation of antigens by macrophages, which consequently prevent the migration of dendritic cells and the activation made by toll receptors with less effect [ , ] . ewers et al. [ ] mention the decline and proliferation of t cells, in addition to the increased production of pro-inflammatory cytokines il- , il- and tnf-α in the elderly. another point is the imbalance between th and th cytokines, generating an increase in the susceptibility of these individuals as infections by viruses and extracellular bacteria [ ] . in view of this, aging is associated with a constitutive pro-inflammatory environment due to persistent and low-grade immune activation, which can lead to increased tissue damage caused by infections [ ] . also, there should be taken into account the positive regulation of the ace-ang-ii-at axis that leads to proinflammatory and pro-fibrotic effects. although not very detailed, it is still suggested the occurrence of a greater number of eca receptors associated with aging, which would increase this imbalance. after all, it is through this receptor that sars-cov- infects humans and thus contributes to the development of covid- in older people [ ] . the precarious metabolic health is considered the main risk factor for the development of severe forms of covid- . this may occur in t dm, obesity and ms, possibly due to immune dysfunction in synergism with pathophysiological complications of these comorbidities [ ] . increased ace expression is a protective adaptive mechanism in t dm; however, it may facilitate the viral entry and spread of sars-cov- in the body [ ] . adipose tissue also exhibits high expression of ace , so the population with obesity may present greater vulnerability to covid- [ ] . it was observed that the expression of ace in the adipose tissue of obese patients allows viral entry in adipocytes and makes this tissue a reservoir for the viral dissemination of sars-cov- , since it is viscerally distributed [ , ] . in addition, obesity is an important factor for the development of t dm-especially when associated with low levels of physical activity and poor physical conditioning-and as mentioned, both diseases are related to higher expression of ace , increasing the risk of advanced infection by sars-cov- [ ] . metabolic disorders lead to immune activation of tissues such as the adipose, increasing the concentration of lowgrade chronic inflammation plasma markers, called metabolic inflammation or meta-inflammation [ ] . in this sense, the release of pro-inflammatory adipokines such as leptin, tnf-α, il- and il- b is observed, with a reduction in antiinflammatory action through the suppression of adiponectins [ , ] . the presented relationship is directly proportional to the presence of adipose tissue, the same that can be regulated through the practice of physical activity [ ] . the pro-inflammatory state found in metabolic syndrome and t dm may increase the probability of an unbalanced inflammatory response in covid- , like the cytokine storm described in patients with severe disease [ ] . similarly, as obesity is a state of low-grade chronic inflammation, it shows a potential for immune amplification of pathogens, as the regulatory elements of the immune response are absent or dysfunctional and this may contribute to the cytokine storm, which is already in greater concentration in obese individuals and which sustains and activates multiple cytokine pathways for a long time after the viral insult [ , ] . damage to blood vessels caused by chronic diseases, such as t dm, associated with hypercoagulability present in covid- may intensify the risk of infection complications [ ] . obesity can also aggravate endothelial dysfunction present in covid- due to inflammation triggered by perivascular and vascular adipose tissue, combined with changes in the synthesis of endogenous vasoactive agents, leading to platelet hyperactivation, leukocyte adhesion and other modifications related to endothelial inflammation, prothrombosis and proatherogenesis [ ] . hypercoagulability is directly proportional to the severity of overweight in obese patients. among the pathophysiological mechanisms are the action of adipocytokinins, with leptin and adiponectin, overactivity of coagulation factors, reduction of fibrinolytic function and, once again, increased inflammation (tnf and il- ). other contributors include elevated oxidative stress, lipid and glucose tolerance disorders, ms and venous stasis. thus, it is considered a synergic effect of obesity and ms on the state of hypercoagulability in covid- , aggravating the risk of vte and dic even more [ ] . there is also a greater amount of macrophages in the adipose tissue of obese individuals due to areas of microhypoxia, which lead to the nuclear factor-κb (nf-κb) pathway activation, thus increasing the expression of genes involved in inflammation [ ] . this condition is the result of the attraction of monocytes in the circulation made by chemokines; when they infiltrate the adipose tissue, they transform into macrophages, which, in turn, release tnf-α and il- which induce the tissue's resistance to insulin [ ] . insulin resistance is also related to the host's immune response, as it can inhibit the resolution of t cell-mediated inflammation [ ] . the adipose tissue is not the only one affected by the deposition of fats, because of that, bone marrow and thymus also present significant changes to the immune system of obese individuals and those with ms [ ] . thus, there is a marked deregulation of immune responses, which leads to a lower presence of circulating t cells, reducing the response to pathogens [ , , ] . despite the lack of accurate data on how physical activity improves the immune response against the new coronavirus, there is evidence of lower rates of ari incidence, duration and intensity of symptoms and risk of mortality from infectious respiratory diseases in individuals who exercise at high levels appropriate. furthermore, different studies suggest that regular physical exercise is directly related to decreased mortality from pneumonia and influenza, improvements in cardiorespiratory function, vaccine response, metabolism of glucose, lipids and insulin [ , ] . increased immune surveillance against infections has been proposed as a mechanism responsible for improving the immune response related to physical exercise. moderateintensity physical activity is responsible for providing an increase in the anti-pathogenic activity of macrophages, at the same time as elevations in the circulation of immune cells, immunoglobulins and anti-inflammatory cytokines occur, thereby reducing the burden of pathogen on organs such as the lung and the risk of lung damage due to the influx of inflammatory cells [ ] . during regular physical exercise practices, inflammatory responses and stress hormones are decreased; in contrast, lymphocytes, nk cells, immature b cells and monocytes are at high levels. thus, there is an improvement in immunovigilance, as well as a reduction in the systemic inflammatory process, factors that corroborate that regular physical activity helps to improve the immune system, while helping to prevent respiratory diseases and thus protect against infections such as covid- [ ] . dynamic exercises that generate cardiorespiratory overload promote the mobilization and redistribution of effector lymphocytes, mediated by catecholamines. this action primarily stimulates subtypes of lymphocytes capable of migrating from reservoirs-such as blood vessels, spleen and bone marrow-to lymphoid tissues and organs-such as the upper respiratory tract, lungs and intestines, aiming at recognizing and fighting pathogens and, thus, increasing immune surveillance and improving the antiviral response [ ] . similarly, regular exercise practices at moderate levels favor the function of the human body's immune surveillance against pathogens, as they stimulate an exchange of white blood cells between the circulatory system and tissues, a fact that reduces morbidity and mortality from acute respiratory disease and infections viral. they are also capable of promoting protection against infections triggered by intracellular microorganisms, as viral agents, given that the predominant immune response is mediated by th cells [ ] . regular exercise of moderate intensity has already been associated with a reduction in respiratory infections compared to sedentariness. however, exhaustive physical practices before or during an infectious condition, such as influenza or covid- , can trigger severe illness due to changes in the immune system [ , ] . this occurs due to the production of th anti-inflammatory cytokines in order to reduce muscle tissue damage, but in strenuous activities this effect can reach immunosuppression levels, thus providing the opportunity for infections [ , ] . therefore, attention should be paid to the importance of developing physical training at appropriate levels of execution. to the detriment of the world demographic change and the habits arising from the technological revolution, the population is aging more, becoming more obese and, consequently, less active when it comes to physical exercise. in this way, the immune system undergoes negative changes; that is, there is a functional impairment of innate immunity and adaptive immunity called immunosenescence, which results in greater susceptibility to infectious diseases and systemic inflammatory processes, decreased response to antibodies and, therefore, compromised immunological surveillance [ ] . therefore, for the elderly population, physical activity is even more essential, as these individuals generally have greater comorbidities and, in relation to the new coronavirus, are more vulnerable to contracting the disease [ ] . damiot et al. [ ] suggested that individuals who have remained active throughout their lives have less pronounced immunosenescence characteristics, which may be a possible protective factor against the development of complications caused by covid- . in this sense, beneficial effects of regular physical exercise have been reported in the elderly population, including reduction in oxidative stress, improvement in immune competence and reduction in cellular changes related to immunosenescence [ , ] . elderly individuals who maintain continued physical activity have levels of tcd + and tcd + lymphocytes similar to younger individuals, in addition to not having harmful defects in the recruitment of lymphocytes during the infectious process [ ] . according to the study by ferrer et al. [ ] with elderly volunteers, through physical activity there is a decrease in the current levels of il- , as well as an increase in the expression of il- in active individuals [ ] . a low presence of circulating pro-inflammatory cytokines is observed in contrast to the increase in anti-inflammatory cytokines. thus, there are positive changes in the immune system of these individuals, including enhancements in host response and vaccine immunoprotection [ ] . similarly, while prolonged maintenance or worsening of obesity and ms perpetuates deregulation of immune responses, promoting greater risks of the individual developing diseases and increasing their vulnerability to infection by the novel coronavirus [ , , ]. an association between physical activity and reduction of inflammatory markers in obese and overweight patients is suggested [ ] . luzi and radaelli [ ] add the lack of physical activity as an important factor among obese patients, as it impairs the immune response against microbial agents, from the activation of macrophages to the inhibition of pro-inflammatory cytokines. on the other hand, both metabolic health and immune health benefit from the practice of physical activity, which reduces the risk of infectious complications. thus, regular physical exercise appears as a preventive measure in the defense of the host against viral infection [ ] . muscle contraction is responsible for the transient increase in circulating levels of il- cytokine, in proportion to the duration of physical activity and the amount of muscle mass recruited. the elevation of this interleukin seems to be followed by increases in antinflammatory cytokines, such as il- , released by cells of innate immunity and responsible for promoting an antinflammatory environment, inhibiting inflammatory mediators to limit tissue damage. this effect may be beneficial in cases of chronic inflammation, such as obesity, t dm and ms, and may reduce the risk of a pathogenic inflammatory response such as the cytokine storm present in severe covid- [ , ] . in addition, il- is associated with enhanced insulin sensibility and glycemic metabolism [ ] . physical practice is able to reduce the excessive concentration of pro-inflammatory adipocin leptin and improve sensitivity to leptin and insulin [ , ] . in patients with t dm and new coronavirus infection, good glycemic control has been associated with better prognosis in covid- [ ] . thus, physical exercise is shown to be an immunomodulatory and non-pharmacological intervention, achieving positive immunomodulation through exercises of light to moderate intensity [ ] . through exercise, there is an improvement in the response to infection in obese individuals, due to immune and cellular restoration [ ] . although covid- is not primarily a metabolic disease, there is a need to maintain metabolic control of glucose, lipid levels and blood pressure in order to prevent metabolic and cardiovascular complications, as well as to reduce the local inflammatory response and block the virus entering the cells [ ] . as seen, innate immunity has an important role in the pathogenesis of covid- and ards, due to inflammatory cascades, recruitment of neutrophils, macrophages and dc cells and increased production of ross. in turn, by the modulation mechanism of chemokine production, physical training and therapeutic exercises can attenuate alveolar neutrophilia in the face of lung injury [ ] . furthermore, the expression of the extracellular superoxide dismutase enzyme (ecsod), an important antioxidant in the body and highly present in the lungs, is enhanced by resistance physical activity and was associated with inhibition of endothelial activation and inflammatory adhesion, with potential benefit to reduce oxidative stress and tissue damage in covid- [ ] . the amplification of the antioxidant defense generated by routine physical activity also contributes to immunological surveillance [ ] . moreover, womack, nagelkirk and coughlin [ ] point out that through the intensity of physical exercise, a change in the potential for coagulation, platelet aggregation and fibrinolysis can be seen. as an example, there is long-term training through aerobic exercises where a decrease in the clotting potential is observed in healthy individuals. thus, it is suggested that the practice of physical exercises contributes to reducing the risk of ischemic events depending on their intensity and duration [ ] and may contribute to attenuating coagulation disorders associated with sars-cov- infection. the anti-inflammatory, antioxidant and endothelial activation inhibitor benefits may also be linked to the reduction in hypercoagulability related to covid- . this is because, as previously mentioned, the exacerbated activation of the immune system increases the expression of the tissue factor and, consequently, the predisposition to the formation of thrombi; in addition, metabolic disorders, oxidant stress and changes in senescence positively stimulate the vasoconstrictor axis eca/ang/at r, contributing to endothelial imbalances [ , , [ ] [ ] [ ] [ ] [ ] ] . therefore, the immunometabolic improvements promoted by physical exercise may help in the control of coagulation disorders in the covid- (fig. ) . finally, in view of the quarantine status adopted in several countries as a measure to prevent and control the spread of sars-cov- during the covid- pandemic, social isolation and restrictions on the movement of people reduced the practice of physical activity, predisposing the population to adopt sedentary behavior [ ] . social distancing is a sine qua non in reducing the speed of contagion of covid- and associated deaths. however, due to these measures, sports clubs, gyms and fitness spaces have suspended their activities in order to reduce agglomerations; thus, difficulties regarding physical exercise were imposed [ ] . therefore, despite being one of the main strategies against covid- , social isolation has been related to behavioral and physiological changes, including the increased prevalence of sedentarism and eating disorders (food compulsion, hyperphagia), resulting in negative consequences for metabolic health, such as weight gain, growth of fat tissue, hyperglycemia and insulin resistance and loss of muscle tissue [ ] . since this condition can harm the body's defenses and contribute significantly to the reduction in individuals' physical condition, functional and health loss, the adoption of healthy habits and an exercise routine can help in maintaining health [ , ] . it is significant to consider that contexts like this increase the susceptibility to stressful events and elevations of glucocorticoids (cortisol), with consequent inhibition of the functions of nk cells and tcd + lymphocytes in the antiviral response. however, good physical conditioning was associated with lower risks of reactivation of latent viral infections in situations of isolation and confinement, indicating a favored immune system compared to individuals with less physical fitness [ ] . physical activity is considered a non-medication practice for the prevention and treatment of diseases of psychological, physical and/or metabolic origin [ ] . regular physical exercise should be encouraged during social isolation as a preventive measure for health, given that exercise is essential during the period of fight against the spread of coronavirus [ ] . the american college of sports medicine (acsm) recommends that the practice of moderate physical exercise should be maintained during the quarantine period, since it helps in the immune reinforcement against sars-cov- . the who recommends that asymptomatic and healthy individuals should exercise at least min per week for adults and min per week for children and adolescents. these times can be distributed during the days of the week and according to the person's routine [ ] . it is important to emphasize that physical activity should be interrupted and a health professional should be consulted in case symptoms such as fever, dyspnea at rest and dry cough are manifested, because these symptoms can be related to covid- [ , ] . in social isolation, the home environment has become the ideal and necessary place for physical activity. activities that are satisfactory and that allow better exploring the home space should be sought. activities of daily living such as organization of spaces, cleaning and maintenance also help in coping with covid- . in environments with children, playing and exercising with them is a great way to promote energy expenditure, thus leaving the beginning of sedentary rest. meditation, stretching and relaxation are allies in combating a sedentary lifestyle. it is important to avoid long rest periods, which should be intercalated with active practices [ ] . the acsm has published guidelines for moderate-intensity activities that can be practiced during the pandemic period, including aerobic exercises and strength training, indoors, like at home, or outdoors, when permitted by fig. benefits of regular moderate-intensity physical activity on factors that influence the response against to covid- . source: the authors ( ) government authorities. options for aerobic activities to be performed at home include walk briskly around the house, up-and downstairs, dancing and jumping rope. when possible, walking or running outdoors, cycling, gardening work and family games are interesting alternatives, as long as infection prevention measures are maintained [ ] . among the strength activities, acsm indicates downloading a strength workout app that does not require any equipment and suggests exercises such as squat, sit-ups, push-ups, lunges and yoga practice, which can also help in anxious states [ ] . oliveira neto et al. [ ] suggest resistance exercises based on acsm recommendations to be performed at home, including exercises involving the muscles of the lower body, upper body and limbs, and lower limbs, which can be adapted for beginners in physical practice or experienced people. activities that make use of the individual's own body weight, associated with resistance training as well as the use of elastic bands, provide excellent health results, results similar to those achieved by traditional gyms. thus, objects such as backpacks, books, market bags and water bottles can be used as an auxiliary tool in resistance physical activity. exercises such as squats, jumping jacks and going up and down steps can be effective in physical training [ ] . as for the intensity and volume of physical exercise practices, these must be moderate, as they exceed both the volume and the intensity, effects such as momentary immunosuppression are achieved, thus providing greater vulnerability as to the contagion of the novel coronavirus. if individuals want to practice high-intensity exercises, a reduction in exercise volume should be adopted as a preventive measure, in order to avoid strenuous exercises [ ] . in addition, technological tools can contribute to the better performance of these activities in the home environment, as video calls with a physical education professional facilitate the orientation of the exercises to be performed, providing support that has, as a consequence, better results and greater safety in the execution of the exercises. regardless of whether or not you are in the risk group for covid- , regular exercise, according to the acsm, should be regularly performed, given that it aims to improve the immune system, reduce stress perceived and decrease anxiety disorders [ ] . there are still gaps in the knowledge regarding the pathogenic mechanisms involved in sars-cov- infection. however, there is consensus in the scientific literature about the important involvement of the immune system in the susceptibility, progression and outcome of covid- . the imbalance in innate and adaptive immune responses, characterized mainly by changes such as cytokine storm and lymphopenia, in addition to the disorders in coagulation-and host-related conditions, including obesity, metabolic syndrome and aging (immunosenescence), is among the factors notoriously associated with a worse prognosis of infection. the benefits of exercise-regular and at appropriate intensity levels-for the immune system in respiratory infections such as covid- include increased immunovigilance and improved immune competence, which help in the control of pathogens, a fact that becomes more important considering the immunosenescence and susceptibility of the elderly population to severe infection. other favorable effects in relation to host factors, such as prevention or reduction of overweight, increased physical and cardiopulmonary conditioning, attenuation of the systemic pro-inflammatory and pro-thrombotic states, decrease in oxidative stress, improvements in glycemic, insulinic and lipidic metabolisms, besides the enhancement of the vaccination response, also indicate how adequately physical activity can help the organism's immune response against covid- . in the covid- pandemic situation, adopting mitigation practices is an essential strategy to reduce the risks related to the novel coronavirus infection. these interventions include the use of personal protective equipment (ppe), adherence to hygiene procedures and social isolation measures, as well as actions that lead to a healthier lifestyle, minimize stress factors and strengthen the immune system, such as regular physical activity. however, remaining active at appropriate levels seems to be a challenge in a context of confinement and social isolation, which emphasizes the importance of developing training with recommendations adapted to the new routine of the population. fortunately, there are viable alternatives 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immune response, and evolution endocrine and metabolic link to coronavirus infection exercise-induced changes in coagulation and fibrinolysis in healthy populations and patients with cardiovascular disease exercise in the time of covid- praticar exercícios físicos é fundamental para a saúde física e mental durante a pandemia da covid- . braz j health rev page_docum ents/eim_rx% for % hea lth_% sta ying% act ive% dur ing% cor onavi rus% pan demic traininginhome-home-based training during covid- (sars-cov ) pandemic: physical exercise and behavior-based approach authors' contributions dtrs had the idea for the article. es, kksf e mrb performed the literature search, data analysis and translation. mps drafted and revised the work.funding not applicable. conflict of interest the authors declare that they have no conflict of interest.human and animal rights statement and informed consent this article does not contain any studies with human participants or animals performed by any of the authors, and informed consent is not a standard required. key: cord- -cb u s s authors: bedford, juliet; farrar, jeremy; ihekweazu, chikwe; kang, gagandeep; koopmans, marion; nkengasong, john title: a new twenty-first century science for effective epidemic response date: - - journal: nature doi: . /s - - -y sha: doc_id: cord_uid: cb u s s with rapidly changing ecology, urbanization, climate change, increased travel and fragile public health systems, epidemics will become more frequent, more complex and harder to prevent and contain. here we argue that our concept of epidemics must evolve from crisis response during discrete outbreaks to an integrated cycle of preparation, response and recovery. this is an opportunity to combine knowledge and skills from all over the world—especially at-risk and affected communities. many disciplines need to be integrated, including not only epidemiology but also social sciences, research and development, diplomacy, logistics and crisis management. this requires a new approach to training tomorrow’s leaders in epidemic prevention and response. when nature published its first issue in , a new understanding of infectious diseases was taking shape. the work of william farr , ignaz semmelweis , louis-rené villermé and others had been published; john snow had traced the source of a cholera epidemic in london (although robert koch had not yet isolated the bacterium that caused it ). the science of epidemiology has described patterns of disease in human populations, investigated the causes of those diseases, evaluated attempts to control them and has been the foundation for public health responses to epidemic infections for over years. despite great technological progress and expansion of the field, the theories and practices of infectious disease epidemiology are struggling to keep pace with the transitional nature of epidemics in the twenty-first century and the breadth of skills needed to respond to them. epidemiological transition theory has focused mostly on the effects of demographic and socioeconomic transitions on well-known preventable infections and a shift from infectious diseases to non-communicable diseases . however, it has become clear that current demographic transitions-driven by population growth, rapid urbanization, deforestation, globalization of travel and trade, climate change and political instability-also have fundamental effects on the dynamics of infectious diseases that are more difficult to predict. the vulnerability of populations to outbreaks of zoonotic diseases such as ebola, middle east respiratory syndrome (mers) and nipah has increased, the rise and spread of drug-resistant infections, marked shifts in the ecology of known vectors (for example, the expanding range of aedes mosquitoes) and massive amplification of transmission through globally connected, high-density urban areas (particularly relevant to ebola, dengue, influenza and severe acute respiratory syndrome-related coronavirus sars-cov). these factors and effects combine and interact, fuelling more-complex epidemics. although rare compared to those diseases that cause the majority of the burden on population health, the nature of such epidemics disrupts health systems, amplifies mistrust among communities and creates high and long-lasting socioeconomic effects, especially in low-and middle-income countries. their increasing frequency demands attention. as the executive director of the health emergencies program at the world health organization (who) has said: "we are entering a very new phase of high-impact epidemics… this is a new normal, i don't expect the frequency of these events to reduce." . we have to act now but act differently: a broader foundation is required, enhancing traditional epidemiology and public health responses with knowledge and skills from a number of areas ( table ) . many of these areas have long been associated with epidemic preparedness and response, but they must now stop being seen as esoteric 'nice things to have', and instead become fully integrated into the critical planning and response to epidemics. this will require considerable changes by the global public health community in the way that we respond to epidemics today and how we prepare for and seek to prevent those of tomorrow. it will mean reshaping the global health architecture of the response to epidemics and transforming how we train new generations of researchers and practitioners for the epidemics of the future . the modern research culture-often shaped by the behaviour of funders-has required many researchers to specialize in narrow fields, with less emphasis on translation than on field-specific innovations. although this siloed landscape has brought major advances in global health, it is not fit for the transitional phase of epidemic diseases: rapidly evolving, high-impact events bring together communities, responders and researchers who do not routinely interact. different assumptions, cultures and practices, each of which may be widely accepted within a particular community, make working together in outbreak situations more challenging. fundamental to success is respect and understanding of the contribution each party brings. in a successfully integrated approach, we each have to realize that our knowledge and skills are a small part of a rapidly expanding toolkit (box ). we need to understand major trends in research and how and when they may influence the response to an epidemic, develop new research to strengthen the support that we can provide across other areas and learn to operate in multi-stakeholder situations-including, at times, as part of a critical debate to bring better practices to the fore. central to this approach must be the communities who are at risk and those affected by epidemics: local people are the first responders to any outbreak and their involvement in the preparation and response activities is essential. from communities, through local and regional health authorities, national public health institutes and international organizations-including many essential partners in sectors beyond public health-the integrated approach must be supported. the who, in particular, has a critical part to play, using its unique mandate not to lead every aspect of preparation, response and recovery, but to change its practices, facilitate integration with and among others, and ensure accountabilities are built in from the bottom to the top. a wave of cholera epidemics across europe in the s and s catalysed a new era of 'infectious disease diplomacy' globally. nations recognized that infections do not stop at borders and that therefore multilateral collaboration is essential to protecting citizens from lethal epidemics. the development of germ theory through the second half of the nineteenth century transformed ideas about the causes of infections, informing scientific research as well as clinical responses. scientific understanding translated into vaccines and antibiotics, while programmes for child health, hygiene, clean water and sanitation became common in the twentieth century. as a result, childhood diseases such as measles and mumps became rare, smallpox was eventually eradicated and polio was eliminated from all but a handful of countries . many people thought that infectious diseases would soon be history. sir frank macfarlane burnet is often cited for his remark in the s that, with the emergence of new diseases being a distant prospect, "the future of infectious diseases will be very dull" . although the focus in high-income nations turned to non-communicable diseases, which constituted a considerable and increasing burden on the health of their citizens, infectious diseases did not disappear. some endemic infections such as malaria and tuberculosis were not susceptible to elimination strategies, and new diseases with epidemic and pandemic potential emerged. ebola virus disease was first identified in the s, hiv/aids in the s, nipah virus in the s, sars and mers at the start of the twenty-first century, and many more have since been identified. far from becoming 'very dull', the field of infectious disease epidemiology has sometimes struggled to adapt: as late as , respected researchers used a nineteenth century 'law' of epidemiology to make predictions about the aids epidemic-these turned out to be vast underestimates . advances in other fields gave epidemiology the chance to evolve. in , when the editors of the international journal of epidemiology provocatively asked whether it was time to 'call it a day' given the putative power of genomics to explain diseases over the capacity of epidemiologists to describe them, their conclusion was that it had the potential to positively transform epidemiology as much as the rise of germ theory a century earlier. at least pathogens that affect humans have been identified as emerging, re-emerging or evolving since the s , while increasing rates of antimicrobial resistance threaten to make formerly controlled infections, such as malaria, untreatable -this also limits our ability to control their epidemic potential. the demographic transition is driving much of this: human society is becoming more urban than rural for the first time in our history, bringing large numbers of people (and often animals) together in densely populated areas . agricultural and forestry practices are changing the relationships between people, animals and our respective habitats . travel is more accessible around the world, advances in computer science and computing speeds have led to a number of applications of artificial intelligence across society . applications in epidemiology include tracking online searches about disease symptoms to aid early detection of epidemics, although more sophisticated methods may be required before artificial intellegence becomes a reliable detection tool . crystallography modern x-ray diffraction and electron microscopy can reveal structures of viruses and antibodies in such detail that it is possible to identify specific sites of vulnerability on the virus. a previous study showed how such techniques identified an antibody that was much more potent against respiratory syncytial virus than the only currently available intervention . developing vaccines for emerging infectious diseases has many challenges, including the time it takes, a limited market and strict regulatory requirements for products that will be given to healthy people . platform technologies use one underlying approach with standardized processes and some antigen-specific optimization to speed up both development and manufacture of vaccines. for example, vector-based platforms combine an antigen, or a gene for an antigenic protein or peptide, in a virus-like particle or liposome. such platform technologies have the potential to deliver vaccines a few months after an emerging pathogen is identified and sequenced, rather than years . review so migration, trade and tourism bring more people into contact and thus affect disease transmission . climate change has many effects on ecosystems and environments, not least in changing the habitats and migratory habits of disease vectors . states with weak health systems are far less likely to cope with or recover from multiple emergent demands without damaging routine services . inequalities , inequities and distrust in national structures and institutions compound people's vulnerabilities . conflict increases the risk of epidemics and makes responding to them close to impossible . since , there have been several outbreaks of ebola (including the two biggest in history), not to mention outbreaks of sars, mers, nipah, influenza a subtype h n , yellow fever, zika and the continued spread of dengue. epidemics overlap and run into each other, yet the world is not currently equipped to cope with this increasing burden of multiple public health emergencies. preparing for epidemics, therefore, requires global health, economic and political systems to be integrated just as much as infectious disease epidemiology, translational research and development, and community engagement. epidemics represent shared risks that cross borders and all of society. health systems, routine care, trust in governments, travel, trade, business-all are disrupted during an epidemic. with such broad risks, the preparation and response must be nationally owned and led, internationally supported and undertaken with a whole-of-society approach. some initiatives have started to build frameworks for this to happen in a coordinated way. for example, the who's pandemic influenza preparedness framework brings together nation states, industry, other stakeholders and the who to implement a global approach to pandemic preparedness and response . a focus must be building coordinated regional and country expertise, resources and capacity through national and regional public health institutions . this brings its own challenges-governance of institutions, leadership, collaborations and interventions have to be impeccable or misconduct can thrive . unwelcome in itself, misuse of funding, resources or people within efforts intended to support an epidemic response will also undermine trust in the organizations that respond to an outbreak and, in turn, prolong the outbreak. key governance components include drafting policies in advance and being willing to implement those policies for data collection and sharing during epidemics. they must be flexible enough to enable affected communities and nations to retain ownership of the response, while drawing on international expertise to find the best possible response. governance should also include processes for vaccine and therapeutic approvals during outbreaks. however, it is clear that the centre of gravity for leadership, governance and implementation must be where the need is greatest if these are to truly deliver. in , julian tudor hart proposed the inverse care law: "the availability of good medical care tends to vary inversely with the need for it in the population served." . an analogue of the inverse care law can be applied to public health and epidemiology. expertise in these fields has traditionally gravitated towards centres of excellence in europe and the united states. of course, high-income countries are not immune to the disruption associated with epidemics, especially in an era of misinformation and growing mistrust in authorities and public health initiatives. however, the centre of gravity must shift so that globally representative distributed networks of collaborating centres can jointly ensure coverage in the regions that urgently need these skills on the ground . international collaborations remain important; however, strengthening epidemiology, public health and laboratory capacity in low-and middleincome countries is essential . collaborative interventions should not be limited to when there is a major outbreak, but be integrated into regular interactions. capacity, resources, expertise and governance can be supported by the increasing role for regional and national centres of disease control. the us centers for disease control (cdc) lends its expertise all around the world in addition to protecting the us population. in , the european cdc started, followed by the china cdc in and by the africa cdc in . although more can be done to improve data sharing and access to laboratories, the networks and connections between these centres have strengthened all of their work, as well as having a positive effect on public health systems in low-and middle-income countries. during the pan-european wave of cholera in the s, there were riots across the continent: doctors, nurses and pharmacists were murdered, hospitals and medical equipment destroyed . similar reports today usually come from communities that have not had positive prior interactions with public health initiatives, and thus the encounter with national or international teams who arrive only in response to a 'new' disease means that trust can never be assumed and has to be earned on both sides. engagement needs to start before an outbreak-ensuring that patients, their families and their communities are at the centre of all public health is essential for the successful prevention and response to epidemics. there is no public health without the support of the community. for example, the early detection of disease events will be improved if more national and regional public health institutions establish community event-based surveillance systems. communities are the first to know when something unusual happens -therefore training and mobilizing community volunteers to report such occurrences is a costeffective way to rapidly detect diseases and contain them at the source. this will also help to sustain engagement between communities and the organizations that respond to outbreaks. furthermore, improved information flow between the community and the public health system should provide a better understanding of local social networks to complement other means of tracking chains of transmission between individuals and places. this can be the community themselves, or it might be veterinarians who see clusters of sick animals, or nurses and doctors who care for patients in primary care-or it may be teams that are often forgotten in public health initiatives, such as those working in critical care facilities; it is striking how the first cases of nipah, sars, mers and influenza a subtype h n were all first identified by clinical teams in critical care facilities. an inclusive, whole-of-society approach is challenging, and the challenges may be magnified in a conflict or post-conflict zone. wars and conflicts not only increase the risk of epidemics as people move to escape violence and health services become harder to maintain , but also make public health responses vulnerable to interruption, thus making them less effective. then, miscommunication, mistrust, disease and violence can fuel each other in a vicious cycle. engaging local communities remains the highest priority, even in unstable contexts such as north kivu and ituri provinces of the democratic republic of the congo (drc) , where an ebola epidemic started in august . it seems inevitable that responding to epidemics in politically unstable environments will become more common, and skilled negotiators and peacekeepers will have to be better integrated in response teams. equally essential, therefore, will be an improved understanding of these challenging operational contexts among affected communities and external responders alike. social scientists have long applied their skills and knowledge in epidemic responses, although their roles have become more visible in recent years . by focusing on communities, social science humanizes the epidemic response , helps to increase understanding of context and may uncover associations between the context or local practices and the risk of transmission. the social science in humanitarian action platform has successfully produced rapid reports and briefings on regions in which an epidemic has been identified, and the global research collaboration for infectious disease preparedness includes a social science research funders' forum to 'propel research in this area' , acknowledging that its integration in the preparation and response to outbreaks is often missing or added as an afterthought to solve a problem that could have been forseen. there is still much to learn about how epidemic responders and social scientists can make the most of each other's expertise and how data from social science can fit into the wider information architecture of epidemic response. as an example, behavioural surveillance will be critical in twenty-first century responses to disease outbreaks . just as behavioural surveillance to improve the understanding of hiv was crucial in identifying high-risk groups for hiv infection, so human behaviours will continue to be important as we respond to future infectious diseases. for instance, the ebola virus outbreak in west africa probably began before december , but it took several months before hospital transmission and traditional burial practices were found to be the leading causes of its rapid spread. the increasing prevalence of mobile phones, wireless internet connectivity and social media activity raises the possibility of using these tools to gather data for epidemiological studies, diagnostics , population mobility during an ebola epidemic or influenza incidence in real time . future developments in predictive technology, machine learning and artificial intelligence will bring more opportunities to move towards 'precision public health' (box ). the use of data from people is becoming strictly controlled, however, and it will be a challenge to persuade countries to invest in a new surveillance system, for example, before its general effectiveness has been demonstrated at a country level . even then, technology-based solutions should be integrated with community-based programmes and other existing epidemic preparedness and response systems because surveillance is more effective when standardized among different countries, districts and communities. to this end, suites of guidance and open-access standardized tools are being developed for reporting cases of disease, as well as consent forms, standard operating procedures and training materials , properly validated diagnostic assays and access to quality-assurance panels in public and veterinary health. the rising trend of engaging citizens in data gathering is also welcome-the use of mosquito-recognition apps enables the collection of data far beyond the capacity of routine mosquito surveillance . this way, citizens feed information into the public health system and the feedback loop offers a fast and direct way to provide citizens with details of potential actions that they can take. as well as potentially supporting diagnosis and surveillance , the fast-developing field of genomic epidemiology can yield information to track the evolution of a virus such as ebola during an epidemic , . there will be times when it can detect outbreaks better than traditional epidemiology, illustrating the need to have these tools available in the same toolbox. during the large lassa fever outbreak in nigeria in , real-time genomic sequencing provided clear evidence that the rapid increase was not due to a single lassa virus variant, nor attributable to sustained human-to-human transmission. rather, the outbreak was characterized by vast viral diversity defined by geography, with major rivers acting as barriers to migration of the rodent reservoir . these findings were crucial in containing the outbreak. developing and sustaining the capacity to conduct real-time sequencing with adequate bioinformatics analyses at regional and national levels will be challenging in low-and middle-income countries. moreover, investments in relatively high-tech capacity (such as real-time sequencing) are competing with other, arguably more fundamental needs, such as equipment and training in primary laboratories. political engagement must be nurtured between epidemics: it is not enough to offer technological and laboratory support during a crisis, even with the promise of building capacity, if the political will is not there. however, with proper preparation, and accessible and trusted data sharing and governance mechanisms, laboratories with limited resources may be able to leap-frog into the twenty-first century , . vaccination is one of the most effective public health interventions and innovative strategies for research and development of vaccines, such as using ring vaccination as a trial design during ebola epidemics since - , must be encouraged. at the start of the - epidemic in west africa, vaccine candidates were already in development, based on a long history of preclinical research, although a lot of work was still required to get clinical trials underway in time to be useful . in , when zika was first internationally recognized as a pathogen that could cause birth defects , there was hardly any research and no vaccines in late-stage development. two-and-a-half years later, results from three phase i clinical trials had been reported , although challenges remained for further development. the lack of a profitable market for such products means that pharmaceutical companies lack the incentives to push this work between epidemics. initiatives such as the coalition for epidemic preparedness innovations are attempting to positively disrupt financing models for vaccines against epidemic diseases , and stockpiles of meningococcal vaccine, yellow fever vaccine and oral cholera vaccine are maintained by the international coordinating group to minimize potential delays due to limited manufacturing capacity . similarly, if investigational treatments or vaccines are to be used as part of the response to an epidemic, ethical protocols for managing informed consent and introducing them in clinical settings must be planned in advance with at-risk communities (box ). trial designs precision medicine refers to the use of genomic sequencing to retrace the specific course of a disease in individual patients, with the aim of being able to choose the best treatment option for each person. in public health, the analogous idea of precisely directing the right intervention to the right population is equally appealing. the potential of such an approach has been illustrated by the identification of two areas in the united states in that were at risk of zika transmission . rather than the whole country, or even only florida, being declared at risk, these two areas each measured less than km , and the response focused only on these specific neighbourhoods. by contrast, a campaign against yellow fever, also in , defined risk 'at the level of entire nations'. a broad interpretation of precision public health incorporates many different types of data to increase the power of epidemiology . such data would not only include genomic information, but also satellite imaging, mobile phone data, social media use data and so on. for example, a study published in combined epidemiological surveillance data, travel surveys, parasite genetics and anonymized mobile phone data to measure the spread of malaria parasites in southeast bangladesh . a retrospective analysis of mobile phone call data in sierra leone from showed how it might have been used to assess the impact of travel restrictions on mobility during the ebola epidemic . the principle of selecting the most relevant information from all available data seems within the scope of good epidemiological practice already. the challenge is recognizing and incorporating new types of data when they become available. should be created as soon as the option becomes viable. the essential consideration is how the resulting data can add to previous trials and influence the approach to trials in future epidemics. for example, research during the - ebola epidemic enabled progress on therapeutic agents that are now being trialled in the ongoing outbreak in drc . scientific progress during and between epidemics must be matched by other workstreams, such as the preparation of supply chain logistics and communication with at-risk populations. plans have to be made for a series of future outbreaks, enabling adaptive, multi-year, multi-country studies . similar plans are needed for continual preclinical research to ensure that future vaccine and therapeutic pipelines will be filled. the term 'one health' is used to acknowledge that human, animal and ecosystem health are tightly interconnected and need to be studied in the context of each other (fig. ) . changes in the environment-whether natural or anthropogenic-affect interactions between pathogens, vectors and hosts in multiple and complex ways, making the emergence or decline of endemic, epidemic and zoonotic diseases difficult to predict, while epidemics of animal diseases can challenge a community's access to food. the fact that pools of viruses, bacteria and parasites are maintained in wild and domesticated animals makes surveillance of potentially zoonotic diseases an intrinsic part of one health epidemic planning. many agencies and nations around the world now use prioritization tools such as those developed by the us cdc or the united nations (un) food and agriculture organization (fao) to identify and prioritize zoonotic diseases of concern. an early precedent was a joint consultation on emerging zoonotic diseases by the who, the fao and the world organisation for animal health in . understanding disease ecology in the zoonotic reservoir could potentially lead to ways to predict the risk of human disease, thus providing the basis for smart early-warning surveillance systems. individual countries with limited resources for epidemiological studies and epidemic preparation and response must decide their own priorities. however, infectious diseases do not respect borders. similarly, the interdisciplinary nature of one health means there are several different lenses through which different sectors assess risks and priorities. for one health approaches to work, these multiple perspectives must be taken into account, whether human health or animal health, ecology or social sciences . epidemics do more than cause death and debilitation: they increase pressure on healthcare systems and healthcare workers and draw resources from services not directly linked to the epidemic. this can leave a legacy of distrust between people, governments and health systems, although more-positive outcomes have been found to strengthen relations between communities and public authorities. the full social and economic costs of the ebola outbreak in west africa have been estimated to be as high as us$ billion when including the effect on health workers, long-term conditions suffered by , ebola survivors, and costs of treatment, infection control, screening and deployment of personnel beyond west africa. as healthcare resources became increasingly allocated to the ebola response, hospital admissions fell and deaths from other diseases rose markedly, adding us$ . billion to the estimated cost. such pressure can be withstood in high-income countries with strong health systems, but in low-income countries the pressure can quickly reach a breaking point. ebola killed almost . % of doctors, nurses and midwives in guinea, . % in sierra leone and just over % in liberia . this is compared to mortality between . % and . % of the whole population of these countries. estimates of the effect of this loss on maternal mortality suggest that thousands more women may have died in childbirth each year since the epidemic ended. beyond the tragic deaths of so many healthcare workers, people were less likely to use health services for children or adults during the epidemic, suggesting decreased trust or even fear of healthcare settings . more recently, in some areas affected by the ebola outbreak in drc, the introduction of free non-ebola healthcare led to unprecedented demand. however, healthcare facilities box in , the prevent project received wellcome funding to provide ethics guidance "at the intersection of pregnancy, vaccines, and emerging and re-emerging epidemic threats" . this was in response to the newly recognized association between infection with zika virus during pregnancy and microcephaly in the newborn. developing a vaccine was an obvious route to explore, but many researchers felt that they could not conduct clinical trials with pregnant women because it is generally assumed that the risk to the woman, the fetus or both outweighs any potential benefit. however, as heyrana et al. argue: "preventing pregnant women from participating in clinical trials is well intentioned but misguided." . prevent rapidly developed guidance for including pregnant women and their babies in zika vaccine research , and has since extended their scope to "a roadmap for the ethically responsible, socially just, and respectful inclusion of the interests of pregnant women in the development and deployment of vaccines against emerging pathogens." . integrating ethics in the preparation and response to epidemics does not close off avenues of research; it opens up possibilities and expedites progress. were not given sufficient additional resources to care for the number of people, which may have contributed to nosocomial infections. survivors, too, need to be cared for long after the epidemic is declared over. a cohort of more than , children is growing up in brazil after being born with microcephaly because their mothers were infected with zika during pregnancy. tracking the development of these children increases understanding of the effects of zika infection and helps to define what medical and social support the affected families may need as many of the children will grow up with severe developmental delays . the challenges posed by twenty-first century epidemics are real and changing: future epidemics will be fuelled by conflict, poverty, climate change, urbanization and the broader demographic transition. in our response we must consider epidemics not as discrete events, but rather as connected cycles for which we can prepare, even if we cannot predict specific outbreaks. the challenge is then to choose the right response at the right scale in the right area at the right time. there needs to be a greater emphasis on absorbing and using positive lessons from each episode and avoiding those that led to negative outcomes . the way that we train practitioners and researchers working in all fields relevant to today's epidemic landscape has to change. a modern approach that is capable of characterizing epidemics and the best ways to control them must go beyond a narrow definition of epidemiology that sustains artificial barriers between disciplines. instead, it must be able to integrate tools and practices from a diverse range of established and emerging scientific, humanistic, political, diplomatic and security fields. we believe that such an approach needs to become the norm for the curriculums of schools of public health around the world. as well as training new generations of epidemiologists so that they have the skills, knowledge and networks to recognize and make use of every tool available to help them to do their work effectively, the entire architecture of the response to epidemics has to be adapted. only then will we be able to maintain the comprehensive and effective response-including prevention and research-needed to stop epidemics and protect people's lives, no matter what the circumstances. celebration: william farr ( - )-an appreciation on 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epidemics war and infectious diseases: challenges of the syrian civil war pandemic influenza preparedness framework for the sharing of influenza viruses and access to vaccines and other benefits (who how africa can quell the next disease outbreaks the ability to prevent, detect and respond to any health issues will always depend on the local capacity and although international partners can bring complementary expertise and resources, it is the local capacity that is critical; in this article, the authors argue for national investment in public health, health systems, science and local leadership un health chief orders probe into misconduct the inverse care law agenda setting, research questions and funding for biomedical research has historically been led from northern hemisphere countries in an unequal northern-southern hemisphere relationship science granting councils in sub-saharan africa: trends and tensions international federation of red cross and red crescent societies. 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protocols & data tools enhancing early warning capabilities and capacities for food safety real-time, portable genome sequencing for ebola surveillance infection control in the new age of genomic epidemiology genomic surveillance elucidates ebola virus origin and transmission during the outbreak genetic diversity and evolutionary dynamics of ebola virus in sierra leone genomic analysis of lassa virus during an increase in cases in nigeria the integration of genomics and other types of data into the surveillance, prevention and response of epidemics is critical and can help to transform the ability to enhance public health; although the tools are now available, it will be key to ensure that these new approaches are fully integrated and not seen as esoteric ivory tower research, but instead as an essential component of twenty-first century epidemiology, public health and epidemics-the next generation of leaders need to be efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola Ça suffit!) a seminal study that shows that ring vaccination could be used in the midst of a devastating ebola epidemic and, furthermore, that innovation research can be conducted in an epidemic, trial designs can be adapted without compromising scientific integrity and that ebola can be prevented through vaccination ethical rationale for the ebola "ring vaccination" trial design ebola vaccination in the democratic republic of the congo insights from clinical research completed during the west africa ebola virus disease epidemic gone or forgotten? the rise and fall of zika virus current status of zika vaccine development: zika vaccines advance into clinical evaluation cepi: preparing for the worst the development of global vaccine stockpiles the ebola clinical trials: a precedent for research ethics in disasters it is an ethical imperative to consider and implement research in an epidemic setting as, for many epidemic diseases, it is the only time at which to conduct the research that will inform and improve the lives of the individuals affected during epidemic and to ensure that future generations are better prepared; however, such research is challenging at many levels and it is critical to have an ethical framework that guides the research, places individuals and communites at the heart of the research and facilitates the maximum benefit for the maximum number of people improving vaccine trials in infectious disease emergencies progression of ebola therapeutics during the - outbreak ebola therapies: an unconventionally calculated risk performance of different clinical trial designs to evaluate treatments during an epidemic the one health concept: years old and a long road ahead surveillance of zoonotic infectious disease transmitted by small companion animals prioritizing zoonoses: a proposed one health tool for collaborative decision-making evaluation of the emergency prevention system (empres) programme in food chain crises report of the who/fao/oie joint consultation on emerging zoonotic diseases (who european centre for disease prevention and control. towards one health preparedness the economic and social burden of the ebola outbreak in west africa epidemics cause enormous disruption to countries, regions and the world; however, the focus is often on the epidemic itself, the pathogen and its immediate effect rather than the much broader effect that the epidemic has not only on the healthcare system-which lasts long after the epidemic itself-as routine vaccination programmes often collapse, maternal-child health suffers, and malaria, hiv and tuberculosis clinics and surgery-all aspects of healthcare-are disrupted, but also on the wider society, as mistrust and tension occurs between citizens, authorities and governments, and education, investments, businesses, trade and tourism inevitablely suffer leading to an economic impact that can health-care worker mortality and the legacy of the ebola epidemic patterns of demand for non-ebola health services during and after the ebola outbreak: panel survey evidence from further pieces of evidence in the zika virus and microcephaly puzzle responding to the ebola virus disease outbreak in dr congo: when will we learn from sierra leone? artificial intelligence in medical practice: the question to the answer? artificial intelligence and big data in public health structure of rsv fusion glycoprotein trimer bound to a prefusionspecific neutralizing antibody vaccine platforms: state of the field and looming challenges platform technologies for modern vaccine manufacturing four steps to precision public health precision" public health -between novelty and hype offline: in defence of precision public health mapping imported malaria in bangladesh using parasite genetic and human mobility data pregnant women & vaccines against emerging epidemic threats: ethics guidance for preparedness, research, and response increasing the participation of pregnant women in clinical trials the ethics working group on zikv research & pregnancy. pregnant women & the zika virus vaccine research agenda: ethics guidance on priorities, inclusion, and evidence generation acknowledgements we thank m. regnier at wellcome for editing the manuscript.author contributions all authors developed the scope and focus of the review and contributed to the writing of the manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to j.f. reviewer information nature thanks peter byass, sharon peacock and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. reprints and permissions information is available at http://www.nature.com/reprints. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -qfdxri authors: wack, andreas; rappuoli, rino title: vaccinology at the beginning of the st century date: - - journal: curr opin immunol doi: . /j.coi. . . sha: doc_id: cord_uid: qfdxri today, the main challenges for vaccinologists include improving vaccines against as yet undefeated pathogens, rapid identification and response to emerging diseases and successful intervention in chronic diseases in which ongoing immune responses are insufficient. reverse genetics and reverse vaccinology are now used to generate rapidly new vaccine strains and to mine whole genomes in the search for promising antigens. the rational design of adjuvants has become possible as a result of the discovery of the receptors that recognize microbial patterns and lead to dendritic cell activation. antigen-loaded dendritic cells, dna in naked, formulated or viral form, and other delivery systems are used to maximize immune responses. although work on the ‘easy’ vaccines has already been completed, it is hoped that a combination of conceptual and technical innovation will enable the development of more complex and sophisticated vaccines in the future. vaccinology at the beginning of the st century andreas wack and rino rappuoli today, the main challenges for vaccinologists include improving vaccines against as yet undefeated pathogens, rapid identification and response to emerging diseases and successful intervention in chronic diseases in which ongoing immune responses are insufficient. reverse genetics and reverse vaccinology are now used to generate rapidly new vaccine strains and to mine whole genomes in the search for promising antigens. the rational design of adjuvants has become possible as a result of the discovery of the receptors that recognize microbial patterns and lead to dendritic cell activation. antigen-loaded dendritic cells, dna in naked, formulated or viral form, and other delivery systems are used to maximize immune responses. although work on the 'easy' vaccines has already been completed, it is hoped that a combination of conceptual and technical innovation will enable the development of more complex and sophisticated vaccines in the future. although the eradication of smallpox in the s and of poliomyelitis hopefully in the coming years mark two of the most important milestones in medical history, we now face an unprecedented succession of new pathogens which jump species barriers to infect humans, and the frustration deriving from the inability to control devastating diseases such as hiv, malaria and tuberculosis. this review will cover the recent progress in vaccinology (figure ), largely resulting from dramatic technical innovation that is now reaching the clinic, as well as the huge challenges posed by old and new pathogens that are facing us. the three 'big killers', the pathogens that most heavily afflict global health, are hiv, mycobacterium and plas-modium. whereas the latter two represent long-term companions of the human species, hiv is a virus that spread in the human population only about years ago and has a nonhuman primate origin [ ] . in fact, most, if not all, of the recently emerging diseases go back to animal reservoirs, from which they infect humans through close contact during hunting and farming, in live animal markets or through food processing, preparation or consumption. an example is ebola virus, which, after three outbreaks between and , has appeared in the human population nine times since , with each outbreak resulting from the handling of dead gorillas, chimpanzees or duikers, which in turn are thought to have been infected by an unknown natural host [ ] . the variant creutzfeldt-jacob disease reached the population through the food chain, from 'rendered' sheep and cow carcasses fed to cows which were subsequently consumed by humans. the severe acute respiratory syndrome (sars)-coronavirus sequences from the earliest identified cases are identical to those found in palm civets and raccoon dogs in animal markets and farms [ ] , strongly suggesting an animal origin for this disease. rapidly changing ecosystems and human behavior, an ever-increasing density of human and (farmed) animal populations and their close vicinity, poverty, a high degree of mobility and many other factors contribute to the more frequent occurrence and often rapid dissemination of new diseases. another example of this is the arrival and persistence of west nile virus in the usa, which is probably a result of increased mobility, an exceptionally broad host range and climatic changes [ ] . among the reemerging diseases of the past few years, diphtheria and cholera should be mentioned [ ] , as well as the frequent appearance of multidrug-resistant bacteria and the cases of anthrax infection as a result of deliberate release in . in the case of influenza, concern for the advent of a new pandemic has been fuelled by reports of human infections by the avian h n virus strain (where h stands for hemagglutinin and n for neuraminidase, the two major surface glycoproteins of the virus) in , leading to deaths (http://www.who.int/csr/disease/avian_ influenza/en/). this was the third time in the space of a few years (after the previous outbreaks of infection in and ) that h n avian flu viruses caused disease and death in humans. an h n avian strain caused infection in humans in and . because the world's population has not been exposed to these strains, it is immunologically naïve, and these or any other avian flu strains with new surface glycoproteins would meet no possible intervention points for vaccine improvement. schematic view of a pathogen-or vaccine-induced immune response. vaccines or pathogens cross epithelial barriers and are taken up by antigen-presenting cells such as dcs. interaction between pattern recognition receptors (prrs) and their agonists activate dcs, resulting in increased antigen presentation, cytokine production and co-stimulation. cd + and cd + t cells recognize antigen presented by dcs and are activated. cognate interaction between primed cd + t cells and b cells activates the b cells, resulting in clonal expansion and antibody production. dc activation also leads to inhibition of the regulatory effect of cd + cd + treg cells. fully activated cd + cells can target tumor cells and pathogen-infected cells. the letters (a-i) indicate processes where improved vaccines can lead to more efficient immune responses: (a) the site of administration influences the type of immune response and enables the usage of lower vaccine doses [ , ] . (b) particulate antigen is taken up more easily by macrophages and dcs than soluble antigen [ ] . (c) tlr agonists and other immunostimulants binding to prrs increase the activation of dcs [ - , , , , ] . (d) dcs matured and loaded with antigen in vitro are efficient vaccines [ , , ] . (e) dna vaccination leads to efficient antigen presentation on mhc class i [ , , , , , ] . crosspresentation of antigen on mhc class i of host dcs is facilitated after vaccination with antigen-loaded dcs undergoing delayed apoptosis [ ] . (f) cytokines can be added in protein or dna form as natural adjuvants [ , ] . (g) recruited nk cells can be an early source of th -driving cytokines [ ] . (h) vaccines can break tolerance when the suppressive effect of cd + cd + treg cells is overcome [ ] . resistance provided by existing immunity and could rapidly expand. once an avian strain has developed a more efficient means of human-to-human transmission, devastating pandemics, such as those arising in , and , could ensue. it should be noted, however, that older studies found seroprevalence rates for avian flu strains among chinese rural populations to be between % and % for h viruses and between % and % for other avian strains [ , ] . thus, it might in fact be the quality of surveillance, rather than the frequency of outbreaks, that has increased over the past few years [ ] . however, the contact between humans and pathogens in animal reservoirs is likely to intensify, and, consequently, the emergence or re-emergence of diseases will occupy vaccinologists frequently in the future. one of the most important future challenges will be to respond promptly to emerging diseases such as those mentioned above. a striking example for rapid reaction was in the case of the sars outbreak, where the genome sequence was publicly available in less than a month after the virus was identified [ ] . this enabled the speedy development of diagnostic tools, as well as the identification and recombinant expression of targets for vaccines and therapeutic agents [ ] [ ] [ ] . for the influenza virus, in addition to the annual definition of the relevant strains to be included in the vaccine for the following season, the world health organization closely monitors cases of avian flu (for further information, see the world health organization website indicated above), and prototype vaccines for these strains are being developed. apart from almost complete lack of protection in the population, an additional threat of the avian flu h n isolate from is that it kills embryonated eggs, the traditional virus growth substrate used for influenza vaccine production. such problems can now be solved, owing to the discovery some years ago that influenza virus can be generated entirely from transfected dna (reverse genetics [ , ] ). in this particular case, webby et al. [ ] have used polymerase chain reaction-based mutagenesis to replace the hemagglutinin cleavage site (which was shown to be the cause of high pathogenicity) of the h n strain with the sequence from a nonpathogenic strain. vero cells were then transfected with plasmids encoding the neuraminidase and mutated hemagglutinin from the circulating strain, together with the plasmids encoding the remaining proteins from the laboratory-optimized pr strain. the resulting vaccine strain was successfully grown in eggs and shown to be nonpathogenic and stable. thus, the use of reverse genetics enables rapid production of a reference vaccine virus in response to the emergence of a new influenza variant [ , ] . in addition, reverse genetics can be used for more far-reaching vaccination strategies, such as the construction of 'consensus' strains expressing conserved amino acid sequences or more than one version of the surface glycoproteins, or additional immunoenhancing molecules, such as cytokines [ ] . much research effort is also being invested into the development of improved cell culture systems that can replace completely the use of embryonated eggs in vaccine production and would render the production process more flexible and controllable. several reports have addressed the question of how to stretch the available supply of vaccine doses in cases of shortage or in the face of a pandemic. two studies indicated that intradermal, rather than intramuscular, application of % [ ] the genomic revolution has opened up a completely new approach to vaccine discovery. for pathogens that do not grow in vitro, the availability of the genome sequence has enabled the development of recombinant vaccines, as has been carried out for hepatitis b virus (hbv) and is underway for hepatitis c. in regard to bacteria, group b meningococcus posed insurmountable obstacles to conventional vaccinology approaches; these were eventually overcome by mining the information from the sequenced genome [ ] . a total of potential vaccine candidates were predicted by computer analysis, of which were expressed and tested for immunogenicity [ ] . some of these candidates are now in clinical trials. this genomebased approach, called reverse vaccinology, is now used routinely in vaccine development, and is a major tool in the quest for vaccines against pneumococcus, group b streptococcus and chlamydia (see also update). recently, genome sequencing of both plasmodium falciparum [ ] and its main vector, anopheles gambiae [ ], has sparked off new hopes for an efficient vaccine against malaria. for the rodent models of this disease, subtractive cdna techniques were used to identify genes that are only expressed in pre-erythrocytic stages of the parasite [ , ]. plasmodium mutants deficient in one of these genes, uis , are blocked in their early liver-stage development and all subsequent stages, and therefore do not lead to disease. when uis -deficient sporozoites are used as genetically attenuated vaccines in mice, they confer long-lasting, stage-specific protection [ ] . this is a promising example of how molecular approaches are employed for the rational design of new vaccines. another example of encouraging progress towards a malaria vaccine was reported by alonso et al. [ ] . they describe phase iib trials of a subunit vaccine consisting of a recombinant protein (expressed in yeast) composed of the carboxy-terminal of the p. falciparum circumsporozoite protein and the hbv surface antigen. this fusion protein, together with unfused hbv surface antigen proteins, forms particles. the final vaccine formulation includes the adjuvant as a, an oil in water emulsion containing the immunostimulants monophosphoryl lipid a (mpl) and quillaja saponaria fraction . it had previously been shown that, indeed, a cd + t cell response to an epitope contained in the vaccine correlates with protection from infection and disease [ ] . in this trial in children, the vaccine showed an efficacy of % and % in preventing clinical episodes and severe episodes, respectively [ ] . the past ten years have changed our vision of the immune response to pathogens. it has become clear that the degree and type of antigen-specific, clonal b and t cell responses (acquired immunity) depend crucially on the prior action of a more ancient system of pathogen detection (innate immunity). this system relies on the activation of antigen-presenting cells such as dendritic cells (dcs) upon recognition of patterns common to viruses and bacteria and largely absent in mammals. with the discovery of the involved pattern recognition receptors, among which the toll-like receptors (tlrs) represent an important subgroup, immune-enhancing molecules or adjuvants can no longer be considered as the alchemistic 'immunologist's dirty secret' but have become amenable to rational design, providing a huge potential for manipulating the immune response. as different tlr agonists elicit different types of immune responses (reviewed in [ ] ), future adjuvants might be able to tailor the immune response so that optimal protection to a given pathogen is induced. in fact, the number of clinical trials involving tlr agonists as new adjuvants is ever increasing [ , ] (table ). the adjuvant function of nonmethylated cytidine-phosphate-guanosine (cpg) sequences, which are frequent in microbes but under-represented in humans and are agonists of tlr , has been extensively demonstrated in animal models [ ] and is currently being tested in several clinical trials. when cpgs are coadministered with licensed hbv or flu vaccines, the combination leads to increased antibody titers or increased (interferon-g) ifn-g production, as compared with the response to the vaccine alone [ , ] . an additional effect of cpg oligonucleotides appears to be the promotion of affinity maturation and, as a result, a higher overall affinity of the vaccine-specific antibody pool [ ] . similarly, the tlr agonist mpl has been shown in the past to enhance the immune response to hbv vaccination in humans [ ] . in addition, both the malaria subunit vaccine mentioned above [ ] and a licensed melanoma vaccine contain mpl. another experimental vaccine that includes a tlr agonist is described below, in the section on synthetic vaccines. activation of dcs increases their ability to process and present antigen and to attract and activate t cells through cytokine secretion; consequently, several cytokines are host-pathogen interactions table human tlr agonists used as adjuvants in vaccine formulations in clinical trials or licensed vaccines a . known currently being tested for their adjuvant function. as mentioned above, the way the innate immune system is activated influences the type of the ensuing acquired immune response. martin-fontecha et al. [ ] showed that the ability of adjuvants to elicit a th type response depends crucially on the recruitment of, and ifn-g production by, natural killer (nk) cells, which indicates a possible mechanism of the way in which adjuvants direct the type of adaptive immune response induced downstream. another vaccine approach is the use of heat shock proteins, which bind specifically and activate dendritic cells and, because they are loaded with endogenous peptides, can be purified from tumor cells and function as a combined antigen delivery system and adjuvant [ ] . because the main targets of adjuvants are dcs, it is a logical step to evaluate their direct use as a vaccine. despite the labor-intensive necessity of individual cell culture for each patient, this approach can be attractive where other approaches have failed, for instance as a therapeutic vaccine in hiv or cancer patients. when hiv patients were treated with autologous dcs loaded with autologous, inactivated hiv, both virus-specific cd + th and cd + responses were induced and the plasma viral loads were reduced [ ] . these results closely reflect previous findings from a similar vaccination of rhesus macaques [ ] , except for the lack of induction of neutralizing antibodies in the human study. dc vaccination is also being tested in a variety of cancer treatments [ ] . in a study comparing the immunogenicity of dcs transfected with cytopathic or noncytopathic viral rna, the former regimen was shown to be more efficient at inducing protective immune responses [ ] . this suggests that reprocessing of dying dcs by endogenous antigen-presenting cells enhances immunogenicity, either through additional danger signals triggered by the cell damage or by the increased level of crosspresentation by endogenous dcs. the same mechanism might be at work in mycobacterium infection of macrophages, where apoptosis was shown to enhance crosspresentation by bystander dcs [ ] . interestingly, following fractionation of the cytoplasm of dying cells, uric acid was identified as a highly efficient endogenous danger signal that enhances immunogenicity [ ] , and might explain the above results. the high expectations associated with dna vaccination, as a result of promising data obtained in mice, were somewhat tempered by disappointing early results when dna was tested as a vaccine in humans. therefore, the latest generation of dna vaccines rely on improved delivery either through use of microparticles [ ] or through viral vectors. a particularly promising approach is a heterologous prime-boost strategy, where adminis-tration of plasmid dna is followed by recombinant virus (modified vaccinia virus ankara [mva] or adenovirus) expressing the same antigen. this regimen induced strong t cell responses against p. falciparum in naïve adults [ ] and enhanced the response in gambian men who are constantly exposed to p. falciparum [ ] . although partial protection against challenge with a different p. falciparum strain was observed, no significant differences in the infection rate was found in the gambian trial [ ] . in spite of these setbacks, a very similar regimen has been shown also to be highly immunogenic against hbv, tuberculosis and hiv, and vaccination only with mva expressing the mycobacterium a antigen elicited strong t cell responses [ ] . because the induction of t cells is considered to be crucial in anti-tumor immune responses, a huge number of trials are presently being conducted to test dna vaccination regimens for anticancer treatment [ , ] . when viral vehicles (vaccinia or adenovirus) were compared with loaded dcs in terms of their ability to overcome established tolerance and induce immune responses in a transgenic mouse model, the viral formulations were able to do so, whereas dcs required repeated administration of tlr agonists or irrelevant virus, or removal of suppressive cd + cd + treg cells [ ] . such models of established tolerance might prove useful for testing the success of vaccines in the face of long-term antigen exposure, as in the case of cancer or chronic diseases. two studies analyzed in detail the t cell response after vaccination with a recombinant canarypox virus expressing melanoma-specific t cell epitopes [ , ] . focusing on the blood and metastases from a patient with complete regression, these studies reconfirmed earlier observations that the frequency of anti-tumor t cells can be relatively high. although vaccination leads to a slight increase in vaccine-specific t cells, these remain only a small fraction of total anti-tumor t cells. by contrast, t cells directed against epitopes not contained in the vaccine represent the vast majority of anti-tumor cells, and their frequency increases both in the blood and in metastases. thus, it appears that with the appropriate vaccine regimen, the inefficiency of pre-existing specific t cells to combat the tumor was reversed in an indirect manner, presumably by activating another subset of t cells. it remains to be clarified, however, how much of this reactivation is due to the action of vaccine-induced t cells and how much is a general, antigen-independent immune-enhancing effect. in any case, for the development of therapeutic vaccines, it will be vital to understand how the balance can be tipped back from a state of tolerance to a successful immune response. the development of vaccines aimed at the polysaccharide (ps) capsule of bacteria is one of the great achievements in vaccinology. so far, the ps used in largescale vaccine production has been purified from the pathogen itself, grown in large quantities -an approach that is costly and difficult to control. through great simplification of the carbohydrate chemistry involved, verez-bencomo et al. [ ] have now demonstrated the first large-scale production of an anti-haemophilus influenzae type b vaccine, consisting of synthetic ps conjugated to tetanus toxoid protein carrier. this vaccine has been shown to be as efficient as commercially available vaccines in inducing protective levels of antibody titers in infants. an entirely synthetic vaccine with a branched structure containing a tlr ligand, a cd + t cell epitope and either a cd + t cell or a b cell epitope has been shown to elicit strong cd + t cell and b cell responses, respectively [ ] . here, the minimal requirements for an efficient vaccine are met in a single molecule: targeting to and activation of dcs, t cell help and activation of antigen-specific cd + t cells or b cells. the world of vaccines is undergoing dramatic changes. never before have such sophisticated techniques and an in-depth knowledge of immunological processes been at hand to exploit fully the potential of protecting from, as well as curing, diseases through vaccination. a formidable task in the future will be the development of effective therapeutic vaccines, in situations where chronic antigen exposure by itself does not elicit a sufficiently strong immune response, as is the case in cancer and chronic infectious diseases. compared with vaccines against selflimiting infections, where the aim is to be as good as the real pathogen (but less harmful), this requires the development of vaccines that are better than the natural antigens in inducing immunity. all of our knowledge will be necessary to succeed in this challenge. a recent article describes the use of multigenome analysis and screening against a large panel of strains to identify a universal group b streptococcus vaccine. although none of the single antigens contained in the vaccine elicits protection against all strains, the combination of four proteins is able to cover a wide range of strains [ ] . mueller ak, labaied m, kappe sh, matuschewski k: genetically modified plasmodium parasites as a protective experimental malaria vaccine. nature , : - . in this study, reverse genetics was used to generate a plasmodium knockout strain with a developmental block at the early liver stage. if used as a live attenuated vaccine in a malaria mouse model, this strain confers protection against infectious sporozoite challenge. origin of hiv- in the chimpanzee pan troglodytes troglodytes multiple ebola virus transmission events and rapid decline of central african wildlife human ebola virus outbreaks consist of parallel epidemics caused by several different viral strains which appear to be transmitted to humans through handling of gorilla, chimpanzee or duiker carcasses molecular evolution of the sars coronavirus during the course of the sars epidemic in china the chinese sars consortium demonstrates that virus sequences from the earliest patients are identical to those found in palm civets, and seven out of cases had documented contact with wild animals. in addition, the isolated case in december was caused by a virus with highest sequence homology to palm civets west nile virus: where are we now? from pasteur to genomics: progress and challenges in infectious diseases pandemic influenza: a zoonosis? semin respir infect serological evidence of human infections with avian influenza viruses influenza: old and new threats sars -beginning to understand a new virus a dna vaccine induces sars coronavirus neutralization and protective immunity in mice severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus rescue of influenza a virus from recombinant dna generation of influenza a viruses entirely from cloned cdnas responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines this paper demonstrates the rapid generation of a genetically attenuated influenza h n reference vaccine strain by use of reverse genetics evaluation of a genetically modified reassortant h n influenza a virus vaccine candidate generated by plasmid-based reverse genetics serum antibody responses after intradermal vaccination against influenza see annotation to efficacy of the rts, s/as a vaccine against plasmodium falciparum infection and disease in young african children: randomised controlled trial this vaccine contains a yeast-expressed hybrid molecule composed of a part of the circumsporozoite protein and the hbv surface antigen, formulated in a complex adjuvant. significant efficacy, in terms of prevention of clinical episodes and of severe malaria a cd (+) t-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural plasmodium falciparum infection and disease using an enzyme-linked immunospot assay on t cells previously expanded in vitro, the authors identified a conserved cd + t cell epitope in the circumsporozoite protein with predictive value: an ifn-g response of cd + t cells to this epitope positively correlated with protection from malaria third meeting on novel adjuvants currently in or close to clinical testing world health organization-organisation mondiale de la sante th meeting on novel adjuvants currently in/close to human clinical testing world health organizationorganisation mondiale de la sante fondation merieux use of cpg oligodeoxynucleotides as immune adjuvants a phase i study of the safety and immunogenicity of recombinant hepatitis b surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant cpg , an immunostimulatory tlr agonist oligodeoxynucleotide, as adjuvant to engerix-b((r)) hbv vaccine in healthy adults: a double-blind phase i/ii study see annotation to co-administration of cpg oligonucleotides enhances the late affinity maturation process of human anti-hepatitis b vaccine response in these two clinical trials, cpg was shown to be an efficient adjuvant when used in combination with licensed vaccines the immunogenicity and reactogenicity profile of a candidate hepatitis b vaccine in an adult vaccine non-responder population induced recruitment of nk cells to lymph nodes provides ifn-gamma for t(h) priming the authors showed that the ability of some adjuvants to induce th priming correlates with the degree of recruitment of nk cells, which represent an early source of ifn-g. this might provide a rationale for the search for efficient th therapeutic cancer vaccines: using unique antigens therapeutic dendritic-cell vaccine for chronic hiv- infection this study followed a previous one in macaques [ ] and showed that injection of autologous dcs loaded with autologous inactivated hiv induces hiv-specific cd + and cd + t cell responses and leads to a prolonged tenfold reduction in virus therapeutic dendritic-cell vaccine for simian aids dendritic cells: a journey from laboratory to clinic dendritic cells transfected with cytopathic self-replicating rna induce crosspriming of cd + t cells and antiviral immunity the surprising observation in this study is that induction of immunity and crosspresentation of antigen is greatly facilitated by the death of the injected antigen-carrying dcs. this suggests that danger signals from dying cells might have an adjuvant effect in vaccinations, as well as in immune responses apoptosis facilitates antigen presentation to t lymphocytes through mhc-i and cd in tuberculosis molecular identification of a danger signal that alerts the immune system to dying cells microparticles for the delivery of dna vaccines enhanced t-cell immunogenicity of plasmid dna vaccines boosted by recombinant modified vaccinia virus ankara in humans safety and immunogenicity of dna/modified vaccinia virus ankara malaria vaccination in african adults a randomised, double-blind, controlled vaccine efficacy trial of dna/mva me-trap against malaria infection in gambian adults recombinant modified vaccinia virus ankara expressing antigen a boosts bcg-primed and naturally acquired antimycobacterial immunity in humans the authors observed antigen-specific ifn-g responses of short duration induced by vaccination with mva expressing the conserved mycobacterial antigen a. this t cell response is substantially higher and sustained in vaccinees who received bacillus calmette-guerin followed by mva three weeks later gene-based vaccines and immunotherapeutics dna vaccines to attack cancer persistent toll-like receptor signals are required for reversal of regulatory t cell-mediated cd tolerance this transgenic adoptive transfer model of tolerance enables the comparison of different vaccine regimens in their ability to break tolerance. viral vaccines are most efficient in overcoming established tolerance. dcbased vaccine strategies that are contrasting frequencies of antitumor and anti-vaccine t cells in metastases of a melanoma patient vaccinated with a mage tumor antigen high frequency of antitumor t cells in the blood of melanoma patients before and after vaccination with tumor antigens these two studies showed that in the blood and metastases of vaccinated cancer patients, t cells specific for the vaccine antigen might represent only a small fraction of total tumor-specific t cells, both before and after vaccination a synthetic conjugate polysaccharide vaccine against haemophilus influenzae type b the large-scale production of a conjugate vaccine containing synthetic polysaccharides has been achieved by simplification of the polysaccharide chemistry involved a totally synthetic vaccine of generic structure that targets toll-like receptor on dendritic cells and promotes antibody or cytotoxic t cell responses this elegant study showed how all of the components required for an efficient vaccine can be included in one synthetic molecule: a t helper epitope, a tlr agonist as an adjuvant and either a cd + t cell or a b cell epitope. the resultant vaccines confer protection and are able to break tolerance in mice variegation of the immune response with dendritic cells and pathogen recognition receptors identification of a universal group b streptococcus vaccine by multiple genome screen the use of different group b streptococcus strains for genome analysis and screening enables the development of a universal vaccine. the single vaccine components are not present in all strains, and do not protect against all strains we are grateful to giorgio corsi for artwork. papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest key: cord- -bmahwxbn authors: masi, davide; risi, renata; gnessi, lucio; watanabe, mikiko; mariani, stefania; lubrano, carla title: letter to the editor: [our response to covid- as endocrinologists and diabetologists] date: - - journal: j clin endocrinol metab doi: . /clinem/dgaa sha: doc_id: cord_uid: bmahwxbn nan m a n u s c r i p t we read with interest the article by kaiser et al. highlighting some relevant aspects about the management of patients with endocrine conditions who are simultaneously infected with the novel coronavirus (sars-cov- ). as far as we are concerned, understanding the evidence of the role played by corticosteroids in sars-cov- infection is of immediate clinical importance. the existing literature does not currently provide conclusive evidence regarding their administration for the treatment of covid- . there have been a number of reports stating generally positive outcomes upon corticosteroids use especially in the early acute phase of infection, as they are capable of reducing immune-mediated damage . in this regard, fu y et al. suggest to adopt steroids in combination with the use of intravenous immunoglobulin to block fc receptors (fcr) activation in order to prevent severe lung injury. however, such treatment remains to be clinically tested for effectiveness. on the other hand, patients taking supra-physiologic doses of glucocorticoids may have increased susceptibility to covid- because of the immunosuppressive effects of steroids, which can be ultimately responsible for severe lymphocytopenia, thus promoting a viral rebound. moreover, steroids have also been associated with higher risk of acute respiratory distress syndrome (ards) and may therefore determine exaggerated proinflammatory responses and even worsen the clinical condition of the patients. however, in the event of covid- , patients on long-term steroids, or suffering from adrenal insufficiency and uncontrolled cushing syndrome may be unable to develop a normal stress response, thus requiring a rescue dose of glucocorticoids. in our opinion, this evidence by kaiser and colleagues should be extended to those patients with chronic pain on therapy with opioids. longterm use of opioids has been associated with suppression of the hypothalamicpituitary-adrenal (hpa) axis through the inhibition of corticotrophin-releasing hormone (crh) and antidiuretic hormone (adh) secretion, resulting in decreased adrenocorticotropic hormone (acth) release and central m a n u s c r i p t our response to covid- as endocrinologists and diabetologists covid- and treatment with nsaids and corticosteroids : should we be limiting their use in the clinical setting ? ecancer associations between immune-suppressive and stimulating drugs and novel covid- -a systematic review of current evidence understanding sars-cov- -mediated inflammatory responses: from mechanisms to potential therapeutic tools clinical evidence does not support corticosteroid treatment for -ncov lung injury opioid-induced endocrinopathies endocrine consequences of long-term intrathecal administration of opioids chronic spinal and oral morphine-induced neuroendocrine and metabolic changes in noncancer pain patients a c c e p t e d m a n u s c r i p t key: cord- -e tyv authors: lin, weikang nicholas; tay, matthew zirui; lu, ri; liu, yi; chen, chia-hung; cheow, lih feng title: the role of single-cell technology in the study and control of infectious diseases date: - - journal: cells doi: . /cells sha: doc_id: cord_uid: e tyv the advent of single-cell research in the recent decade has allowed biological studies at an unprecedented resolution and scale. in particular, single-cell analysis techniques such as next-generation sequencing (ngs) and fluorescence-activated cell sorting (facs) have helped show substantial links between cellular heterogeneity and infectious disease progression. the extensive characterization of genomic and phenotypic biomarkers, in addition to host–pathogen interactions at the single-cell level, has resulted in the discovery of previously unknown infection mechanisms as well as potential treatment options. in this article, we review the various single-cell technologies and their applications in the ongoing fight against infectious diseases, as well as discuss the potential opportunities for future development. five months since the first reported infection cluster, covid- has turned into a vicious worldwide pandemic that infected more than . million people and caused over , deaths [ ] . the pandemic will also have large spillover effects in terms of economic damage both in the form of healthcare costs and in monetary losses from the disruption of global supply chains, with world trade expected to fall between % and % in [ ] . the covid- pandemic serves as a grim reminder that infectious disease is, and will always be, a major threat to the continued existence of mankind. to date, there are about microorganisms known to be pathogenic to humans. these pathogens can be broadly classified as viral, bacterial, fungal, and parasitic pathogens [ ] . in particular, there have been infectious diseases that have been persistently difficult to eradicate, namely, human immunodeficiency virus and acquired immune deficiency syndrome (hiv/aids), tuberculosis, and malaria. aids, due to hiv, is responsible for nearly million deaths per year [ ] . the death toll from tuberculosis, caused by mycobacterium tuberculosis (mtb) bacteria, is the highest amongst all infectious diseases, which is a problem that is exacerbated by the rise of antimicrobial resistance variants of the disease [ ] . malaria, a parasitic infection, has afflicted humans for thousands of years and continues to do so today [ ] . in light of the above-mentioned examples, among other infectious diseases, further efforts have to be directed for the continued management of the global burden of these diseases. the covid- pandemic has highlighted many questions that are relevant in the context of infectious disease as a whole. why are certain people more susceptible to infections? why are some infected individuals asymptomatic or display only mild symptoms? why are there differences in terms of disease progression and outcomes among patients? this diverse response to infection could be explained by the interactions of inherently heterogeneous populations of pathogens, host cells, and immune cells. however, discerning this heterogeneity is difficult in conventional bulk analyses, as they fail to recognize the following: ( ) the genomic variability of pathogens, ( ) the coexistence and interactions of infected host cells and bystanders, and ( ) the diverse functional roles of immune surveillance participants. aside from the limited resolving power in pathophysiological studies, bulk analyses often fall short in terms of the level of precision and the amount of derived information needed for early diagnostics and high-efficacy vaccine development against infectious diseases. just as how microscopy revolutionized our understanding of biology, the enhanced resolution, precision, and breadth of information offered by single-cell technologies has brought an exciting overhaul to our perception of infectious diseases in recent years. the use of single-cell genomics, transcriptomics, proteomics, and epigenetics (referred to as omics altogether in this article) has flourished in many areas of the battlefield against infectious diseases. table presents commonly used single-cell technologies in infectious disease studies alongside several of other non-single-cell systems. a scoring heatmap is used to represent the complexity of information in various aspects that they can provide (e.g., genetic, epigenetic, proteomic, spatial). the heatmap also provides an overall ranking of throughput, cost, and downstream assay compatibility amongst all the listed techniques. hence, it serves as a general guide for future users to select the methods that match their desired outputs. for instance, if the primary target for the study is to collect genetic information (e.g., analysis of invading virus gene heterogeneity in single host cell), single-cell sequencing might be the best candidate. likewise, if the focus is on proteomics orchestrating the immune responses, mass cytometry can furnish the most detailed insight. for infectious disease models that involve the interplay of genomics and proteomics, both cite-seq and reap-seq can be the suitable candidates. while flow cytometry has the highest throughput and lowest cost per experiment amongst the listed single-cell methods, it yields very limited aspects of information. other single-cell assays capable of providing more complex data typically come at the expense of a decreased throughput and increased cost. it is also worth mentioning that microfluidics has made great success in boosting the throughput and cost efficiency of existing single-cell assays. one prominent example would be the transition of well plate systems into microchambers or microdroplets, which ultimately reduces the required amount of reagent required per experiment and in turn reduces costs. in this review, we identified infection pathophysiology, therapeutic discovery, and disease diagnostics as three major areas in which single-cell omics has contributed substantially in the past decade. in pathophysiological studies of infectious disease, single-cell omics offer excellent spatial-temporal resolution that help to not only reconstruct the uneven subcellular distribution of pathogen across the entire host cell population, but also reveal the sequence of immune events accompanied by the change of immune cell profiles. single-cell omics also extrapolates meaningful molecular details that describe the dynamic host-pathogen interplay and immune activation. furthermore, single-cell omics identifies the rare molecules and cell subtypes that exhibit significant functionality in the pathogen-host immune interactions. insights in fundamental pathophysiology naturally have spillover benefits for translational science, such as in vaccine development where single-cell omics has the capability to enhance the discovery of mechanistic correlates of protection through multi-parameter measurements of the immune state with respect to disease, and it enables precision quality control checkpoints to aid the evaluation of vaccine efficacy. in the field of antibody discovery, single-cell omics can simultaneously interrogate antigen specificity and recover the b cell receptor gene sequences, which in turn shortens the previously prolonged and labor-intensive research cycle in the search for effective therapeutic or diagnostic antibodies. in the application of infectious disease diagnostics, single-cell omics are on the verge of practical clinical deployment, as demonstrated by some examples of automated and miniaturized devices. the diagnostic power of single-cell omics can be further enhanced by incorporating digital assays or integrating with other label free single-cell technologies. while there are many merits of single-cell analysis, we also discuss the new sets of challenges that need to be addressed in these systems. finally, we will conclude with our insight on future prospects of single-cell research in infectious disease and highlight several emerging single-cell technologies that may further enrich our arsenal against infections. understanding the pathophysiology of infection is critical to the rational design of prophylactic and therapeutic strategies to tackle infectious diseases. the course of infection, determined by the encounter of pathogens and host cells, is often measured as population-averaged results, leaving the important cell-to-cell heterogeneity out of the picture. the heterogeneity arises from both the pathogens and the infected cells. for example, pathogen heterogeneity can be reflected in the case of viruses, as a mixture of mutated viral particles displaying different infection ability [ ] , or in the cases of bacteria, as a population of cells having different resistance to the same antibiotics [ ] . host cellular heterogeneity is a combined result of variances in metabolism, composition, activation status, cell cycle, or infection history [ ] . recent advances in single-cell analysis provide an attractive approach to probe the cellular population diversity and characterize infection pathophysiology at single-cell resolution. in this section, we will review how the recent advancement of single-cell technologies has helped deepen the understanding of pathogen and host cell heterogeneity and how the complex immune system reacts against infectious pathogens, with a focus on the contributions of single-cell sequencing. pathogen heterogeneity can be inherent or as a result of heterogeneous host-pathogen interactions. it is a favorable feature for pathogens because varied genomic sequences or functional properties enable immune evasion, colonization in novel hosts, and drug resistance acquisition; therefore, they increase the possibility of survival. besides, stochastic fluctuation in biochemical reactions may also contribute to cell-to-cell variability. single-cell technologies provide high-resolution insights into different aspects of intracellular pathogen replication. one area of virology that has benefited from the enhanced resolution of single-cell technologies is the study of variation in infection across single cells and the reasons for such variation. in the study by heldt et al., cells were infected in a population, isolated into microwells, and incubated. the supernatant was subjected to viral plaques measurement, and viral rna was quantified from lysed single infected cells [ ] . it was shown that cells infected by influenza a virus (iav) under the same conditions produced largely heterogenous progeny virus titers, ranging from to plaque-forming units (pfu) and intracellular viral rna (vrna) levels varied three orders of magnitude. similarly, using scrna-seq, another study determined the percentage of viral transcripts in the total mrna generated from iav-infected cells, and it revealed that while most cells contained less than % of viral transcripts, some cells generated more than %, demonstrating infection heterogeneity from the angle of viral load [ ] . reasons for this variation can be further explored through the use of high-throughput imaging technology. for instance, akpninar et al. used virus expressing red fluorescence protein (rfp) to study the effect of defective interfering particles (dip) on viral infection kinetics. dip are noninfectious progeny particles lacking genes essential for replication, and they are commonly produced during infection due to the high mutation rate. when participating in infection along with viable viral particles, they compete for host cellular machinery and result in viral replication inhibition. in this study, cells in a bulk population were infected with a mixture of vesicular stomatitis virus (vsv) expressing rfp and vsv-dips, and they were either untreated or isolated by serial dilution. rfp expression was observed during incubation as a surrogate for viral replication levels. the results showed that dip inhibited viral replication times more on single cells, suggesting that the inhibition of viral replication is mitigated by cell-cell interactions when infection happens in a population [ ] . table . overview of commonly used single-cell technologies and their respective characteristics: a higher color intensity corresponds to a higher score (e.g., higher throughput, ease of moving cells of interest onto subsequent assays, higher information content, higher cost). the genomic mutation of pathogens during infection can be also detected directly. the sequencing of transcriptome and viral genes in single infected cells showed that iav is highly prone to mutation during infection [ ] . detected mutations can cause consequences include viral polymerase malfunction and failure to express the interferon (ifn) antagonist protein, which is correlated to heterogeneous immune activation among infected cells [ ] . the sequencing of plaques from vsv-infected cells detected parental single nucleotide polymorphism (snp) and snp generated during infection ( figure a -e) [ ] . although extremely low multiplicity of infection (moi) was adopted, resulting in % of the cells statistically infected with only one pfu, % contained more than one parental variant, indicating that pre-existing differences in viral genomes can be spread within the same infectious unit, in this case, the host cell population. moreover, by measuring the viral titers produced by each infected cell, a significant correlation was found between the number of mutations in the viral progeny and the log yield of the initially infected cell. (e) correlation between the abundance of each type of substitution in single-cell-derived plaques and natural isolates. all panels adapted with permission from [ ] . copyright , elsevier. genomic variability also widely exists among bacteria populations. fluorescence labeling enables the quantification of bacterial growth in single host cells [ ] [ ] [ ] , and by correlating the heterogenous growth with host response, it was found that the salmonella population exhibits different induction levels of the phop/q two-component system, which modulates lipopolysaccharides (lps) on the surface of individual bacteria [ ] . to understand the pathophysiology of infectious diseases, it is important to study the identities of targeted cells. mounting evidence has shown that even under identical conditions, individual host cells manifest differential susceptibility and responses to infection in a population. how does this preference arise? do they share similar features that might be reasons for their susceptibility of infection? how do the states of infected cells affect pathogen replication and infection outcome? furthermore, how are host cells' phenotypes influenced by infection individually and temporally? answers to these questions are critical for the identification of target cells and individuals of novel pathogens, as well as for the understanding of infection pathophysiology. analysis of cells exposed to pathogens at single-cell resolution requires, first and foremost, strategies to distinguish infected cells from uninfected ones. pathogen-specific proteins, such as viral glycoproteins embedded in the cell membrane, or intracellular proteins such as viral capsid or polymerases, as well as pathogen nucleic acids, including genomic dna/rna and transcripts, can serve this purpose. these microbial elements can be labeled with specific antibodies or oligonucleotide probes for detection and quantification. alternatively, pathogen nucleic acids can be directly captured in deep sequencing. by combining tools for pathogen identification with host cell phenotyping assays, infected cells can be profiled at the single-cell level. xin et al. investigated the effects of host cell heterogeneity on both acute and persistent infection by foot-and-mouth disease virus (fmdv) [ ] . by sorting single infected cells with facs based on cellular parameters, and quantifying viral genome replication with rt-pcr, they showed that the host cell size and inclusion numbers affected fmdv infection. cells with larger size and more inclusions contained more viral rna copies and viral protein and yielded a higher proportion of infectious virions, which is likely due to favorable virus absorption. additionally, the viral titer was -to -fold higher in cells in g /m than those in other cell cycles, suggesting that cells in the g /m phase were more favorable to viral infection or for viral replication. such findings have also been reported for other viruses [ , , ] , revealing a general effect of heterogeneous cell cycle status in a population on virus infection. golumbeanu et al. demonstrated host cell heterogeneity using scrna-seq: they showed that latently hiv-infected primary cd + t cells are transcriptionally heterogeneous and can be separated in two main cell clusters [ ] . their distinct transcriptional profiles correlate with the susceptibility to act upon stimulation and reactivate hiv expression. in particular, genes were identified as differentially expressed, involving processes related to the metabolism of rna and protein, electron transport, rna splicing, and translational regulation. the findings based on in vitro infected cells were further confirmed on cd + t cells isolated from hiv-infected individuals. similarly, enabled by scrna-seq and immunohistochemistry, several candidate zika virus (zikv) entry receptors were examined in the human developing cerebral cortex and developing retina, and axl was identified to show particularly high transcript and expression levels [ , ] . scrna-seq can also be used to identify potential target cells of novel pathogens and facilitate the understanding of disease pathogenesis and treatment. the spike protein of the virus sars-cov- , the pathogen responsible for the covid- pandemic, binds with the human angiotensin-converting enzyme (ace ) [ , ] . this binding, together with a host protease type ii transmembrane serine protease tmprss , facilitates viral entry [ , ] . by analyzing the existing human scrna-seq data, it was identified that lung type ii pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells co-express ace and tmprss , which suggests that they might be the putative targets of sars-cov- [ ] . in the preparation of scrna-seq library, standard poly-t oligonucleotide (oligo-dt) is commonly used to capture mrna from single cells, which can also capture polyadenylated viral transcripts from dna virus or negative-sense single stranded rna virus. a simultaneous analysis of host transcriptome profiles and viral dna/rna offers information on the presence of the studied pathogen and its activities and allows a more accurate characterization on the dynamics of host-pathogen interactions. wyler et al. profiled the transcriptome of single human primary fibroblasts before and at several time points post-infection with herpes simplex virus- (hsv- ), and they described a temporal order of viral gene expression at the early infection stage [ ] . more importantly, by simultaneously profiling the host and viral mrna, they identified that transcription factor nrf is related to the resistance to hsv infection. the finding was verified with the evidence that nrf agonists impaired virus production. steuerman et al. performed scrna-seq of cells from mice lung tissues obtained days after influenza infection [ ] . facs was applied to sort immune and non-immune cells based on cd expression. nine cell types were clustered (figure a) , and viral load was determined by the proportion of reads aligned to influenza virus gene segments, with higher than . % considered infected. the authors found that viral infection can be detected in all cell types, and the percentage ranges from % in epithelial cells to % in t cells. however, the high variability of viral load was only observed among epithelial cells, while the majority of infected cells of other cell types showed to have low viral load (less than . %) ( figure b ). for positive sense rna virus whose transcripts lack polyadenylation and cannot be captured by oligo-dt, a reverse complementary dna oligo probe to the positive-strand viral rna was employed. zanini et al. described this method and correlated gene expression with virus level in the same cell to study the infection of dengue virus (denv) and zika virus (zikv). they identified several cellular functions involved in denv and zikv replication, including er translocation, n-linked glycosylation, and intracellular membrane trafficking [ ] . interestingly, by contrasting the transcriptional dynamics in denv versus zikv-infected cells, differences were spotted in the specificity of these cellular factors, with a few genes playing opposite roles in the two infections. genes in favor of denv (such as rpl , tram , and tmed ) and against denv infection (such as id and ctnnb ) was also validated with gain/loss-of-function experiments. analysis methods have been advancing for the detection of genetic variant-based scrna-seq data [ ] [ ] [ ] . they could contribute, in the study of infectious diseases, to the characterization of temporal changes in viral mutational prevalence [ ] . moreover, viral mutation can be correlated with host gene expression status at the single-cell level to further investigate their potential mutual effect on one another throughout the course of infection and reveal the dynamic host responses and pathogen adaptations in the progression of infection [ ] . in spite of the above-mentioned examples characterizing virus presence with scrna-seq, it is worth noticing that viral mrna or genome occurrence is not necessarily equivalent to viral progeny, due to reasons such as missing essential genes caused by mutations. experimental techniques enabling the joint analysis of host transcriptional responses and viral titers will be needed to reveal the underlying mechanisms of virus production levels and host cell heterogeneity. another challenge of analyzing viral rna data is distinguishing infected cells with intracellular viral transcription from uninfected cells acquiring exogenous viral rna. combining single-cell transcriptomics data with flow cytometry or mass cytometry by time-of-flight (cytof) to measure the intracellular viral protein may help overcome this issue. immune and non-immune single cells were isolated from the whole lung of control and influenza-treated mice for massively parallel single-cell rna sequencing. host and the viral mrna were simultaneously measured, allowing the identification of infected as opposed to bystander cells, the quantification of intracellular viral load, and the profiling of transcriptomes. nine cell types were distinguished based on their transcriptional identities (b) the single-cell heterogeneity of intracellular viral load during influenza infection. percentages of low (yellow), medium (light brown), and high (dark brown) viral-load states (y axis) within the population of infected cells are shown for each of the nine cell types (x axis; total numbers of infected cells are indicated). (c) host genetic responses across all cell types. differential expression in influenza-treated and control mice (color bar) of nuclear-encoded genes (rows) across the nine major cell types (columns). right column indicates membership in four type i interferon (ifn)-related categories. all panels adapted with permission from [ ] . copyright , elsevier. immune responses activated by infection, since it is the innate immune responses that are primarily initiated in infected cells, or adaptive immune responses by lymphocytes carrying specific roles, are dynamic and complex, and they often happen in specific tissue microenvironments. heterogeneity in immune responses is also a long-recognized phenomenon. for instance, the activation of antiviral responses in dendritic cells (dcs) by bacterial lps starts with a small fraction of cells initiating the reaction, followed by the response by the rest of the population via paracrine responses [ ] . technologies that enable the simultaneous measurement of multiple parameters facilitate high-resolution characterization of transcripts and protein at the single-cell level and boost our understanding of how host immune responses are initiated and orchestrated against infection. although pathogens usually dominate the war with host immune responses, hence the prevalence of infectious diseases, in-depth understanding of the interplay provides valuable information for the design of strategies to fight against infectious diseases. in this section, we cover the single-cell characterization of both innate immune responses from infected cells and adaptive immune responses activated in infected units. type i interferon (ifn), a key cytokine in innate immunity, orchestrates the first line of host defense against infection. its production is initiated upon host cells sensing pathogen-specific molecules, and it turns on the antiviral state of host cells by activating the transcription of hundreds of ifn-stimulated genes (isgs), some of which are crucial for coordinating adaptive immune responses. many studies have shown a large variability of ifn expression among infected cells. in the case of influenza virus infection, this can be partially explained by the high mutation rate during replication, revealed by sequencing viral genes in single infected ifn reporter cells [ ] . however, such viability was also found to exist in infected cells expressing unmutated copies of all viral genes, which might be a result of the stochastic nature of immune activation irrelevant to viral genotypes [ ] . in another study, pbmcs from patients with latent tuberculosis infection (ltbi) or active tuberculosis (tb), and from healthy individuals were analyzed with scrna-seq [ ] . t cells, b cells, and myeloid cells were distinguished, and subsets were clustered. the novel finding in this work is the consistent depletion of one natural killer (nk) cell subset from healthy individual samples to samples from ltbi and tb, which was also validated by flow cytometry. the discovered nk cell subset could potentially serve as a biomarker for distinguishing tb from ltbi patients, which is valuable for predicting disease outcome and developing treatment strategies. by analyzing scrna-seq data of pbmcs derived from individuals before and at multiple time points after virus detection, kazer et al. investigated the dynamics of immune responses during acute hiv infection [ ] . after identifying well-established cell types and subsets in pbmcs, the authors examined how each cell type varies in phenotype during the course of infection. genes involved in cell-type specific activities, including monocyte antiviral activity, dendritic cell activation, naïve cd + t cell differentiation, and nk trafficking manifested similar changes with plasma virus levels: peaking closer to detection and gradually descending with time. phenotypic variations in bacteria populations were shown to influence host cell responses. avraham et al. investigated macrophage responses against salmonella infection with fluorescent reporter-expressing bacteria and scrna-seq on host cells [ ] . transcriptional profiling revealed the bimodal activation of type i ifn responses in infected cells, and this was correlated with the level of induction of the bacterial phop/q two-component system. macrophages that engulfed the bacterium with a high level of induction of phop/q displayed high levels of the type i ifn response, which was presumably due to the surface lps level related to phop/q induction. with a similar setup, saliba et al. studied the salmonella proliferation rate heterogeneity in infected macrophages [ ] . the varied growth rate of bacteria, indicated by fluorescent expression by engineered salmonella in single host cells, influenced the polarization of macrophages. those bearing nongrowing salmonella manifested proinflammatory m macrophages markers, similar with bystander cells, which were exposed to pathogens but not infected. in comparison, cells containing fast-growing salmonella turned to anti-inflammatory, m -like state, showing that bacteria can reprogram host cell activities for the benefit of their survival. the above-mentioned strategy to simultaneously profile host cell transcriptome and viral rna also plays an important role in characterizing immune responses against infection by identifying infected immune cells and analyzing the transcriptomes simultaneously. for instance, it was applied to study the heterogeneous innate immune activation during infection by west nile virus (wnv) [ ] . high variability was revealed for both viral rna abundance and ifn and isgs expression. interestingly, the expression of some isgs, with tnfsf , ifi l, and mx being the most prominent examples, was found to be negatively correlated with viral rna abundance, which could be a direction for future studies on wnv-mediated immune suppression in infected cells. similarly, zanini et al. studied the molecular signatures indicating the development of severe dengue (sd) infection by analyzing single pbmcs derived from patients [ ] . facs was employed to sort pbmcs into different cell types (t cells, b cells, nk cells, dcs, monocytes), and then scrna-seq was performed. the majority of viral rna-containing cells in the blood of patients who progressed to sd were naïve immunoglobulin m (igm) b cells expressing cd and cxcr receptors, as well as monocytes. transcriptomic profiling data indicated that various ifn regulated genes, especially mx in naive b cells and cd in cd + cd + monocytes, were upregulated prior to progression to sd. comparison of the single-cell transcriptomes of lung tissue from health and influenza-infected mice revealed that genes, among which the majority are isgs and targets of antiviral transcription factors, were consistently upregulated among all nine identified infected cell types, including both immune and non-immune cells [ ] . this finding suggested that antiviral innate responses against influenza infection generically exist ( figure c ). moreover, by contrasting the expression profiles among infected, bystander, and unexposed cells, it was shown that the non-specific ifn gene module is a result of extracellular exposure and responses of environmental signals. while single-cell transcriptomics analysis provides an unbiased determination on host cell states, proteomics analysis offers direct characterizations of proteins expressed upon pathogen activation. going beyond traditional flow cytometry, mass spectrometry, or cytometry by time-of-flight (cytof) offers vastly increased numbers of parameters that can be investigated simultaneously, exponentially increasing the depth of the dataset collected. for instance, to investigate the effect of a precedent dengue virus infection on the outcome of subsequent zika infections, pbmcs derived from patients with either acute dengue infection or health individuals were incubated with dengue virus or zika virus, and the treated pbmcs were assessed by multiparameter cytof [ ] . cytof in this study allowed the simultaneous detection of changes in the frequency of immune cell subpopulations and quantification of functional activation markers and cytokines in distinct cell subsets. while secondary infection with dengue virus led to increases of cd + t cells and t cell subsets, which are involved in adaptive immunity, secondary infection with zika virus induced the upregulation of several functional markers including ifnγ and macrophage inflammatory protein- β (mip- β) in nk cells, dcs, and monocytes, indicating an intact innate immunity against zika virus in the cases of possible concurrent dengue infection. hamlin et al. compared two denv serotypes (denv- and denv- ) in their infection in human dcs using cytof, which allowed simultaneous analysis on denv replication, dc activation, cytokine production, and apoptosis [ ] . the tracking of intracellular denv proteins and extracellular viral particles showed different replication kinetics yet similar peak viral titers by these two serotypes, as well as the percentage of infected dcs. moreover, denv- infection was found to induce a higher expression of cd , cd , and greater production of tumor necrosis factor-α (tnfα) and interleukin- β (il- β), compared to denv- infection. additionally, bystander cells, which were identified by the absence of intracellular viral proteins, were identified to produce less tnfα and il- β, but show more activation of interferon-inducible protein- (ip- ), which is a member of isgs. besides cytof, host cell secretomes can also be measured with customized miniatured systems, and the level of multiplexing and flexibility of sample handling is often improved. for instance, lu et al. showed the co-detection of secreted proteins from immune effector cells stimulated with lps [ ] . in a similar setup, chen et al. performed a longitudinal tracking of secreted proteins from single macrophages in response to lps treatment [ ] . these studies provide valuable insights into the dynamic and comprehensive responses to pathogen over time. notably, such methods require microfabrication tools and skills, which is not always available and thus hinder their accessibility, compared with flow cytometery and cytof. epigenetic profiling at the single-cell level is also important, especially for elucidating the influence of host immune responses in chronic infection. the assay for transposase-accessible chromatin with high throughput sequencing (atac-seq) utilizes tn transposase to insert sequencing adapters into regions of open chromatin, in order to study genome-wide chromatin accessibility. buggert et al. applied atac-seq and established the epigenetic signatures of hiv-specific memory c + t cells resident in lymphoid tissue [ ] . yao et al. used chromatin immunoprecipitation followed by high-throughput sequencing (chip-seq) to examine the histone modification of progenitor-like cd + t cells from mice chronically infected with lymphocytic choriomeningitis virus (lcmv) [ ] . they found that progenitor-like cd + t cells showed distinct epigenomic features compared with memory precursor cells, exhibiting more abundant active histone markers (h k ac modification) at genes co-expressed with tox, which encodes the thymocyte selection-associated high mobility group box protein tox. this might promote the long-term persistence of virus-specific cd + t cells during chronic infection. in some cases, deep sequencing can be implemented together with other single-cell technologies for a comprehensive and systematic profiling of immune responses against infection. for instance, michlmayr et al. performed -plex cytof on peripheral blood mononuclear cells (pmbcs), rna seq on whole blood, and serum cytokine measurement of blood samples from patients with chikungunya virus (chikv) infection [ ] . moreover, samples collected at acute and convalescent phases were compared to study the disease progression. such multidimensional analysis allows the large-scale, unbiased characterization of gene expression, cytokine/chemokine secretion, and cell subpopulation changes in response to infection. one important result of this study is revealing monocyte-centric immune response against chikv, with the frequency of two subsets both related to antibody titers and antiviral cytokine secretion. in addition, significant viral protein expression was found in two b cell subpopulations. while multiple assays can be done on the same bulk sample to obtain different data parameters (e.g., transcriptomic, proteomic), such datasets are not able to correlate the data parameters at the resolution of a single cell. newer advances allow the simultaneous collection of multiple types of parameters for the same cell. for instance, cellular indexing of transcriptomes and epitopes by sequencing (cite-seq) and rna expression and protein sequencing (reap-seq) are techniques for the simultaneous collection of transcriptomic and high-dimensional information on specified proteomic targets. by using antibodies tagged with unique nucleotide sequences, the subsequent transcriptomic sequencing simultaneously sequences these tags to allow the quantification of the antibody targets. corresponding transcriptomic and proteomic data at the single-cell level allows the opportunity to study the role of post-translational gene regulation in the immune response. the increased dimensionality of the information obtained may also allow more accurate machine learning to identify signatures of healthy or dysfunctional immune responses. for instance, using cite-seq, kotliarov et al. were able to identify a common signature of activation in a plasmacytoid dendritic cell-type i interferon/b lymphocyte network that was associated both with flares of systemic lupus erythematosus (sle) and influenza vaccination response level [ ] . as noted above, the ability to study biological processes at the single-cell level gives an unprecedented to attribute bulk phenotypes in immunology and host-pathogen interaction to specific cell subpopulations, including rare cell populations, in a relatively unbiased fashion. apart from basic science discovery, how do these insights affect clinical practice in infectious disease? biomarker discovery is one obvious area of impact-the molecular differences found to underpin broader disease phenotypes can be used to diagnose or even predict disease. in particular, diagnosis is a notable problem in infectious disease, where identification of the causative pathogen can take days to weeks for culture-based systems, which may delay appropriate, targeted treatment [ ] . apart from biomarker discovery, single-cell technology is also revolutionizing the discovery of vaccines and therapeutics, which will be elaborated upon in the sections below. other clinical uses of single-cell technology may require an increased uptake of such technologies within the hospital setting. for instance, one potential area of impact is antimicrobial resistance. the bulk genotype or phenotype of a pathogen population may not accurately identify its ability to become resistant to antimicrobials, since antimicrobial resistance can involve the selection of a previously rare, resistant population. should single-cell technology become routinely used in hospitals, the increased resolution could enables the identification of such rare populations, which can inform the choice of antimicrobials prescribed. to generalize, this similarly applies to any disease phenotype that can be triggered by a rare host or pathogen cell population. the complexity of current single-cell technologies hinders their implementation in the clinic, and in the section titled diagnostics, we highlight various steps that have been taken toward simplifying single-cell technology platforms to allow their clinical use. the first step of the vaccine development pipeline would be to identify a promising disease antigen, which could be in the form of a recombinant protein or inactivated/attenuated virus. unlike traditional vaccinology where vaccines were generated via pathogen growth and inactivation, the reverse vaccinology approach relies on predicting antigen features that are likely to trigger protective functions and engineering the antigen accordingly [ ] . to predict these antigen features, two main approaches have been used: via whole genome sequencing and more recently, identifying and mapping the structural epitopes of neutralizing antibodies using the methods discussed later in this review [ ] . after identifying a vaccine candidate, the next step would be to verify its efficacy. this efficacy is quantified based on its ability to bring about a set of specific immune responses which are specifically linked with protective functions, which are known as correlates of protection (cops) [ ] . it is important to identify the cops for each vaccine for multiple reasons, including the following: ( ) to understand the mechanisms of vaccine protection for improvement of vaccines, ( ) to understand the mechanisms of vaccine protection for improvement of vaccines, ( ) to determine the consistency of the vaccines produced, ( ) to evaluate the levels of protection to patients before and after treatment, and ( ) for the licensure of said vaccine [ ] . historically, most of the cops in commercial vaccines typically involve quantifying the titer of neutralizing antibody produced by antigen-specific memory b cells. in the past decade, better understanding of the in vivo vaccine response has led researchers to identify several relevant memory t-cell responses as cops, and these t-cell responses are usually quantified by measuring the expressed cytokines via techniques such as elispot, flow cytometry, and elisa [ ] . however, it remains difficult to define vaccine cops for a number of diseases. these include those diseases that cannot yet be eliminated by vaccine or infection-elicited immune responses (e.g., hiv- infection, tuberculosis), since a suitable end point of protection is not attainable. they also include those diseases for which vaccines do not yet exist but vaccine cops may be expected to differ from infection-related cops, including diseases for which natural clearance occurs via the innate immune response or early adaptive immune response (e.g., . even when immune parameters that correlate with disease risk are found, the causative mechanism of immune protection, or mechanistic cop, may remain elusive if multiple immune parameters are elicited in parallel by a protective response. as seen in the excellent review by plotkin [ ] , the cops may not always be as obvious or limited to humoral immunity, and since vaccines typically elicit multiple immune responses. this is especially true for the case of vaccines against complex pathogens such as hiv and malaria, where the resultant network of immune responses may not always be easily identifiable. single-cell approaches may define a greater space of immune parameters to be explored as cops. furthermore, the increased breadth of data that can be obtained from a single sample is useful in increasing the number of hypotheses that can be probed, especially in longitudinal analyses, which are most useful for mechanistic immune studies but where the sample volume is often limited. furthermore, using a systems vaccinology approach via omics technology, researchers have begun to uncover these potential cops early in the vaccine development process [ ] . in one of the earliest proof-of-concepts, querec et al. successfully identified a cop for vaccine efficacy on humans vaccinated against yellow fever. a gene marker present in cd + t cells which could predict for protection was discovered by using a multivariate analysis of the immune response via a combination of flow cytometry and microarray techniques [ ] . with the rapid developments in single-cell omics technology, a deeper understanding of vaccine response can be obtained through an even more detailed mapping of the interactions between the various immune cell populations at the single-cell level, as well as identify the causes of heterogeneous vaccine response in individual immune cells [ ] . this could be seen from the recent work by waickman et al. [ ] where a dengue vaccine elicited a highly polyclonal repertoire of cd + t cells that was identified using scrna-seq. combined with transcriptional analysis of the cd + t cells, the authors established a set of metabolic markers that could be potential cops for vaccine efficacy evaluation. combining the simultaneous analysis of single-cell transcriptomic and tcr sequence data, tu et al. identified preferential transcriptional phenotypes among subsets of expanded tcr clonotypes. this is a strategy that may be highly valuable in assessing the functionality of t cells and their correlation to protection in vaccine responses [ ] . antibodies are widely used in therapeutics and diagnostics due to their high specificity and generally low toxicity. antibodies are capable of mediating protective functions against infectious diseases, including pathogen neutralization, antibody-mediated phagocytosis, antibody-mediated cellular cytotoxicity, and complement-dependent cytotoxicity. antibody-containing sera remains in use for diseases where there are no other therapeutic options, including for viruses such as hepatitis a or b, rabies, vaccinia, sars-cov- at the point of writing, and for toxins (e.g., snake venom). however, there are limitations to this approach: serum therapy from animal sources causes a risk of serum sickness due to immune reaction against animal protein, while pooled hyperimmune sera from humans is difficult to collect and standardize. instead, the appropriate b cell clone that secretes antibody with protective activity can be isolated, and its antibody sequence can be obtained and expressed in culture to obtain monoclonal antibodies as therapeutics. similarly, in diagnostics, monoclonal antibodies provide the specific recognition of pathogen antigens that allow the rapid diagnosis of infection. in order to identify the correct b cell clone from thousands or millions of b cells, its antigen specificity and/or protective activity must be interrogated. this is classically done by cell immortalization (such as by hybridoma production or epstein-barr virus infection to generate b lymphoblastoid cell lines), followed by single-cell plating and expansion to obtain sufficient antibody from a single clone, and then the well-based screening of the antibody-containing cell supernatants. however, these techniques are low in throughput and efficiency, losing more than % of potential cells [ , ] for hybridomas, and - % of potential cells for b lymphoblastoid cell lines [ , ] . moreover, there remains a bottleneck in throughput at the subsequent stage of subcloning and screening the resulting clones to determine which clones are antigen-specific and functional for the desired purpose-even large experiments are limited to screening several thousand cells [ , ] , or up to , cells for robot-assisted operations [ ] , whereas a single ml human blood draw contains an order of magnitude more (approximately , ) candidate cd + igd-class-switched memory b cells [ ] . more recently, techniques that avoid the need for cell expansion have been developed-these speeds up the life cycle for monoclonal antibody discovery. primary b cells expressing antigen-specific b cell receptors (bcrs) are labeled using fluorescent antigens, allowing flow cytometry-based single-cell sorting to isolate these antigen-specific b cells [ ] . this technique is useful especially for the interrogation of memory b cells, which express the bcr on their surface. the interrogation of plasmablasts and plasma cells, which secrete antibodies but have low or no surface expression of the bcr, require other procedures such as the formation of an ig capture matrix on the b cells [ ] , or alternative methods of screening that allow the physical separation of single cells such as droplets [ ] , nanowells [ ] [ ] [ ] [ ] , or microcapillaries [ ] . following the isolation of the desired b cells, they are lysed and their rna is interrogated to recover the antibody heavy and light chain genes. with these techniques, both antigen interrogation and antibody gene recovery do not require large clonal cell populations, removing the need for inefficient and time-consuming cell expansion processes. for the recovery of antibody genes, rt-pcr is commonly used, but recovery rates are typically low (< % success rate for each pair of heavy and light chains) due to the large variability across the v gene families. single-cell rna-seq (smart-seq ) is an alternative to rt-pcr, which results in improved recovery rates (> %) [ ] . bcr recovery can also be done in the same step as antigen-specific sorting via the use of dna-barcoded antigens, such that both the antigen barcodes and bcr sequence are recovered simultaneously during single-cell ngs [ ] . this has been used to successfully isolate broadly neutralizing hiv- -specific antibodies and influenza-specific antibodies simultaneously from a single sample, although the resulting antibody candidates had variable neutralization functions, which required subsequent in vitro confirmation. another method for monoclonal antibody discovery is the use of phage display libraries, where phages expressing antibody genes are selected for using an antigen-coated surface in an iterative process of biopanning [ ] . this has been a fast and effective method for monoclonal antibody discovery. the main limitation of phage library display is the random, largely non-native pairing of vh and vl genes, which may cause problems in subsequent antibody expression and production, and it may also have a higher likelihood of triggering anti-idiotypic allergic responses. more recently, a single-cell emulsion technique has been used for the interrogation of antigen specificity and high-throughput sequencing, allowing the interrogation of a yeast library utilizing natively paired human antibody repertoires [ ] . using it, rare broadly neutralizing antibodies against hiv- could be identified, albeit with the correct antigen required for identifying the desired b cell clones. antigen binding is the most common form of screening for monoclonal antibodies due to its compatibility with high-throughput methods including flow cytometry, biopanning, and nanowell-based elisa. however, antigen binding may not correlate with functional activity against the intended target. for example, this may occur if the protein antigen used does not accurately mimic the native form of the antigen; monoclonal antibodies generated against the protein antigen may not be active against the native target [ ] [ ] [ ] [ ] . another example would be if functional activity requires binding in a specific orientation, such as virus neutralization requiring the monoclonal antibody to disrupt the receptor binding site [ , ] . assays for monoclonal antibody function include assays for virus neutralization, opsonophagocytosis, antibody-dependent cellular cytotoxicity, and receptor agonism/antagonism [ ] . currently, these assays are typically done in bulk with relatively low throughput, creating a bottleneck in monoclonal antibody screening. microfluidic technologies, such as water-in-oil emulsions or nanowells, are being developed to increase the throughput of such assays. for instance, a high-throughput screen for enzyme antagonism using a droplet-based assay has been reported [ ] . using water-in-oil microdroplets, el debs et al. co-encapsulated single hybridoma cells with an enzyme (ace- ) and an enzyme substrate that emits a fluorescence signal upon enzyme hydrolysis, and they were able to sort out hybridomas secreting ace- -inhibiting antibodies through fluorescence-activated droplet sorting. another group has recently also reported assays that are capable of assaying cellular internalization, opsonization, and the functional modulation of cellular signaling pathways [ ] , and several companies have also reported proprietary platforms that may be able to carry out some other functional assays [ ] . however, the specificity and sensitivity of these assays have not been reported. the ability to immobilize single cells in nanowells allows repeated longitudinal profiling, which is a property that was utilized by story et al. to obtain antibody-antigen binding curves that can classify related populations of b cells [ ] . the characterization of diverse bacterial populations, including microbiome studies, has traditionally been done at the bulk level. for instance, the selection of particular organisms out of a diverse population has been done by the plating and amplification of single colonies. however, this method is limited in throughput. the enhancement of throughput can be done via miniaturization-for instance, one study isolated antibiotic-resistant e. coli mutants by encapsulating and culturing single bacteria in nanoliter-scale droplets containing the antibiotic [ ] . this approach can be applied to accelerate the identification of targets acted upon by antibiotics of unknown mechanisms. apart from being limited in throughput, traditional microbial selection systems also require the ability to culture the microorganism of interest in vitro. however, it is estimated that the bulk of microorganisms cannot be cultured and expanded in typical cell culture media [ ] . one potential solution is to use microfluidic devices to physically separate and phenotype individual bacteria while immersing them in media derived from their natural environment. this method was adopted to identify a new antibiotic, teixobactin, from a previously unculturable β-proteobacteria belonging to a group of gram-negative organisms not previously known to produce antibiotics [ ] . in the clinical setting, single-cell analysis techniques are currently rarely routinely used in infectious disease diagnostics and monitoring. it is still impractical to apply most of the other conventional single-cell analysis techniques for diagnostic applications due to the associated high costs, long workflow durations, and high degree of technical expertise required. one notable exception would be flow cytometry, where aside from its high initial equipment cost, its fast turnaround times, high sensitivity, and ease of operation make it a staple tool in clinical institutions worldwide [ ] . flow cytometry is mainly used to perform the immunophenotyping of blood cells against various disease-specific biomarkers [ ] [ ] [ ] . the most prominent example would be in the routine monitoring of human immunodeficiency virus (hiv) progression by counting the number of cd + t cells in a patient's blood sample [ ] . the other single-cell technique that has seen some use in diagnostics against pathogens would be fluorescence in situ hybridization (fish). as a diagnostic tool, fish has numerous advantages that include low cost and complexity; its rapid turnaround time allows the diagnosis of fastidious bacteria and the ability to distinguish between mixed populations of pathogens at a single-cell resolution [ ] . while fish has been successfully used for the direct identification of panels of pathogens from blood samples [ , ] , its reliance on image analysis as the readout limits the throughput of this technique, and the results are subject to user-to-user variation and bias [ ] . to resolve these issues, a variant of the technique, fish-flow, was developed. fish-flow combines fish with flow cytometry to achieve higher throughputs as well as automates the signal readout through the cytometric system [ ] , and it has been used to detect hiv reservoirs in t cells [ ] as well as bacteria from blood [ ] . while the ability to identify biomarkers at a single-cell resolution is certainly invaluable in the fight against infectious diseases, current flow cytometer systems are typically bulky and expensive, thereby limiting their use in a laboratory setting [ ] . fortunately, advancements in microfluidics and low-cost electronics have given rise to the development of portable platforms that can perform single-cell analysis in a point-of-care (poc) setting. recent examples of portable cytometric systems that are relevant to infectious disease diagnosis include a miniaturized modular coulter counter capable of label-free detection and the differentiation of particles of varying sizes [ ] , a low-cost and portable image-based cytometer for the quantification of malaria-infected erythrocytes [ ] (figure a) , and a portable miniaturized flow cytometer that is capable of multi-channel fluorescence interrogation of whole blood samples [ ] . the portability of such cytometers could mean faster turnaround test timings through on-site diagnostics and disease monitoring, hence expediting clinical decisions and improving healthcare outcomes in general [ ] . in addition, the portability of such microfluidic systems lends to other practical applications of flow cytometry, especially in pathogen detection in water and food sources. particularly, diarrheal diseases (a leading cause of death for children under the ages of ) are closely linked to the consumption of contaminated water sources and could be mitigated via regular, on-demand pathogenic testing of drinking water [ ] . however, adapting current single-cell technologies into a portable format holds its own set of unique challenges. most of the existing literature surrounding such technologies still report separate sample enrichment or staining steps prior to cell analysis [ ] [ ] [ ] ; such additional preparatory steps increase assay complexity, which may not be desirable in a poc setting [ ] . while a gamut of existing microfluidic technology has already been established for sample purification as well as for reagent addition and mixing, integrating the various modules into a single platform is typically not a trivial process [ ] . for single-cell technology to make the successful transition from the lab to the bedside, such practicalities must be considered and successfully implemented. digital assays are a relatively recent assay format comprised of the following steps: ( ) the discretization of a single initial larger sample volume into multiple smaller volumes (typically via microwell, microvalve, or droplet emulsion partitioning techniques [ ] ), and ( ) performing the chemical or biological assay on each individual volume to obtain a quantifiable signal [ ] . due to the ability to individually assay a large number of cells at the single-cell level, the digital assay format has been widely employed in single-cell omics studies [ ] . in the field of infectious disease research, while most of the applications of digital assays have been centered on answering fundamental questions relating to pathophysiology, there are other single-cell diagnostic applications that can benefit tremendously from such an assay format. an example mentioned earlier in the review would be rapid antimicrobial-susceptibility testing (ast) to address the surge of antimicrobial-resistant infections worldwide as a result of the misuse of antimicrobials. phenotypic ast, which involves the culture of the pathogen in the presence or absence of antibiotics, may help guide treatment options, but existing conventional assays have low sensitivity and require a long time of - h for cell regrowth to achieve measureable assay outcomes [ , ] . higher-sensitivity single-cell digital assays that have been recently reported can obtain measurable signals without requiring cell regrowth and could be the answer to reducing ast turnaround times ( figure b ) [ ] [ ] [ ] . another application of digital assays could be in quantifying viral reservoirs in patients at a single-cell resolution. in hiv eradication studies, latent reservoirs are reactivated using latency-reversing agents (lras) for subsequent inhibition via antiretroviral therapy [ ] . the ability to isolate and individually assay the patients' blood to obtain the distribution of reactivation states in the heterogenous cell population can give clinicians an idea of antiretroviral treatment efficacy in the future [ , ] . pump-free droplet emulsion generation system that is capable of performing antimicrobial-susceptibility testing (ast) of different species of bacteria with a turnaround time of ≈ h. image reproduced with permission from reference [ ] . copyright royal society of chemistry. (c) microfluidic impedance cytometry is able to differentiate between healthy and malaria-infected red blood cells at a single-cell resolution based on the difference in electrical impedance measured across two electrodes. image reproduced from [ ] under a creative commons license. the development of label-free single-cell analysis techniques has been gaining considerable attention over the last decade with the advent of microfluidics because of their numerous advantages over their counterparts that require cell labeling. some of the advantages include: ( ) lower technical complexity and turnaround times in assay workflow because the preparatory step is omitted, ( ) not requiring knowledge of cell biomarkers beforehand, making them suitable for assaying novel cell populations, and ( ) by avoiding the use of labels that might affect the natural state of the cells, results might be more representative of actual in vivo cellular conditions [ ] . coupled with the precise fluid handling capabilities afforded by microfluidics systems, there is a burgeoning number of label-free single-cell analysis platforms that have been reported in recent years that are able to measure infection based on the inherent properties of the cells. one prime example would be the identification of cells via their electrical properties, specifically electrical impedance. this impedance is derived from the change of voltage or current signal when single cells flow across a pair of miniaturized electrodes, and it has been shown to be able to differentiate between healthy and malaria-infected erythrocytes ( figure c ) [ ] , as well as the viability and species of parasitic protozoa [ ] . another promising direction for label-free single-cell analysis is via measuring the inherent optical properties of cells. this has been shown in recent work such as the single-cell identification of parasites through their raman spectra [ ] , the quantification of single-cell viral infection titer through laser force cytology [ ] , and single bacteria detection via refractive index measurements [ ] . lastly, the mechanical and size properties of cells have also been exploited for identifying infected single cells. for example, using inertial microfluidics, white blood cells could be hydrodynamically isolated from lysed blood containing ring-stage malaria parasites as a result of the white blood cells' larger sizes [ ] . other recent works that demonstrate potential applications for single-cell label-free infectious disease analysis includes cell identification via their acoustophoretic responses [ ] , as well as their deformability and hydrodynamic resistance [ ] . evidently, there is a host of promising label-free single-cell analysis technology that could be translated to clinical diagnostic applications. in the near future, these technologies would be useful complement poc applications where labeling steps in the assay workflow would increase the technical complexity and hinder the transition from the lab to bedside. among the methods discussed, scrna-seq is the primary tool for single-cell studies. in the following section, we briefly cover some important points that needs to be considered when designing and conducting such experiments. for a more in-depth coverage of this subject matter, the reader is invited to read other excellent reviews from luecken [ ] , see [ ] , and lähnemann [ ] . as covered earlier in this review, the main applications of scrna-seq in infectious disease study comprise of the following: ( ) studying effect of host cell heterogeneity on infection, ( ) identifying host immune responses, and ( ) antibody discovery. however, the number of sequenced cells and depth of sequencing ultimately depend on the end goal of each experiment as well as the amount of financial resources at hand. the availability of a variety of commercial platforms for single-cell analysis with different throughput and sensitivity can provide users with different options to best suit the purpose of their studies [ ] . for studies that involve identifying the cell types of a heterogeneous sample, a minimum of , reads per cell would be sufficient [ ] , while testing on a significantly large number of cells would ensure that rare subpopulations do not get missed out. one such application in infectious disease studies would be the systemic characterization of immune cell populations in response to an infection, wherein a large number of cells has to be screened in order to encompass the extensive diversity of b and t cells [ ] . on the other hand, for studies which the main goal is to obtain a high resolution readout of the transcriptome for a small number of cells, , , reads per cell would be a reasonable estimate [ ] . in typical bulk analysis, multiple biological and technical replicates can be performed in order to ensure the reproducibility of data. however, for single-cell experiments, particularly for scrna-seq, there are two main issues to contend with. firstly, measurements typically have high technical variability as replicate measurements cannot be performed on the same cell, which is lysed as part of the rna extraction process. secondly, the resulting single-cell data are typically noisy due to technical variations from the multitude of steps in scrna-seq, as well as biological variation stemming from cell heterogeneity [ ] . as such, great care has to be taken at each step of the scrna-seq workflow (i.e., sample preparation, library preparation and sequencing, data analysis) to minimize such technical variability and batch effects. one of the major sources of such variability arises from the initial sample preparation process. regardless of how the cells are dissociated, purified, or enriched, cell expression is likely to change in response to the stress induced from these processes. to minimize such undesired changes which might affect downstream data analysis, the sample preparation protocol should be optimized iteratively for each cell type [ ] . to reduce technical variability, one common method would be to spike = in known quantities of synthetic rna into the samples as controls to normalize read counts prior to data analysis [ ] . a recent advancement in such rna spike-in normalization methodology would be the bearscc (bayesian ercc assessment of robustness of single-cell clusters), which generates simulated technical replicates based on the readout signal variation from spike-in measurements [ ] . an alternative to rna spike-ins would be the use of unique molecular identifiers (umis) incorporated into the primers during reverse transcription, which essentially act as unique barcodes that allows the identification and subsequent tracking of transcribed mrna. then, the resulting data can be normalized against the umi levels to account for amplification bias during the library generation step [ ] . however, both rna spike-in and umi have their own set of limitations to consider; rna spike-ins are unsuitable for protocols that utilize poly-t priming and template switching, and since they are typically used in large amounts relative to the endogenous rna, they could potentially occupy a lot of reads. protocols utilizing umi need to ensure that library sequencing is sufficiently deep to cover all umi transcripts; otherwise, there will be a risk of incorrectly quantifying the initial sample rna [ ] . another source of error for scrna-seq comes from batch effects, which are brought about by unavoidable variations between batches of experimental runs due to changes in environmental conditions, temperature, reagent lot, etc. in response, several computational methods have been developed to mitigate said batch effects from the scrna-seq data. for example, one of the more commonly used batch-effect correction methods, combat, utilizes an empirical bayesian framework that removes batch effects via a linear model, which factors in both the mean and variance of the scrna-seq data [ ] . for a more in-depth study on the comparative performance between the various batch-effect correction methods, we urge readers to consult a recent study by tran et al. [ ] . while single-cell platforms have indeed come a long way in the past two decades, the plethora of existing techniques still face a few general concerns that could present themselves as opportunities for development in the near future. one of the inherent challenges in single-cell studies stems from the simple fact that the total amount of biological material present in a single cell is pretty limited and as a result, the resulting data are typically noisy from multiple biological and technical sources. making sense of the data requires downstream data pre-processing and analysis, which are non-trivial components of the workflow that limits the accessibility of such studies to groups with the essential background. additionally, with the increasing number and complexity of parameters at which single-cell assays are being performed, the curse of dimensionality is a pertinent problem that still requires further examination [ ] . another concern for single-cell platforms would be inter-experiment variability, as mentioned in the previous section. single-cell technologies innately have high measurement sensitivity and thus are more susceptible to variations in results obtained from technical replicates, and methods to bioinformatically correct for such differences are required. coupled with the fact that single-cell studies are typically expensive and therefore sample sizes are small, ensuring that results are comparable between each sample becomes an even more important issue. finally, the inability to maintain viable cells after analysis, particularly for high-throughput methods such as flow cytometry or scrna-seq, gives rise to a couple of problems. firstly, a majority of the conventional single-cell studies are limited to a single time point of study, following which the cells are discarded. secondly, the irrecoverability of the cells makes it difficult to integrate back-to-back assays, which required measuring different parameters. as such, improvements in cell handling to improve cell viability would be invaluable in obtain multi-parametric datasets required for a more holistic understanding of cellular behavior. to date, the applications of single-cell technology have revolutionized our understanding of host-pathogen interactions. while many studies have focused on immune cells from the blood, the study of immune responses in the context of solid tissues or foci of infection (in both acute and chronic disease phases) is important to understand the local context of host-pathogen interaction. for this, techniques allowing the integration of spatial information with other single-cell technologies will be useful. for instance, imaging mass cytometry has been used to obtain quantitative information on proteins at a spatial resolution of µm [ ] . this is done by systematically ablating a formalin-fixed tissue sample spatially line by line. the increased number of markers allows the fine distinction of cell subsets and activation states, providing valuable information on cellular roles in immune effector function or immunopathogenesis. it may even be possible to simultaneously obtain information on specific dna and rna targets via in situ hybridization. similarly, several techniques have been recently developed to obtain simultaneous spatial and transcriptomic data, including multiplexed error-robust fish (merfish) [ ] , laser capture microdissection sequencing (lcm-sequencing) [ ] , tomo-seq [ ] , slide-seq [ ] , and spatial transcriptomics [ ] . these techniques may similarly be helpful in infectious disease to better define the interplay of immune cells, susceptible cells, stromal cells, and pathogens. another important gap that remains to be bridged is the ability to comprehensively access the state of a single cell across time. both immune and infection processes are highly dynamic, but because most of the single-cell technologies listed above are destructive, changes over time must be assessed either by careful time-point studies, or by assuming the presence of a range of cells in a population that represent early and late stages of the process (e.g., cellular activation or infection stage). with the advent of microfluidic devices that can immobilize single cells for continued study, the same cell can be assessed at multiple points for longitudinal study. in addition to typical proteomic marker analyses and rna or dna in situ hybridization techniques with live cell imaging, it is already possible even to measure more complex phenotypes such as bioenergy metabolism [ ] . since microfluidic devices are also used for d organoid growth to simulate in vivo conditions, it is conceivable that future developments in technology will allow similar types of information to be collected in the context of organoids. this will represent one approximation toward high-dimensional in vivo data, which remains impossible with current methods. most of the single-cell studies reviewed in this article are based on end-point assays that are destructive and can therefore only measure a single time point of these single-cell targets. however, several recent studies outside the sphere of infectious disease have highlighted time as a prominent variable that influences the level of heterogeneity in host cells, immune cells, and pathogens. to that end, microfluidic platforms that enable the automated and precise control of media and reagents are ideal for performing such dynamic studies. for example, wu et al. [ ] , using a customized microwell-microvalve system, performed a continuous measurement of a disintegrin and metalloproteinases (adams) and matrix metalloproteinase (mmps) secretions by single hepg cells upon a phorbol -myristate -acetate (pma) challenge. using their microfluidic platform, heterogenous changes in the secretion rates of adam and mmps were observed in response to pma stimulation, which may be used to predict hepg cell fates. in another recent study, a microfluidic platform that combined mutation visualization (mv) and microfluidic mutation accumulation (µma) enabled real-time tracing of mutations of single bacteria [ ] . evidently, the utilization of microfluidic technology could enable high temporal resolution single-cell studies suited for uncovering the pathophysiology of infectious diseases. the advantages offered by microfluidics technologies include the efficient capture and compartmentalization of single cells, the precise control of fluid exchange, and ensuring a viable microenvironment for cell survival. these characteristics enable the dynamic study of large populations of single cells in parallel, which may eventually provide us with a more comprehensive understanding of the causes and effects of single-cell and single-pathogen heterogeneity. through the various applications of single-cell technology, we have gained a more thorough understanding of infectious disease pathophysiology at an unprecedented resolution. revealing the heterogeneity within populations of pathogens has allowed a finer dissection of virulence factors, and similarly, heterogeneity within populations of infected cells has given us a deeper understanding of host immune defenses. in addition, the high-dimensional single-cell information that can be collected even from primary cells has allowed us to identify rare but important cell subtypes, and it has shed light on the complex interplay between the different cells of the immune system. with the advent of antibody and t cell-based therapeutics, and antibody-based diagnostics, the contributions of single-cell technology to the high-throughput identification of candidate b and t cell receptor sequences that are target-specific have also accelerated the development of new therapeutics and diagnostics for both newly emerging and existing diseases. the adoption of single-cell technologies is likely also to revolutionize clinical studies for both drugs and vaccines, given its immense potential for biomarker discovery. with the field of single-cell technology only just taking off in the last decade, there remain vast prospects in both the increased adoption of existing technologies and the development of new technologies. the authors declare no conflict of interest. covid- coronavirus pandemic trade set to plunge as covid- pandemic upends global economy microbiology by numbers history of the discovery of the malaria parasites and their vectors the use of single-cell rna-seq to understand virus-host 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multiplexed rna profiling in single cells laser capture microscopy coupled with smart-seq for precise spatial transcriptomic profiling genome-wide rna tomography in the zebrafish embryo slide-seq: a scalable technology for measuring genome-wide expression at high spatial resolution visualization and analysis of gene expression in tissue sections by spatial transcriptomics a platform for high-throughput bioenergy production phenotype characterization in single cells high-throughput protease activity cytometry reveals dose-dependent heterogeneity in pma-mediated adam activation real-time visualization of mutations and their fitness effects in single bacteria this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - i gk et authors: bachmann, maría consuelo; bellalta, sofía; basoalto, roque; gómez-valenzuela, fernán; jalil, yorschua; lépez, macarena; matamoros, anibal; von bernhardi, rommy title: the challenge by multiple environmental and biological factors induce inflammation in aging: their role in the promotion of chronic disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: i gk et the aging process is driven by multiple mechanisms that lead to changes in energy production, oxidative stress, homeostatic dysregulation and eventually to loss of functionality and increased disease susceptibility. most aged individuals develop chronic low-grade inflammation, which is an important risk factor for morbidity, physical and cognitive impairment, frailty, and death. at any age, chronic inflammatory diseases are major causes of morbimortality, affecting up to – % of the population of industrialized countries. several environmental factors can play an important role for modifying the inflammatory state. genetics accounts for only a small fraction of chronic-inflammatory diseases, whereas environmental factors appear to participate, either with a causative or a promotional role in % to % of patients. several of those changes depend on epigenetic changes that will further modify the individual response to additional stimuli. the interaction between inflammation and the environment offers important insights on aging and health. these conditions, often depending on the individual’s sex, appear to lead to decreased longevity and physical and cognitive decline. in addition to biological factors, the environment is also involved in the generation of psychological and social context leading to stress. poor psychological environments and other sources of stress also result in increased inflammation. however, the mechanisms underlying the role of environmental and psychosocial factors and nutrition on the regulation of inflammation, and how the response elicited for those factors interact among them, are poorly understood. whereas certain deleterious environmental factors result in the generation of oxidative stress driven by an increased production of reactive oxygen and nitrogen species, endoplasmic reticulum stress, and inflammation, other factors, including nutrition (polyunsaturated fatty acids) and behavioral factors (exercise) confer protection against inflammation, oxidative and endoplasmic reticulum stress, and thus ameliorate their deleterious effect. here, we discuss processes and mechanisms of inflammation associated with environmental factors and behavior, their links to sex and gender, and their overall impact on aging. the inflammatory response is different in men and women. adult females develop stronger innate and adaptive immune responses than males. these sex-related differences can determine the ability of immune cells to generate an effective inflammatory response, which translates into epidemiological differences on the prevalence of various pathologies, including allergies ( ), asthma ( , ), autoimmune diseases ( ), anaphylaxis ( ), neonatal sepsis ( ), and cancer ( ), among several pathologies. the immune response of women is polarized towards an increased production of th cells, t regulatory cells (treg), m macrophages, il , il , and gata- cytokines, and decreased th , th , tbet, and rorgt lymphocytes ( - ). on the contrary, men show an immune response that depends on th lymphocytes ( , ), high il production ( ) and low levels of reactive mast cells ( ) . men have also an increased response of microglia in the central nervous system (cns) and an increased presence of tnfa and prostaglandins in response to inflammatory stimuli ( ). differences in inflammatory response between men and women vary among specific tissues. in the cns inflammation, women show greater levels of b-cell (cd +, cd +, cd d hi b ) migration from the spleen to the site of injury than men, followed by an increase of macrophages/microglia (cd b+, cd ), which appears to generate a lower neuroinflammatory response in female compared with male mice ( ). in addition, women develop an increased immunoreactivity due to high numbers of ifn-producing dendritic cells ( , ). female mice tend to have m phenotype and activated eosinophils and mast cells show a higher reactivity than in male mice ( , ) . however, in response to an acute inflammatory stimulus, males produce higher amounts of inflammatory cytokines, cd a+ neutrophil and t cells infiltration of the injury site ( ). conversely, the inflammatory microenvironment in female mice is characterized by an increased production of antibodies ( , ) and a differential pattern migration of antibody-secreting cells ( ). the immune system responds differently in men and women not only because of the influence of sex hormones, but also differences in the patterns of autosomal methylation and x chromosome methylation, which determine distinctive profiles of gene expression ( , ). sex hormones exert antagonist effects on the immune system: both estradiol and testosterone have a suppressive effect on the immune response ( ). estrogen is the sex hormone with the greatest impact on the immune response, being described as one of the non-modifiable regulators of the immune system, due to its immunoregulatory and protective effects in many inflammatory models ( ). however, this is contradictory with the fact that women have a higher prevalence of autoimmune diseases than men, although estrogens should be a protective condition ( ). the sex-dependent difference in the immune response is time-, and estrogen dose-dependent ( ). variations on the estrogen concentration during the ovulatory cycle, puberty or menopause, can promote the development of immune-related diseases ( ). mice exposed to chronic estrogen-treatment generate hormone resistance, decreasing the clonal expansion of treg lymphocytes in autoimmune diseases ( , ) . estrogen regulates immune response primarily through aand b-estrogen receptors (era/b), mitogen-activated protein kinase (mapk) pathways, estrogen-dependent ′- ′-cyclic adenosine monophosphate (camp) response element-binding (creb), and modifications in the production of camp in immune cells ( ). in addition to estrogen receptors, the presence of il receptors influences the type of immune response; female macrophages express greater amounts of il receptors than males. il receptors favor the m phenotype when stimulated by estrogen. in agreement with that, estrogen induces an increased expression of il on naive cd + t cells ( , ) . for a better general view of estrogen´s mechanisms and effect on the innate immune system cells we recommend reviews that have extensively covered those topics ( - ). sex regulates gene expression in multiple human tissues, in fact, one third of the autosomal genes that are expressed in a sexbiased manner exhibit androgen or estrogen hormonal response elements ( , ). sex hormones play a strong role in sexually dimorphic gene networks ( ), inducing aberrant expression in immune response genes via differential methylation ccl cxcl il ( ) . there are changes in the methylation pattern of sex-dependent immune response genes during embryonic development, which are reinforced in puberty by the estrogenmediated induction of active forms of chromatins that are maintained during adulthood ( ) . immune response-related genes located in chromosomes and x are differently expressed in b lymphocytes depending on the sex of the individual ( ). among the differentially expressed genes that are relevant for the immune/inflammatory response, can be mentioned the toll-like signaling, cytokine receptors, jak-stat pathway and genes related to the activation of t-cell receptors ( ) . phenotypically, the different pattern of gene expression may explain the greater female t-cell expandable capacity when exposed to an antigen ( ) . female t cells present higher activation and division capacities than their male counterparts. however, male t cells can develop greater infiltration potential and a lower self-reactive phenotype than female ones ( , ) . these differences could be due to the high expression of peroxisome proliferator-activated receptors (ppars) ( ) , prostaglandins, and cyclooxygenase- (cox- ) in males ( ) . the influence of sex on the immune response is observed throughout life and is accentuated with aging. in the neonatal stage, women have a lower concentration of regulatory t lymphocytes than men ( ) . during childhood, men develop a more intense immune response and are more likely to develop infections by various pathogens compared with women ( , ) . with increasing age, the dynamics and proportion of lymphocytes and myeloid cells differ depending on the sex due to the differential expression of genes of the immune response in men and women ( ) . also, in aged individuals, epigenomic changes generate a more robust innate and pro-inflammatory response in men and an increased activity in the adaptive immune response in women ( , ) . in recent times, during the covid- pandemic, it has been observed that the infection by sars-cov- in older adults shows conspicuous differences; men have elevated plasma levels of il and il and a high amount of monocytes whereas women develop a robust activation of t lymphocytes ( ) . this differences in the immune response could explain the higher mortality of covid in men than in women ( , ) . to recapitulate, sex hormones and genetic expression patterns in men and women can generate distinct immune and inflammatory responses that determine singularities in the epidemiological distribution of immune diseases. research protocols in immune response and inflammation must be redefined to avoid results biased by sex. furthermore, research in women is urgently needed to define the efficacy for women of several therapies that were originally tested in men. the increase in noncommunicable diseases (ncds), such as obesity, hypertension and cancer as well as the low-grade chronic inflammation that characterizes most ncds ( ) can be affected by environmental factors that change the immune response. lifestyle factors like nutrition can modulate the immune system. it has been reported in mice that western diet-induced systemic inflammation and reprogramming of myeloid cell precursors is mediated by the activation of the nlrp inflammasome, which is a key sensor of the innate immune system for metabolic danger signals, such as uric acid and cholesterol ( ) . metabolic regulation appears to be very robust and long lasting, being reported that proper nutrition during pregnancy can reduce the risk for ncds in the offspring even at adult age ( , ) . the impact of the diet on the immune response and inflammation some diet types can result in metabolic and epigenetic changes that affect immune function ( ) , as reported in populations that consume a high-fat and low-fiber western diet, who show a prevalence of ncds higher than populations that consume a mediterranean diet or a diet based on bioactive compounds, like the hydroxytyrosol in olive oil ( ) ( ) ( ) . there is evidence supporting the anti-inflammatory activity of phenolic extracts from olive oil, such as their ability to reduce lipopolysaccharide (lps)-stimulated nitric oxide (no) production by the raw- . macrophage cell line. the hydroxytyrosol stearate and the hydroxytyrosol oleate decrease no production in a concentration-dependent manner ( ) . in addition, olive oil extracts increase total plasma glutathione concentration ( ) , increasing the antioxidative response of the individual. nordic diet has many similarities with the mediterranean diet, but its effects on low-grade chronic inflammation are less known. both diets include abundant fruits, vegetables, whole grain products, fish and vegetable oil, but restrict saturated fat and red and processed meats ( , ) . observational ( , ) and interventional ( , ) studies report an inverse association between the adherence to nordic diet and the concentration of high sensitivity c-reactive protein (hscrp). single intervention studies reported beneficial effects, reducing il receptor a (il ra) ( ) and cathepsin s ( ) , and downregulation of inflammatory mediators in the adipose tissue ( ) and peripheral blood mononuclear cells (pbmcs) ( ) . a key nutrient in fish are the n polyunsaturated fatty acids (pufas) ( ) . the greenland inuit population, which has a high dietary intake of n -pufas, have a lower incidence of myocardial infarction than the danish population ( ) . numerous studies associate the cardioprotective effects of n- pufas to their effect on immunomodulation ( ) ( ) ( ) , and control of inflammation, including neuroinflammation during aging ( ) . the mechanism of the anti-inflammatory effects of n -pufas n -pufas can regulate the transcription and expression of inflammatory mediators such as cytokines, chemokines and adhesion molecules in cardiomyocytes, fibroblasts, endothelial cells, and monocyte-macrophages ( ) ( ) ( ) ( ) . anti-inflammatory effect of eicosapentaenoic acid (epa), docosahexaenoic acid (dha) and their biologically active metabolites (d and e resolvinsmediators derived from omega- fatty acids, primarily epa and dha that block the production of proinflammatory mediators and regulate leukocyte trafficking to inflammatory sites) can be mediated through one of the mechanisms capable of reducing inflammation of raw- . cells and of primary intraperitoneal macrophages ( ) . one of the mechanisms is the activation of g-protein coupled receptors (gpr), ea. gpr inhibition of toll-like receptor (tlr )-mediated inflammatory response, which blocks nfkb activation. the other is mediated by nuclear receptors, particularly ppars-a/g. dha binds to ppars with high affinity resulting in the activation of anti-inflammatory cascades ( ) , which appears to be responsible for the beneficial health effects ( ) . the inhibition of nfkb-mediated pro-inflammatory activity ( ) is the common mechanism of immunomodulation by n -pufas, being dha more effective than epa in reducing lps-n -pufas induced inflammatory cytokine production by macrophages ( ) . n -pufas are incorporated into phospholipid bilayers and in human atherosclerotic plaques. their incorporation is associated with a reduction in the number of foam-and t cells, and a decrease in inflammation ( ) . the increased incorporation of n -pufas in membranes affects both the innate and adaptive immune responses, impairing the maturation of dendritic cells and the function of macrophages, as well as the polarization and activation of t and b cells ( ) ( ) ( ) . it is well known that n - pufas compete with n -pufas for being incorporated into cell membranes and for the active sites of cox- and lipoxygenase, resulting in the production of less potent pro-inflammatory or even anti-inflammatory mediators, such as the -series of prostaglandin and thromboxane ( ) . resolvins reduce also neutrophil-derived ros production, favoring neutrophil apoptosis and clearance by macrophages, and inhibit chemokine signaling ( ) . the partial agonist/antagonist activity of resolvin e (rve ) on the leukotriene b receptor on polymorphonuclear cells (pmns), inhibits nfkb activation, reduces release of pro-inflammatory cytokines and reduces infiltration by pmn ( ) . moreover, rve reduces tnfa and ifng presence in the aortic wall, decreases the levels of the inflammatory marker crp and reduces macrophage infiltration of the intima. thus, rve attenuates atherosclerosis and atherosclerotic plaque formation ( ) . aging is associated with the activation of inflammatory signaling pathways ( , ) , which can be targeted by specific nutrients with anti-inflammatory effects, such as n -pufas ( , ) . in the brain, the main n -pufa is dha, representing - % of total fatty acids ( ) . aging and neurological disorders are associated with decreased levels and turn-over rate of brain n -pufas ( ) ( ) ( ) ( ) . in aged mice, n -pufa supplementation and diets enriched in dha have been reported to revert age-induced spatial memory deficits and impairment on learning and memory ( ) ( ) ( ) . in older adults, a low consumption of n -pufas and decreased erythrocyte dha levels are associated with cognitive impairment ( , ) . dietary supplementation with dha is positively correlated with an improvement in declarative memory test performance, improved cognitive function ( , ) and a lower risk of developing neurological disorders ( ) . the probable mechanisms by which n -pufas mediate their effects in the resolution of age-related neuroinflammation are the increased synthesis of n -pufa-derived rvd and decreased n -pufaderived oxylipins, displaying an anti-inflammatory profile ( , ) . to recapitulate, the evidence indicates that n -pufas and their bioactive metabolites have immunomodulatory and antiinflammatory properties. potential cardioprotective lipid mediators, through multiple mechanisms, including changes in cell membranes composition, and modification of both cell signaling and gene expression, shift the pattern of lipid metabolites toward a more anti-inflammatory metabolite profile. dietary habits may be essential regulators of the inflammatory profile and promote healthy aging, reinforcing the recommendation of a n -pufa rich diet. the long term chronic psychological stress is increasing among the world's population ( ) . its circuit arises at high cortical centers through the limbic system to the hypothalamus, where corticotropin-releasing factor (crf) is produced, which is responsible for inducing the pituitary gland to liberate adrenocorticotropic hormone (acth) that signals the adrenal cortex to synthesize and secrete glucocorticoids (gcs) ( ) . stress also activates the sympathetic nervous system (sns), particularly the adrenal medulla, activating chromaffin cells to produce epinephrine (epi), a main stress hormone along with gcs. the latter plays a key regulation feature inhibiting the hypothalamic-pituitary-adrenal (hpa) axis through negative feedback at the pituitary gland, hypothalamus, and medial prefrontal cortex, reducing crf secretion [rewieved in ( ) ]. the interplay of social and environmental stressors induces inflammation through multiple biological mechanisms, including epigenetic factors ( ) . studies in rats show that the methylation patterns of genes involved in the stress response, such as the glucocorticoid receptor (nr c ) and crf, can be modified by psychosocial factors from early childhood ( ) . similarly, early life adversity induces acute and long-lasting epigenetic modifications in nr c genes, regulating hpa axis and cytokine production, reinforcing the importance of the activation inputs during critical periods of development ( , ) . acute short-term emotional stress, such as speaking in public, leads to a transient increase in circulating inflammatory biomarkers and natural killer (nk) cells by the sns catecholaminergic activity ( ) . on the contrary, chronic stress results in a reduction of cytotoxic nk activity, determining a poorer response to cytokines ( ) . therefore, stress appears to have short term beneficial immune effects, whereas chronic stress in the absence of immune challenge has the opposite effect ( , ) , activating constantly the hpa axis with the consequent persistent elevation of systemic gcs and reduction of nk cell responsiveness to cytokines ( ) , affecting the balance of the t helper cell type /type (th /th ) cytokine networks, predisposing to a wide range of diseases ( ) . the stress magnitude has been associated with il b mrna overexpression in peripheral pbmcs, providing a molecular mechanism by which psychological stress is translated into an immune system response ( , ) . when stress becomes chronic, such as in depression, there is a maintained overproduction of inflammatory cytokines, which have been associated with gcs resistance. immune cells become less sensitive to their anti-inflammatory effects because of their persistent secretion, leading to chronic low-grade inflammation ( , ) . activation of the innate and adaptive immune system by chronic mild stressors increases inflammatory cytokines gene expression, maturation and trafficking of dendritic cells (dc), increased macrophage number and t cells recruitment and activation. social stressors can induce an increase in inflammatory responses and a state of gcs resistance at different levels ( , ) . the acute repeated social defeat stress (rsds) and chronic restraint stress (crs) models induce an inflammatory response that results in neuroinflammation and depressive behavior ( ) . stress activates the hpa axis and the sympatho-adrenomedullar (sam) axis causing neuroinflammation by circulating cytokines that crossed the blood-brain barrier (bbb) at the circumventricular organs and by cytokine bbb transporters. an inflammatory response that promotes bbb permeability, allowing more inflammatory factors entering the brain, including crf, metalloproteinase- , il , and tnfa ( ) . additionally, microglia produce chemokines that attract monocytes into the brain ( ) . activation of sns and hpa axis through continuous psychological stress dysregulate cytokine production, and together with the stress hormones corticosteroids and catecholamines, can affect endothelial adhesion molecules, causing endothelial damage ( ) . corticosteroids could facilitate the infiltration of monocytes by increasing the expression of il and il receptors on endothelial cells. these monocytes and lymphocytes, after attaching to such sites, would commence the process of infiltration into the wall vessels, leading to foam cell formation and thrombotic events ( , ) . chronic unpredictable mild stress (cums) decreases body mass and impairs the metabolism of carbohydrates and lipids. a model for cums showed an increased liver and pancreas protein-lipid peroxidation and protein oxidation ( ) . high ros production in both organs could be a result of a response mechanism to stress at the cellular level. in the liver, protein oxidation can be due to the regulation of metabolic impairments by gcs and epi ( ). the antioxidant system of the liver is in general more efficient than the pancreas. however, it is insufficient to clear the reactive species increased as consequence of chronic stress, which could be due to alterations in the antioxidant enzymatic activity ( ) . altogether, stress appears to have short term beneficial effects on the immune function, whereas chronic stress ( , ) activates persistently the hpa, elevating systemic gcs, and impairing the cytokine balance. the overproduction of inflammatory cytokines lead to gcs resistance driven by immune cells that lose their sensitivity to gcs, leading to a state of chronic low-grade inflammation ( , ) . this gcs imbalance, shares common features with aging, mediating an enhanced neuroinflammatory priming ( ) . the presence of psychological stress potentiates the defective immune response observed in aging, which at the same time conditionate an exaggerated sickness response to immune challenges (such as chronic stress). thus, chronic stress contributes to the phenomenon of inflammaging, which promotes the development of several age-related pathologies, including atherosclerosis and diabetes among others [reviewed in ( ) ]. additionally, there is an impairment of the antioxidant defense system to manage ros production after chronic stress, resulting in the damage of various tissues ( ) . in addition, people exposed to chronic stress age rapidly, showing a faster telomere shortening in their cells ( ) ( ) ( ) . on the other hand, epigenetic changes acquired during critical developmental stages could shape chronic stress-response along the lifespan, either promoting or reducing pathological aging ( , ) . substance abuse, such as alcohol and drugs, are important triggers of chronic inflammatory processes ( , ) . the effects of alcohol on human health are complex and depend on multiple factors. however, many of those factors are associated with the generation of immunosuppression and increased morbimortality in heavy users. those effects, which have been previously reviewed by goral et al. ( ) will not be discussed in this review. here, we will describe the effect of cocaine and methamphetamine abuse. both drugs are potent psychostimulants that, when repeatedly consumed, significantly disrupt the functioning of the cns, and modify the regulation of the immune response, leading to a chronic neuroinflammatory state ( ) . in general, it is known that drug abuse, among other factors, increases nfkb transcription of multiple proinflammatory genes that spread across brain cell types further amplifying of nfkb transcription, as has been reviewed by crews et al. ( ) . cocaine (benzoylmethylecgonine according to the international common denomination) is a strong stimulant tropane alkaloid that acts by modulating the catecholaminergic neurotransmitter dopamine. studies of the striatum of mice after the administration of various drugs showed that h after administration of mg/kg cocaine, there is a significant increase in gene arrays for hypoxia-inducible factor (hif- ), transcription factors, and cytokine receptors (il r, tnfa). two hours after cocaine administration, there is an increased gene expression for various tnf receptors, inducible no synthase (inos) and adhesion molecules ( ) . in the nucleus accumbens of mice stimulated with cocaine, there is a significant increase in matrix metalloproteinase (mmp ), macrophage colony stimulating factor (mcsf) and major histocompatibility complex ii (mhc-ii) ( ) . the brain of human subjects consuming cocaine shows an increased density of macrophages and activated microglia ( ) . cocaine induces the activation of microglia through the endoplasmic reticulum stress and autophagy pathways ( ) . studies of human and rodent immune cell populations after cocaine administration show decreased numbers of t lymphocytes, modulation of nk activity and cytokine production ( ) . among brain glial cells, astrocytes are the most abundant, and perform critical functions, being involved in neurogenesis, promotion of neuronal survival, elimination of free radicals, and the production of no to maintain neuronal homeostasis ( ) . nevertheless, astrocytes can also be activated by toxic stimuli, leading to a new phenotype called "reactive astrocytes", similar with the changes observed after inflammatory activation. this phenomenon has been described in various neuropsychiatric disorders, such as alzheimer's and parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis ( ) . the reactivity of astrocytes to toxic stimuli, such as cocaine, infection or disease, potentiates the neuroinflammatory process ( ) . methamphetamine (desoxyephedrine; meth) is a synthetic adrenergic agonist with psychostimulatory effects, structurally related to the ephedrine alkaloid and adrenaline. studies on the effect of meth are limited. however, it has been determined that its abuse affects the immune response. animals exposed to both acute and chronic meth use show alkalization of normally acidic organelles in immune cells, inhibition of antigen presentation, and impairment of phagocytosis ( ) . meth also generates mitochondrial oxidative damage, dysfunction of t lymphocytes and decreased production of antibodies and cytokines ( ) . meth has effects in various tissues ( ) . in the lungs, the number of t lymphocytes decreases compared with that of untreated animals, indicating a reduction in circulating cd + cells, and levels of il and il increases. in the spleen, recruitment of pmn and the number of ly- g+ and f / + are increased, whereas cd + cells are significantly reduced. in addition, levels of tnfa, ifng, il , and il are higher than those of control mice. in the liver, there is an increase of t lymphocytes and macrophages, hepatocellular atrophy, and increased levels of ifng, tnfa, il b, - , - , - , and - in the group exposed to meth compared with control animals ( ) . in the cns, meth can induce the activation of calpains and caspases; the production of ros with the subsequent induction of oxidative stress, and the release of high amounts of glutamate, causing excitotoxicity ( ) . recently, raineri et al. reported that meth induces activation of astrocytes and microglia, increasing the levels of il and tnfa mrna and its receptor (tnfr ) in the mouse striatum and hippocampus ( , ) . medical advances have resulted in the increment of the average life expectancy in developed countries. the aging of the population is associated with an increase in the number of older people using drugs of abuse. from to , the number of cocaine users aged or older that required treatment for drug addiction in the us increased by % ( , ) . aging is associated with low-grade basal inflammation that can be compounded by substance use. as cocaine exposure is associated with elevated inflammation and altered immune functioning, the presence of cocaine use disorder might exacerbate inflammatory processes in aging adults ( ) . a recent report by soder et al, compared the levels of inflammation (through the neutrophil to lymphocyte ratio) in older adults with cocaine use disorder (cud) and in healthy older adults, finding that the group with cud had a significantly higher baseline level of inflammation ( ) . the use of illegal drugs such as cocaine or methamphetamine has not been shown to affect cognitive function in older adults at the clinical level. however, the evaluation of the cognitive function of young drug users reveals a decreased performance compared with healthy young people. in fact, the cognitive function of young drug users is similar to that of adults older than years of age ( , , ) . in summary, both cocaine and meth can directly impair the immune response, induce the activation of glial cells and stimulate the release of pro-inflammatory mediators in the cns. all those effects cause relevant changes in glial cell regulation and inflammatory activation, triggering chronic neuroinflammation and potentiating pathological aging. air pollution has become an important threat to public health. air pollutants consider a mixture of gases such as nitrogen oxides (nox), sulphur oxides (sox), tropospheric ozone (o ), volatile organic compounds (vocs), and particulate matter (pm) ( ) . pm can enter the respiratory tract leading to severe in situ damage as well as inducing additional systemic effects ( ) . the world health organization (who) suggests a maximum annual exposure of μg/m³ of pm . , however, the exposure of % of the world's population exceeds the proposed limit ( ) . exposure to air pollutants is associated with increased morbimortality associated with respiratory, cardiovascular, metabolic, neurological, carcinogenic and autoimmune diseases ( , ( ) ( ) ( ) . inflammation is the main pathophysiological mechanism induced by air pollutants. in terms of the molecular and cellular mechanism induced by pollutants, pm and sox can generate ros, inducing oxidative stress, together with mitochondrial dysfunction and the consequent energy deprivation ( ) ( ) ( ) . as a direct consequence, nfkb and mapk inflammatory pathways are activated, triggering an innate immune activation ( , ) . despite the attempts to resolve the inflammatory event, the outcome appears to be an imbalance in lymphocyte homeostasis and immune system dysregulation, with inhibition of th and treg lymphocytes ( ) . there is also an increase of th lymphocytes and recruitment of eosinophils, resulting in respiratory disorders such as asthma ( , , ) . in parallel, pm deactivates the nuclear factor erythroid pathway (nrf ), involved in antioxidant regulation and prevention of oxidative stress, a necessary process for the resolution of inflammation. therefore, to maintain oxidation-reduction reactions becomes impossible, becoming a breaking point towards increased ros production and the non-resolution of the inflammatory event ( ) . another mechanism of action of pollutants is the activation of the aryl hydrocarbon receptor (ahr) by toxic agents. the binding of pm to ahr increases circulating th and decreases treg lymphocytes. increase in th associates to the release of il , promoting an abrupt increase of th lymphocyte response. these changes promote the dysregulation of the immune response associated with the development of autoimmune processes ( ) . aberrant increases in th may result in increased inflammation, with consequences such as asthma and acute respiratory failure syndrome (ards), due to neutrophil infiltration and tissue damage ( ) . studies suggest the existence of a decline in treg levels and, therefore, an inability to suppress th , th and phagocyte responses ( , ) . in addition, exposure to pm has been associated with fibrotic events, where il increases synthesis and secretion of collagen in the lung parenchyma ( , ) . in addition, it has been described that pm also induces the expression of tgfb, directly promoting fibroblast differentiation, which could also induce collagen deposition followed by a lower antifibrotic process in the liver ( ) . pollutants may promote direct dna damage through oxidation of nitrogenous bases. hu and yu described in a paper different mechanisms and changes in mirna expression that comprise specific targets of dna methyltransferases, which can impair the methylation of tumor suppressor genes ( ) . furthermore, urban populations show increased levels of mitochondrial methylation genes due to pm exposure ( ) . there is evidence of the existence of methylation, acetylation and phosphorylation of histones h and h , markers found in genes involved in the activation of immune cells and cardiovascular diseases ( , ( ) ( ) ( ) ( ) . altogether, air pollutants can generate dna adducts promoting carcinogenesis and deteriorate telomerase activity, as reviewed by martens and nawrot ( ) , and contributing to continuous dna damage and premature aging ( , ) . in vivo studies suggest that the inflammatory activation is doseand time-dependent. mice exposed to pm show that both variables are determinant for the outcome. however, inflammatory effects and major genetic changes appear to be especially dependent on the exposure to high concentrations of pm. one possible explanation is that a prolonged exposure could induce an adaptive response of the inflammatory activation ( ) , which may be mediated by the inactivation of the nrf pathway, generating a loss of antioxidant capacity and deregulation of the immune system ( ) . the resolution appears to depend on the exposure context. acute exposure would result in high levels of ros and damage, whereas prolonged stimulation, even a low-grade one, generates a constant production of ros and chronic low-grade inflammation ( ) , consequent with the potentiation of disease risk and an epigenetic age acceleration ( ) , promoting pathological aging. direct causes of the deregulation of the inflammatory resolution process resulting from inhaled contaminants are still unknown, however, the burden of associated chronic diseases is expected to increase. it is mandatory to intensify environmental policies specifically in lower-middle-income countries in prevention of the development of inflammatory conditions and the subsequent chronic diseases. aging, characterized by a progressive loss of cellular functions, is an inevitable physiologic process inherent to all living beings ( ) . the number of older adults is increasing. during the next years, up to % of the world population will be older than years ( ) . this demographic change is accompanied by a higher incidence of ncds accumulated in the aging population ( ) . therefore, various strategies have been proposed to improve the health and quality of life of older adults ( ), along with recommendations for the development of public policies that support the fiscal expenditure resulting from ncds ( ) . one of the most studied events of aging is the impairment of the immune system, characterized by an aberrant-increased activation of the innate immunity ( , ) , and high levels of circulatory inflammatory mediators that establish an inflammatory environment, and a decrease of the adaptive immune response ( , ) and a decrease of the adaptive immune response ( ) due to this low-grade chronic inflammation ( , ) , which together would promote the inflammaging phenomenon ( ) . interestingly, it is proposed that age would not be the cause per se of these diseases associated with aging ( ) . thus, there is a deterioration of the immune system's response to external stimuli, which depends on the individual's history ( ) . also, several epigenetic mechanisms can modulate the immune response in aging, enhancing changes in intercellular communication that could perpetuate inflammatory events ( ) . on the other hand, it is described that epigenetic clocks would be useful to analyze mechanisms associated with this environmental influence ( ) . finally, they would be capable of modulating the immune response in aging, enhancing changes in intercellular communication that could perpetuate inflammatory events ( ). multiple age-dependent changes play important roles in the promotion of ncds, with increased oxidative stress standing out as one of the main mechanisms. over the last two decades, evidence has revealed that increased oxidative stress and inflammation are involved in various ncds such as alzheimer's disease ( ) , rheumatoid arthritis ( ) , cardiovascular diseases ( , ) , and cancer ( ), among others. also, recent studies propose that the activation of nfkb signaling pathways could be the main driver of these associations ( ) ( ) ( ) ( ) . interestingly, de almeida et al. showed different sources of low-grade chronic inflammation that promote cardiovascular disease ( ) . in the cns, high levels of ros lead to the activation of astrocytes and microglia, further increasing the overproduction of ros and proinflammatory cytokines that promote the development of neurodegenerative changes ( , , ) . in fact, several systemic biomarkers appear to be associated with neuroinflammation and the development of cns diseases associated with aging ( ) . these modifications trigger the phenotype of senescent or aged cells characterized as sasp ( , ) extensively studied in the context of the deleterious effects of aging. however, sasp is also essential for remodeling and promoting wound healing, which requires a strict control of the inflammatory response, thus avoiding the induction of cell aging phenotypes that contribute to the development of chronic inflammatory diseases ( ) . the immune imbalance in aging occurs due to various alterations in cellular behavior and phenotype, which cause functional deficiencies in immune cells ( ) . for example, this context induces polarization of macrophages towards an inflammatory phenotype characterized by strong activation of the inflammasome ( ) . thus, these events could induce il b and tnfa release, changes in the chemoattraction of neutrophils mediated by the reduction of the intercellular adhesion molecule (icam- ) expression, and the aberrant activation of the phosphoinositide lipid kinase- (pi k) ( ) . also, there is a decrease in the expression of pattern recognition receptors (prr), which leads to the activation of proinflammatory signaling promoting tissue damage ( , ) . finally, the reduced level of certain hormones due to the impaired hypothalamic function causes the loss of muscle mass and an increase in adipose tissue, further contributing to the release of inflammatory cytokines and changes in metabolism ( ) . despite the remarkable effort being made to understand the basis of the processes underlying the inflammatory imbalance during aging, it is not fully understood. in aging, there are cumulative epigenetic changes that promote low-grade inflammation ( , ) , including a decrease in the global genome methylation, with increased methylation in specific regions, as those with repressive histone marks of cd + and cd + t cells ( ) and bivalent chromatin domains ( ) and histone acetylation and methylation. however, the influence of genomic methylation during aging remains undetermined ( ) . several studies correlate the methylation of multiple sites on cpg islands with the increase of the low-grade inflammation marker, crp ( , , ) . nonetheless, stevenson et al. propose that the dna methylation could be better associated with the low-grade chronic inflammation than crp ( ) . in addition, the age-related mitochondrial dysfunction, with the resulting oxidative stress and decreased atp production ( ) , affect the expression and activity of dna methyltransferases, which are responsible for maintaining the methylation pattern of dna ( ) . the reduced methylation results in the demethylation of the tnfa promoter in leukocytes and macrophages ( ) and the adhesion of immune cells to the endothelium ( ) . also, many epigenetic events contribute to the differentiation of proinflammatory t cells, th ( ) , which can compromise immunocompetence, associated with repression of differentiation of immune cells, loss of treg function ( ) and the alteration of the hematopoietic stem cells differentiation ( ) . thus, epigenetic mechanisms appear to have a major role in the inflammatory imbalance, which are associated with the accumulation of damage in time that ultimately leads to the perpetuation of a constant inflammatory response. according to the who, % of the world population is sedentary, lacking the benefits of physical exercise ( ) . conditions such as sedentarism, unhealthy diet, overweight, obesity and aging induce chronic low-grade inflammation. physical exercise increases the anti-inflammatory potential and reduces the pro-inflammatory effect ( ) . this equilibrium is partly modulated through tlrs ( ) , which are fundamental for the recognition of prrs, including the damage-associated molecular patterns (damps) and the induction of an inflammatory response in the absence of pathogens. there is evidence that in young people, physical exercise decreases tlrs expression, co-stimulatory molecules cd / cd , and mhcii ( , ) in cd + monocytes. physical exercise also affects the adipose tissue. exercising reduces tlr mrna expression and tnfa production in adipocytes ( , ) in obese mice. chronic physical exercise decreases tnfa and tlr gene expression in the skeletal muscle ( ) . the evidence suggests that obesity-or cerebral ischemiainduced neuroinflammation, which are associated with the overexpression of tlr and tlr , may be reduced by physical exercise through the reduction of tlrs expression as well as their downstream signaling molecules (tnfa, il b, myd , traf , tak , and nfkb), together with the reduced microglial activation ( , ) . there is evidence that cigarette smoking induces inflammatory status [reviewed in ( ) ]. however, exercise training reduces smoke-induced inflammation. in that sense, training for min with endurance exercise for days in smoke-exposed mice demonstrated that therapeutic exercise training significantly reduces the expression of il b and tnfa mrna in rectus femoris ( ) . physical exercise has been used as a therapeutic tool in chronic pathological conditions. in that sense, obese older adults (body mass index ± kg/m ; ± years) undergoing an exercise program consisting in physical therapy, endurance, and resistance for min, days per week, show a reduced expression of tlr , il , and tnfa mrna in skeletal muscle ( ) . in older adults, -week physical exercise reduces the expression of tlr and tlr , as well as tlrs downstream mediators, such as myd , p , pp , trif, ikki/ikkϵ, irf , and pirf in pbmcs ( ) . similarly, dendritic cells from multiple sclerosis patients undergoing an exercise (endurance and resistance) program for weeks reduce tnfa and mmp secretion when stimulated with a tlr ligand (lps in combination with ifng, or a tlr ligand) ( ) , suggesting that long-term physical exercise decrease tlr responsiveness. on the other hand, high-intensity physical exercise in untrained individuals induces inflammation, resulting in the increased expression of tlr , ap , nfkb, and p in mice myocardium and in adipose tissue ( ) ( ) ( ) . physical exercise associated with eccentric contractions causes expression of tlr and nfkb in skeletal muscle and liver in rats ( , ) . furthermore, this phenomenon induces muscle damage, which can increase chemotaxis, attracting nk, cd + t cells, macrophages and neutrophils to the site of injury, promoting the production of cox , inos, monocyte chemotactic protein- (mcp- ), tnfa, il , and il b, in addition to the production of ros and the activation of nfkb ( , ) . in healthy young males, one session of intense endurance exercise ( h intense cycling immediately followed by h intense running), increases plasmatic concentrations of il and il , in addition to increased gene expression of proinflammatory il receptor (il r) and tlr signaling pathways. moreover, plasma myoglobin changes in correlation with neutrophil tlr gene expression (r= . ), suggesting that their transcriptional activity was particularly induced by damps ( ) . therefore, inflammation and muscle damage are mainly associated with the type and intensity of the exercise, with loads that exceed individual physical abilities. chronic physical exercise generates epigenetic modifications. the physical exercise associated with an energy expenditure > kilocalories per week, results in hypomethylation of the il gene and hypermethylation of the tnfa gene ( ) , with an inverse correlation between tnfa methylation and tnfa mrna expression ( ) . the methylation of the caspase recruitment domain (asc) of the apoptosis-associated specklike protein gene, the main regulator of inflammasome and promoter of the activation of il b and il , decreases with aging. however, older adults who maintain physical exercises regularly express higher levels of asc methylation than subjects not exercising, which would imply a decreased release of inflammatory cytokines ( ) ( ) ( ) ( ) . similarly, in review a -month walk training can induce hypermethylation of the nfkb- gene, suppressing inflammation through the inhibition of the nfkb pathway ( ) . as life expectancy increases, age-related diseases thrive. aging is a complex multifactorial process of molecular and cellular decline that renders individuals susceptible to disease and death. maintenance of cell integrity, cell metabolism and host-defense mechanisms are tightly regulated by the surrounding microenvironment. a growing body of evidence in different biological models has contributed towards identifying biological mechanisms that ward off structural and functional deterioration. these data offer us insights into healthy aging. molecular integrity of the genome, telomere length, epigenetic stability, and protein homeostasis are all features linked to more youthful stages (regardless of the age), associated with mitochondrial fitness, metabolic regulation, efficient intercellular communication, stem cell renewal, and regenerative capacity in tissues. a good understanding of the environmental and endogenous mechanisms that underlie agerelated normal and deleterious changes, and how these pathways interconnect, remains a major challenge for slowing pathological aging while extending older adults' healthy lifespan. the study of the environmental influence on the development of complex-chronic diseases shows that in addition to genetic predisposition, the pathogenesis is promoted by changes in metabolism and behavior, cellular environment, and epigenetic regulation patterns. the type of nutrient, or environmental cytokine milieu dramatically affects not only the homoeostasis of tissues but also of complete organs and even of the whole individual. thus, tissue stress, malfunction, and damage may induce inflammation alarm responses, which result either in resolution of tissue damage, restoration of normal cell function or development of chronic disease ( figure ). older adults often present inflammaging, characterized by increased levels of proinflammatory cytokines il , il , il , tnfa/crp ( ) . however, the cellular sources of these cytokines are partially unknown. the increased inflammatory cytokines have been proposed to be a driver of unsuccessful aging (increased morbidity, degenerative processes, or frailty) and shortened health-span. the inflammatory scenario is complex and occurs in response to various internal and environmental stimuli ( figure ) mediated mainly by a high level of proinflammatory cytokines. indeed, in healthy aging, increased production of the anti-inflammatory cytokines tgfb and il , can regulate the pro-inflammatory state ( , ) . research into the impact of environmental factors on inflammaging is at an early stage and the involved mechanisms are not completely understood. several hypotheses have been developed to explain the chronic inflammation: aging-related increase of stress ( ) and oxidative stress ( ) , dna damage in senescent cells [reviewed in ( ) ], and stem cell aging ( ) . the proposed mechanisms are likely interdependent, resulting in the generation of ros causing oxidative damage and amplification of the cytokines secretion, thus perpetuating a vicious circle of systemic inflammation where tissue injury and healing mechanisms proceed in parallel while damage slowly accumulates over the lifespan of the individuals. endocrine and metabolic alterations are linked to the shift towards a pro-inflammatory profile, which could explain some age-related pathologies, such as alzheimer's and parkinson's disease, osteoporosis, diabetes, cancer, and frailty ( , ) . regarding stress-induced immune modifications, new evidence suggests that cross talk signals between the cns, endocrine and immune system are required for optimal response to stress [discussed in ( ) ]. various stressors can affect the activity and regulation of immune cells via direct regulation by the autonomic and peptidergic system or through the release of neuroendocrine mediators. moreover, neuronal catecholamines modulate immune cell functions. these interactions are bidirectional, cytokines produced by immune cells, such as il , can modulate the production of corticotropin-releasing hormone (crh) by the hypothalamus. chronic diseases are favored by some modern living conditions, such as the intake of high-caloric foods and the low level of physical activity, or endogenous signals produced by the chronic stress of modern life. there are many challenges in conducting research on biosocial processes, which will define novel disease-trigger factors. tailor-made approaches will depend on genetics, epigenetics and a constellation of factors depending on the historical as well as the present exposure to the environment. although environmental factors also express themselves as epigenetic changes, the combinatorial effect of the multiple factors generates complex patterns of epigenetic regulation, and the concomitant exposure to environmental factors can further modify the individual response. all authors contributed equally on the conception of the work, the analysis of literature and preparing the content of the review. rbe drafted and organized the manuscript. all authors contributed to the article and approved the submitted version. endogenous and environmental factors can be mostly beneficial (in green) and deleterious (in red) or can have both beneficial and deleterious effects depending on the specific context. the interplay of lifespan endogenous and environmental factors regulates the aging phenotype depending on dna damage, epigenetic changes, and inflammation. these drivers can induce functional aging hallmarks: changes in endocrine and metabolic regulation, and defective immune regulation that will further determine the response of the individual. in yellow we show processes that can participate in both protection and damage. exposure to various alarm signals induce an acute inflammation that, when associated with deleterious environmental and biological factors, potentiates chronic inflammation, which can be further promoted by excess ros production and oxidative stress that results from mitochondrial dysfunction or nox activity, leading to inflammaging and eventually to age-related disease. on the contrary, in the presence of protective environmental and biological factors, the initial inflammatory activation will be resolved and lead to a healthy aging process. ros, reactive oxygen species. sarcopenic obesity and 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longevity emerged from studies in humans role of tgf b signaling in the pathogenesis of alzheimer's disease age-dependent changes in the activation and regulation of microglia stress responses and innate immunity: aging as a contributory factor oxidative stress, inflamm-aging and immunosenescence dna damage response (ddr) and senescence: shuttled inflamma-mirnas on the stage of inflamm-aging emerging models and paradigms for stem cell ageing from inflamm-aging to immune-paralysis: a slippery slope during aging for immune-adaptation aging and parkinson's disease: inflammaging, neuroinflammation and biological remodeling as key factors in pathogenesis impact of stress on aged immune system compartments: overview from fundamental to clinical data the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © bachmann, bellalta, basoalto, goḿez-valenzuela, jalil, lépez, matamoros and von bernhardi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - baxwgc authors: nan title: introduction to the immune response date: - - journal: primer to the immune response doi: . /b - - - - . - sha: doc_id: cord_uid: baxwgc in this chapter we provide an overview of the immune system and its vital role maintaining human health. immune responses require the coordinated action of leukocytes that travel the body to eliminate threats posed by trauma, infection, toxins, and cancer. leukocytes communicate via direct contact and via production and receipt of soluble proteins and intercellular messenger proteins called cytokines. complete clearance of unwanted entities may involve both innate and adaptive leukocyte responses, which influence each other. some innate mechanisms require no induction and are completely non-specific, whereas others are inducible and involve broad receptor-mediated recognition of a limited number of pathogen-associated or damage-associated molecular patterns (pamps/damps). adaptive responses involve the selective activation of lymphocytes via engagement of their antigen receptors by a specific foreign antigen. the three major subsets of lymphocytes are t helper cells (th), cytotoxic t cells (tc) and b cells, which use distinct mechanisms to recognize antigen and carry out different effector functions. immune system malfunction can contribute to many clinical illnesses including autoimmune disorders, allergies, immunodeficiencies, chronic inflammation and cancer. stated that microbes invisible to the naked eye were responsible for specific illnesses, and the first human disease-causing organisms or pathogens were identified in the late s. at about the same time, louis pasteur applied jenner's immunization technique for smallpox to the prevention of various animal diseases. pasteur demonstrated that inoculation with a pathogen that had been weakened in the laboratory could protect against a subsequent exposure to the naturally occurring pathogen. it was pasteur who coined the term vaccination (from the latin vaccinus, meaning "derived from cows") for this procedure, in honor of jenner's work. the research of pasteur and other investigators spurred the evolution of immunology as a science distinct from (but related to) the established fields of microbiology, pathology, biochemistry and histology. today, immunology can be defined as the study of the cells and tissues that mediate immunity and the investigation of the genes and proteins underlying their function. a normal healthy person's body always strives to maintain homeostasis, a natural state of balance of all its organs and the nervous and circulatory systems. when this homeostasis is disturbed by either trauma, pathogens, or the deregulation of body cells (such occurs in cancer), the immune system responds in an attempt to restore balance. at its simplest level, this response involves identifying and clearing damaged and dying cells from the body. more complex responses have evolved to counteract assault on selected landmark discoveries in immunology and immunologists who have won nobel prizes for their work are featured in appendices a and b, respectively. the body by infectious agents, including bacteria, viruses, parasites and fungi. because these foreign invaders are literally everywhere on earth and constantly seeking vulnerable hosts, the immune system is constantly occupied with containing attacks from this quarter. indeed, despite the successful eradication of smallpox and the development of antibiotics, infectious diseases remain among the biggest killers on the planet. longstanding disorders such as malaria and tuberculosis, as well as "emerging" diseases caused by new pathogens such as west nile virus, severe acute respiratory syndrome (sars) virus, and h n influenza virus, present ongoing challenges to the immune system. humans are surrounded by other organisms-in the air, in the soil, in the water, on the skin, and on the mucosae, the protective layers of epithelial cells that line the gastrointestinal, urogenital and respiratory tracts. while most of these organisms are harmless and some are even beneficial, some are pathogenic. like all species, pathogens live to reproduce. in order to reproduce, however, many must penetrate a host's body or one of its component cells. infection is defined as the attachment and entry of a pathogen into the host. once inside the body or cell, the pathogen replicates, generating progeny that spread into the body in a localized or systemic fashion. the manner of this replication determines whether the pathogen is considered extracellular or intracellular. extracellular pathogens, such as certain bacteria and parasites, do not need to enter cells to reproduce. after accessing the body, these organisms replicate first in the interstitial fluid bathing the tissues and may then disseminate via the blood ( fig. - ) . intracellular pathogens, such as viruses and other bacteria and parasites, enter a host cell, subvert its metabolic machinery, and cause it to churn out new virus particles, bacteria, or parasites. these pathogens may then also travel systemically by entering the blood. for both intracellular and extracellular pathogens, if the infectious agent overwhelms normal body systems or interferes with cellular functions, the body becomes "sick." this sickness is manifested as a set of characteristic clinical symptoms that we call an "illness" or "disease." pathogens that are not blocked by epithelial barriers can gain access to underlying cell layers. extracellular pathogens replicate outside host cells, whereas intracellular pathogens must enter host cells to replicate. in both cases, if infection is not controlled, the pathogen may spread locally in the tissues or enter the bloodstream and spread systemically. our bodies are under constant assault by harmful microbes, yet, most of the time, assaults by these organisms are successfully repelled and disease is prevented. this resistance is due to both basic and sophisticated immune responses that combat pathogens. simply put, the job of the immune response is to "clean up" infections in the interstitial fluid, tissues and blood, and to destroy infected host cells so that neighboring host cells do not share their fate. because pathogens are constantly evolving mechanisms to evade or block immune defenses, the immune system must constantly adapt to maintain its effectiveness. it is a continual horse race as to which will be the more successful mechanism: the body's immune surveillance or the pathogen's invasion and infection strategy. we note here that the immune response itself may cause limited collateral damage to tissues as part of its larger battle against pathogens, but such immunopathic effects are usually short-lived in an otherwise healthy individual. at the turn of the twentieth century, there were two schools of thought on what mechanisms underlay immune responses. one group of scientists believed that immunity depended primarily on the actions of cells that destroyed or removed unwanted material from the body. this clearance process was referred to as cell-mediated immunity. however, another group of researchers was convinced that soluble molecules in the serum of the blood could directly eliminate foreign entities without the need for cellular involvement. in this case, the clearance process was referred to as humoral immunity, a term derived from the historical description of body fluids as "humors." today, we know that both cell-mediated and humoral responses occur simultaneously during an immune response and that both are often required for complete clearance of a threat. the cells responsible for cell-mediated immunity are collectively called leukocytes ("leuko," white; "cyte," small body, i.e., a cell) or white blood cells. however, "blood cell" is a bit of a misnomer, because a majority of leukocytes reside in tissues and specialized organs, and move around the body through both the blood circulation and a system of interconnected vessels called the lymphatic system. the soluble molecules responsible for humoral immunity are proteins called antibodies, and antibodies are secreted by a particular type of leukocyte. the production of these antibodies and the mounting of cell-mediated immune responses depend on an elaborate signaling system by which leukocytes communicate with each other as well as with other cell types in the body. this signaling is mediated by small secreted proteins called cytokines, which are mainly produced by leukocytes. the mammalian immune system can mount two types of responses: the innate response and the adaptive response. recently, it has become increasingly clear that these two types of responses are less distinct and more interconnected than once thought. indeed, researchers now believe that vertebrates are capable of a continuum of immune responses that bring more and more precise weapons to bear on a threat as required. more specifically, innate immunity is involved in all levels of immune response, whereas the adaptive response is mounted only when innate mechanisms signal that there is a relatively serious infection. in all cases, the objective is to clear the body of unwanted entities and re-establish homeostasis in the most efficient way possible. innate immune responses are triggered by disruptions to homeostasis caused by either non-infectious or infectious means. with respect to the former, innate responses help to repair tissues injured by trauma (such as that caused by a cut, a blow, or surgery) and remove dead cells and cells too damaged or old (senescent) to be of further use to the body. products synthesized by leukocytes mediating innate responses (innate leukocytes) also help to prevent "bystander injury" of healthy tissues. with respect to invading pathogens, elements of the innate immune system are the first to confront the threat and work to eliminate it from the body. such elements include preexisting anatomical and physiological barriers that attempt to block pathogen entry, and other responses that are induced after the pathogen has gained entry. only if innate responses are insufficient to control the situation and restore homeostasis, as is often the case with infection by a rapidly replicating pathogen, is an adaptive response mounted. together, the innate and adaptive immune responses allow a seamless escalation of countermeasures that maintain homeostasis in the face of cellular aging, tissue trauma and/or pathogen infection. what exactly triggers the initial response by the innate system? in a word: recognition. wherever cells are damaged or dying-either in the presence or absence of infection-particular host macromolecules are released into the extracellular milieu or become accessible on the surface of damaged cells or in cellular debris. these molecules are called damage-associated molecular patterns (damps). when a pathogen attacks, it furnishes common molecular structures on its own surface, or on the surface of cells it has infected, or as part of products it synthesizes. these structures are called pathogen-associated molecular patterns (pamps). cell damage or death in the absence of a pathogen gives rise to damps only, but both damps and pamps will be present when a pathogen invades ( fig. - ) . it is the recognition of damps and pamps by innate leukocytes that initiates innate responses. damps and pamps are recognized by host proteins called pattern recognition molecules (prms), most of which are expressed by innate leukocytes. there is not a large array of different prms, meaning that there is a limited repertoire of molecular patterns that can be recognized. however, each prm recognizes a damp or pamp that is shared by many different damaged cells or pathogens. thus, prms give the innate immune response the property of broad recognition. furthermore, because innate leukocytes expressing prms are present in large numbers, these cells don't need to multiply to work effectively: the response is immediate. finally, because innate leukocytes are activated but don't multiply in response to engagement of their prms, the speed and strength of their response is exactly the same upon a second exposure to the same pathogen or type of damage. the result is a response that is said to have no memory. recognition is also the key to initiating the adaptive immune response, but there are important differences. in contrast to the broad recognition of the prms mediating the innate response, the receptors expressed by the cells participating in the adaptive response (adaptive leukocytes) are equipped with receptors that recognize unique molecular structures on pathogens. these unique structures are called antigens, and the receptors that recognize them are antigen receptors. together as a population, adaptive leukocytes express a vast array of antigen receptors capable of recognizing almost any structure, meaning that they represent a diverse repertoire of antigen specificities. however, the antigen receptor of any one adaptive leukocyte binds to an antigen exclusive to a single type of pathogen. thus, antigen receptors have the property of specific recognition. because adaptive leukocytes are very few in number, these cells must proliferate and expand their ranks after receptor engagement before they can be effective: the response is delayed. in addition, after adaptive leukocytes proliferate and act to eliminate the pathogen, some of these cells become long-lived so that the speed and strength of the adaptive response is increased upon a subsequent exposure to the same pathogen. the adaptive response is thus said to have memory. the distinguishing features of the innate and adaptive responses are summarized in table - . a damp is a common molecular structure that is produced by host cells when they are damaged or dying (with or without pathogen infection). a pamp is a common molecular structure shared by a wide variety of pathogens or their components or products. in the case of pathogen attack, both damps and pamps will be present. cell damage or death antigens got their names because they were first identified as pathogen components that could bind antibodies; i.e., they induced "antibody generation." "antigen" now refers to structures targeted by humoral or cell-mediated responses. from the point of view of the body's leukocytes, a complex pathogen represents a collection of many different pamps, which evoke an innate response, and antigens, which may evoke an adaptive response if the innate response is not sufficient to eliminate the threat ( fig. - ) . anatomically, an innate response can be triggered at any point in the body because the majority of innate leukocytes are positioned where microbes most commonly attempt to gain access: just below the skin surface or at the mucosae protecting body tracts such as the gut and respiratory system. most of the time, the innate response successfully eliminates an invader. only those entities that succeed in overwhelming the innate defenses trigger adaptive responses that are initiated by adaptive leukocytes positioned in specialized anatomical regions called the lymphoid tissues, which include the spleen, tonsils and lymph nodes (see ch. ). the general characteristics of innate and adaptive immune responses primarily as they relate to pathogens are described in more detail in the next two sections. in this section, we present a general introduction to the central elements of innate immunity, namely barrier defenses, complement activation, pattern recognition, inflammation, phagocytosis and target cell lysis. more detailed information about the innate immune response can be found in chapter on innate immunity. innate leukocytes express a small number of pattern recognition molecules (prms) of limited diversity that collectively recognize a wide range of threats adaptive leukocytes express an almost infinite number of extremely diverse antigen receptors, each of which is specific for a particular pathogen response is triggered by prm binding to widely shared damageassociated molecular patterns (damps) or pathogen-associated molecular patterns (pamps) response is triggered by antigen receptor binding to a unique antigen derived from a specific pathogen number of leukocytes responding to a given threat is large, so no proliferation is required and response is fast number of leukocytes responding to a given threat is very small, so proliferation is required and response is slower responding cells do not proliferate and are short-lived, so level of defense is similar upon repeated exposure to the same pathogen responding cells proliferate and are long-lived, so level of defense is stronger and faster with repeated exposure to the same pathogen a given pamp is expressed by a wide variety of pathogens and is recognized by prms whose engagement activates innate leukocytes. an antigen is a molecular structure that is generally unique to a specific pathogen and is recognized by a specific antigen receptor whose engagement activates a particular adaptive leukocyte. pathogen antigens pamps note: elements of an innate immune response can be found in all multicellular organisms, whereas the mechanisms of the more recently evolved adaptive immune response are present only in the higher vertebrates (fish and above). appendix c contains a table of comparative immunology that summarizes the immune system elements present in an evolutionarily diverse range of animals. the first level of innate defense is mediated by a pre-existing collection of physical, chemical and molecular barriers that exclude foreign material in a way that is totally non-specific and requires no induction. these elements include anatomical barriers and physiological barriers. an example of an anatomical barrier is intact skin, while the low ph of stomach acid and the hydrolytic enzymes in body secretions are examples of physiological barriers. should barrier defense prove insufficient, other forms of innate defense are induced. a key player in the induced innate response is complement, a complex system of enzymes that circulates in the blood in an inactive state. once activated, the complement system contributes directly and indirectly to innate and adaptive immune defenses, as described in detail in chapter . when invaders breach anatomical and physiological barriers, innate leukocytes start to take action as a result of pattern recognition mediated by the binding of prms to pamps furnished by pathogens and to damps emanating from damaged host cells. as introduced previously, and as illustrated in figure - , prms come in several different forms, some of which are membrane-bound and others of which are soluble. prms that are expressed by innate leukocytes are called pattern recognition receptors (prrs). prrs are located in the plasma membrane of an innate leukocyte, or are soluble molecules free in the leukocyte's cytoplasm, or are fixed in the membranes of intracellular vesicles called endosomes. other prms are made by non-leukocytes and are present as soluble molecules free in the extracellular milieu. when these latter prms bind to a given pamp or damp, they must then bind to another receptor fixed in a leukocyte membrane in order to trigger the appropriate clearance response. the clearance mechanisms typically induced following prm or prr engagement by a damp or pamp are inflammation, phagocytosis, and/or target cell lysis. a brief description of each of these processes follows, with more detail provided in chapter . when leukocyte prrs are engaged by their ligands, intracellular signal transduction leads to the induction of new gene transcription and the synthesis of various "proinflammatory" cytokines. these cytokines in turn drive events responsible for the influx of first innate and later (if necessary) adaptive leukocytes into the site of injury when pamps on a pathogen bind to the prrs of an innate leukocyte, the cell becomes activated and attempts to clear the pathogen through phagocytosis, target cell lysis, and/ or the induction of inflammation. the prr may be expressed on the leukocyte surface, on its endosomal membranes or in its cytoplasm. or infection. this influx is part of a process called inflammation or an inflammatory response, and the redness and swelling we commonly associate with inflammation are its outward physical signs. this inflammation is normal and helpful, and, when properly regulated, promotes a localized gathering of the cells and molecules necessary to repair tissue damage and clear pathogens. once the threat is eliminated, the inflammation resolves naturally with time. however, if the inflammation fails to resolve and becomes chronic, it may become pathological. the powerful molecules secreted by the innate leukocytes participating in the inflammatory response can eventually cause tissue damage and impair immune system function if left to operate unchecked. thus, properly controlled inflammation is part of a healthy innate response and essential for homeostasis, whereas excessive or prolonged inflammation is immunopathic and undermines homeostasis. innate leukocytes frequently use a sophisticated means of engulfing entities that is called phagocytosis ("eating of cells"). phagocytosis is carried out primarily by three types of prr-expressing cells: neutrophils, macrophages and dendritic cells (dcs); these cell types are consequently known as phagocytes. neutrophils mainly engulf and destroy pathogens, while macrophages and dcs engulf not only pathogens but also dead host cells, cellular debris and host macromolecules. the phagocytosis of foreign entities by macrophages and dcs allows these cells to "present" unique antigens from this material on their cell surfaces in such a way that the antigens can be recognized by adaptive leukocytes that have been drawn to the site of inflammation. these cells are then activated and mediate antigen-specific protection. cancer cells and cells infected with intracellular pathogens frequently express certain damps and/or pamps on their cell surfaces that mark them as "target cells" for destruction by cells of the innate system. when these molecules are recognized and bound by prrs of innate leukocytes, complex processes are initiated that result in the lysis of the cancer cell or infected cell, preventing it from causing further harm to the body. these types of innate responses are carried out primarily by neutrophils, macrophages and another type of innate leukocyte called a natural killer cell (nk cell). in this section, we introduce the main features of adaptive immunity. the adaptive leukocytes referred to in previous sections are called lymphocytes. lymphocytes are categorized as either b lymphocytes (b cells) or t lymphocytes (t cells). the fundamental functional and developmental characteristics of lymphocytes are responsible for the specificity, division of labor, memory, diversity and tolerance of the adaptive response. each of these important concepts is introduced here as a prelude to more detailed discussions of these topics in later chapters. the specificity of an adaptive immune response for a particular antigen is determined by the nature of the antigen receptors expressed on the surfaces of t and b lymphocytes. each lymphocyte expresses thousands of identical copies of a unique antigen receptor protein. interaction of these antigen receptor molecules with antigen triggers activation of the lymphocyte. in contrast to the broad recognition mediated by prms, antigen binding to a lymphocyte antigen receptor usually hinges on the presence of a unique molecular shape unlikely to appear on more than one pathogen. the antigen receptors on the surface of a b cell are called b cell receptors (bcrs), whereas those on the t cell surface are called t cell receptors (tcrs). in both cases, the antigen receptor is itself a complex of several proteins. some of these proteins interact directly and specifically with antigen, while others convey the intracellular signals triggered by antigen binding into the cytoplasm and nucleus of the lymphocyte. these signals are critical for sustaining the activation of the lymphocyte and promoting its proliferation into an army of short-lived daughter effector cells. these effector cells then undertake the effector action appropriate for eliminating the pathogen furnishing the antigen. "division of labor" in the adaptive response refers to the different but equally important contributions of b lymphocytes and two types of t lymphocytes. these two major t cell subsets are called cytotoxic t cells (tc) and helper t cells (th). the roles of b, tc and th cells in the adaptive response are defined by the distinct mechanisms by which they recognize and respond to antigen (table - ) . b cells recognize antigen in a way that is fundamentally different from that used by tc and th lymphocytes. the bcr of a b cell binds directly to an intact antigen, which may be either a soluble molecule or a molecule present on the surface of a pathogen ( fig. - a) . activation of a b cell in this way causes it to proliferate and produce identical daughter effector cells called plasma cells that secrete vast quantities of specific antibody. an antibody is a protein that is a modified, soluble form of the original b cell's membrane-bound antigen receptor. antibody molecules enter the host's circulation and tissues, bind to the specific antigen, and mark it for clearance from the body, establishing a humoral response. since antibodies are present in the extracellular milieu, such a response is effective against extracellular pathogens. however, antibodies are unable to penetrate cell membranes and so cannot attack an intracellular pathogen once it has entered a host cell. unlike bcrs, tcrs are unable to recognize whole, native antigens. rather, a tcr recognizes a bipartite structure displayed on the surface of a host cell. this bipartite structure is made up of a host surface protein called an mhc molecule bound to a short peptide derived from a protein antigen. this complex is referred to as a peptide-mhc complex (pmhc). tc and th cells are activated by the binding of their tcrs to specific pmhcs presented on the surface of a dc. dcs can acquire extracellular pathogen antigens by phagocytosis, and intracellular pathogen antigens either by infection or by phagocytosis of the debris of dead infected cells. peptides from these antigens are bound to one of two types of mhc molecules, either mhc class i or mhc class ii, and then displayed on the dc surface for inspection by t cells. tc cells recognize antigenic peptides that the dc has presented on mhc class i molecules ( fig. - b) . the activated tc cell proliferates and differentiates into identical daughter effector cells called, for historical reasons, ctls (cytotoxic t lymphocytes). ctls can lyse any host cell displaying the same peptide-mhc class i complex recognized by the original tc cell. mhc class i molecules are expressed on almost all body cells, making them all potential targets for ctl-mediated lysis. however, unlike dcs, most host cells cannot phagocytose extracellular entities. it is therefore only after infection with an intracellular pathogen that a host cell displays an antigenic pmhc complex that can be recognized by the tcr of first the tc cell and then its daughter ctls. the cell-mediated immunity directed against such infected host cells is critical to the clearance of intracellular pathogens. th cells recognize antigenic peptides that a dc has presented on mhc class ii molecules ( fig. - c) . the activated th cell proliferates and differentiates into identical th effector cells that can recognize the same pmhc structure that activated the original th cell. however, mhc class ii molecules are expressed only by certain cell types that can act as specialized antigen-presenting cells (apcs), including dcs, macrophages and activated b cells. in the course of a pathogen attack, apcs can capture the pathogen and/or its products or components, and then display peptides derived from its antigens on mhc class ii. when the tcr of a th effector cell recognizes this pmhc structure, the cell is stimulated to produce cytokines that assist b cells and tc cells to become fully activated. in this way, th cells contribute to both humoral and cell-mediated adaptive responses, and are thus crucial for protection against many extracellular and intracellular pathogens. in addition, cytokines secreted by th cells stimulate innate leukocytes, thereby reinforcing this arm of immunity. the combined efforts of tc, th and b lymphocytes and many types of innate leukocytes may be necessary to eliminate a particularly wily invader. the innate response deals with a given threat in a very similar way each time it enters the body. in contrast, the adaptive system can "remember" that it has seen a particular antigen before. this immunological memory means that an enhanced adaptive response is mounted upon a second and subsequent exposures to a given pathogen, so that signs of clinical illness are mitigated or prevented. in other words, immunity is achieved because the body has effectively "adapted" its defenses and acquired the ability to exclude a particular pathogen. immunological memory arises in the following way. a constant supply of resting b and t cells is maintained throughout the body, with each lymphocyte expressing its complement of unique antigen receptor proteins. when a pathogen antigen enters the antibody production by b cells is described in detail in chapters and . "mhc" stands for "major histocompatibility complex," a region on human chromosome that contains genes encoding several types of molecules vital for the adaptive immune response. mhc genes and proteins are described in detail in chapter . body for the first time, a process of clonal selection takes place in which only those lymphocytes bearing receptors specific for that antigen are triggered to respond. the selected cells leave the resting state and multiply to generate daughter cells all expressing the same antigen-specific bcr or tcr. the differentiation of these daughter cells gives rise to the short-lived effector cells required to eliminate the pathogen, as well as long-lived memory cells that persist in the tissues essentially in a resting state until a subsequent exposure to the same pathogen ( fig. - ) . the attack on the pathogen by this first round of effector cells is called the primary immune response. the second (or subsequent) time that the particular pathogen enters the body, it is met by an expanded army of clonally selected, antigen-specific memory cells that undergo much more rapid differentiation into effector cells than occurred during the first antigen encounter. the result is a stronger and faster secondary immune response that eliminates the pathogen before it can cause illness. new populations of memory cells are also produced during the secondary response, ensuring that the host maintains long-term or even lifelong immunity to that pathogen. the degree of diversity of antigens recognized also distinguishes the adaptive response from the innate response. whereas the innate immune system exhibits a genetically fixed and finite capacity for antigen recognition, the recognition capacity of the adaptive immune system is nearly limitless. in fact, our bodies are capable of recognizing totally synthetic antigens that do not occur in nature. this huge diversity arises from the combined actions of several genetic mechanisms that affect the genes encoding the when antigen x enters the body, only those lymphocytes with receptors that specifically recognize x will be selected to proliferate and generate daughter clonal selection is explained in detail in chapter for b cells and in chapter for t cells. note: the generation of protective memory lymphocytes is the basis of vaccination. a healthy person is immunized with a vaccine containing pathogen antigens in order to provoke the production of memory cells that will prevent disease from developing if the individual is ever naturally infected by that pathogen. vaccination is described in detail in chapter . antigen receptors. some of these mechanisms operate before a lymphocyte encounters antigen, and some after. the primary source of antigen receptor diversity is a gene rearrangement process that occurs during the development of b cells and t cells prior to encounter with antigen. the genes encoding antigen receptors are not individual continuous entities. rather, the bcr and tcr genes are assembled from a large collection of pre-existing gene segments by a mechanism called somatic recombination. a single, random combination of gene segments is thus created in each developing lymphocyte. as a result, a lymphocyte population is generated in which the antigen receptor proteins are vastly diverse in specificity because they are encoded by hundreds of thousands of different dna sequences. in the case of b cells, additional mutational mechanisms operate that result in further structural diversification of antibody proteins. the sheer numbers of b and t lymphocyte clones generated in a healthy individual guarantee that there will be at least one clone expressing a unique receptor sequence for every antigen encountered during the host's life span. as alluded to previously, these clones, with their array of antigen receptor specificities, are collectively called the individual's lymphocyte repertoire. the generation of lymphocyte clones that can theoretically recognize any antigen in the universe raises the question of how the body avoids lymphocyte attacks on molecules present in its tissues. this avoidance is called tolerance, the fifth aspect in which the adaptive response exhibits more refinement than the innate response. as stated previously, the specificity of each antigen receptor is randomly determined by somatic recombination during early lymphocyte development. by chance, some of the genetic sequences produced will encode receptors that recognize self molecules (self antigens). these lymphocytes must be identified as recognizing self and then either removed from the body entirely, or at least inactivated, to ensure that an individual has an effective lymphocyte repertoire that does not attack healthy tissue. tolerance is established in two broad stages, each of which involves multiple mechanisms. the first stage, called central tolerance, occurs during early lymphocyte development. the mechanisms of central tolerance are designed to eliminate clones that recognize self antigens, thus establishing a lymphocyte repertoire that targets "non-self." in the second stage, called peripheral tolerance, any b and t lymphocytes that recognize self but somehow escaped the screening of central tolerance and completed their development are functionally silenced by another set of inactivating mechanisms. although we described the innate and adaptive responses separately in the preceding sections, in reality, they occur in a continuum that brings stronger and stronger weaponry to bear when (and only when) it is needed. our bodies normally deal with a pathogen attack in three broad and overlapping phases, two of which involve innate responses and the last of which requires adaptive immunity ( fig. - ) . an individual pathogen, such as a single influenza virus particle, is dealt with by these phases sequentially, but because the actual assault on the host involves billions of such particles, not every particle is in the same phase at the same time. as a result, in the host as a whole, and for an infecting pathogen population as a whole, all three phases may be happening concurrently. the first phase is mediated by the innate physical barriers, which offer immediate protection. the components making up these barriers (skin, mucosae, gut enzymes) are non-inducible in that they pre-exist and do not develop or change in response to the presence of a foreign entity. should these barriers be penetrated, the second, inducible phase of innate defense becomes effective - hours after the threat enters somatic recombination, the process that shuffles the dna of the genes encoding bcrs in b cells and tcrs in t cells, is achieved by specialized gen etic mechanisms that are des cribed in chapter for b cells and chapter for t cells. the establishment of central tolerance during lymphocyte development is described in chapter for b cells and in chapter for t cells. processes regulating mature lymphocytes, including mechanisms of peripheral tolerance, are des cribed in chapter . a pathogen attempting to enter the body first encounters pre-existing barriers and enzymes that block access non-specifically. if the pathogen manages to enter the underlying cell layer, mechanisms mediated by complement and innate leukocytes are induced due to relatively broad recognition of pamps. if a more targeted, pathogen-specific response becomes necessary, elements of innate immunity then facilitate induction of highly specific adaptive responses initiated by engagement of the antigen receptors of b, th or tc lymphocytes. complement activation phagocytosis target cell lysis inflammation skin mucosal barrier ph, saliva, proteases, etc. the body. innate leukocytes activated by the binding of prrs to pamps provided by the attacking pathogen, or to damps present due to host cell injury or death, work quickly to eliminate the invader using the mechanisms of inflammation, phagocytosis and target cell lysis. complement activation also contributes to clearance at this stage. in many cases, the incursion is completely controlled by the two phases of innate defense before adaptive immunity is even triggered. however, the cells of the innate response are ultimately limited both in numbers and in recognition capacity. fortunately, even if the pathogen ultimately manages to thwart complete elimination by innate immunity, some of the invaders and/or their components or products will have been taken up by innate leukocytes and presented as foreign antigens to t lymphocytes. the third and final phase of host defense mediated by adaptive immunity is thus initiated. th, tc and b cells are clonally selected and activated, and then proliferate and differentiate into memory cells and large numbers of daughter effector cells that eliminate the pathogen via humoral and/or cell-mediated mechanisms. because it takes time for the clonally selected lymphocytes to produce effector cells, adaptive responses are not usually observed until at least hours after the start of infection. the second and third phases of immune defense are more tightly interwoven than it may first appear. innate and adaptive immunity do not operate in isolation, and each depends on or is enhanced by elements of the other ( fig. - ) . the lymphocytes of the adaptive response require the involvement of cells and cytokines of the innate system to become activated and undergo differentiation into effector and memory lymphocytes. conversely, activated lymphocytes secrete products that stimulate and improve the effectiveness of innate leukocytes. the interplay between innate and adaptive immunity is sustained by cytokine signaling and through direct intercellular contacts between innate and adaptive leukocytes. by cooperating in this way, innate and adaptive immunity combine to mount an optimal defense against pathogens. normal host homeostasis is re-established when the host wins (at least for that moment) the ongoing horse race between the host's immune system and a pathogen attempting to establish infection. pathogens replicate at widely divergent rates, with some bacteria and viruses able to double their population size within a couple of hours. fast-replicating pathogens therefore initially get ahead of the host's immune defenses, but then are caught and destroyed as the specialized cells of the immune system become activated. the mystery highlighted by the authors of this article is why slowreplicating pathogens are able to establish infections at all, and why so many of the most damaging chronic infections we experience, like those of the hepatitis b and c viruses and the bacteria causing tuberculosis, are caused by some of the slowest-replicating pathogens. the answer may lie in achieving certain threshold levels of infection that supply adequate amounts of the molecular triggers needed to spark first the innate response and then the adaptive response. if a pathogen replicates rapidly, large amounts of damps and pamps are present that immediately activate innate leukocytes, including the apcs needed to initiate t cell responses. these t cells proliferate quickly enough to overwhelm the pathogen and eliminate it. in contrast, a slow-replicating pathogen may generate only low levels of damps and pamps that do not activate large numbers of apcs, delaying the t cell response. even once the t cell response is initiated, without sufficient cytokines produced by activated apcs, t cell proliferation may be slow and insufficient to completely eliminate the pathogen. the pathogen then survives to establish a low level of infection that doesn't kill the host but makes him/her miserable. the memory t cell response is of little help, as it only controls rather than eliminates the slow-replicating pathogen. an uneasy balance is set up between the pathogen and the host immune system that results in chronic infection. the contrast in immune response kinetics between what the authors call "fast" and "slow" pathogens is illustrated in figure from their article. "the race between infection and immunity: how do pathogens set the pace?" by davenport, m.p., belz, g. t. and ribeiro, r.m., ( ) when the immune system is functioning normally, harmful entities are recognized, and the host is protected from external attack by pathogens and internal attack by cancers. some localized tissue damage may result from the normal inflammatory response that develops as innate leukocytes work to eliminate the threat, but these immunopathic effects are limited and controlled. as well, the tolerance of the healthy immune system prevents cells of the adaptive response from attacking normal self tissues. sometimes a person's immune system is incomplete, either because of primary immunodeficiency or acquired immunodeficiency. in primary immunodeficiencies, the failure of the immune system is congenital; that is, an affected individual is born with a genetic defect that impairs his/her ability to mount innate and/or adaptive immune responses. in an acquired immunodeficiency, an external factor such as a nutritional a healthy immune response is crucial for providing an individual with immunity to infectious diseases (ch. ) and may be made more robust through vaccination (ch. ). innate and adaptive immune mechanisms work together to allow a full range of responses of appropriate strength and specificity. in addition to their direct and broadly protective role, innate immune responses recruit adaptive leukocytes and contribute to their activation. once activated, lymphocytes mount humoral and cell-mediated antipathogen responses that are highly specific. cytokines are produced that further stimulate both the innate and adaptive responses. innate and adaptive immunity thus work synergistically to protect the host from all threats. cell-mediated the pace of pathogen replication may have implications for vaccination strategies. currently, vaccination often uses a harmless pathogen antigen to induce the host to respond and generate large populations of memory lymphocytes, which stand guard ready to mount a faster and stronger response should the real pathogen attack. for a fast-replicating pathogen, this tactic is successful because these types of pathogens readily generate enough pamps, damps and antigen to surpass the thresholds of apc and t cell activation. with the "head start" provided by memory lymphocytes, the pathogen may not get to replicate at all. however, a slow-replicating pathogen may not provide sufficient antigen to reach even the lower threshold required by memory t cells, meaning that the vaccine will not have offered an advantage to the host. worse, if the large numbers of memory t cells present after vaccination compete for the miserly quantities of antigen present, and so proliferate only slowly, the pathogen may in fact get a better foothold than if the host had not been vaccinated. these considerations should encourage us to experimentally test the threshold hypothesis and to refine our approach to vaccination accordingly. imbalance or a pathogen may cause the loss of an immune system component. for example, in acquired immunodeficiency syndrome (aids), the human immunodeficiency virus (hiv) destroys the t lymphocytes needed to fight infection. in both primary and acquired immunodeficiencies, patients show a heightened susceptibility to recurrent infections and sometimes develop tumors. the prognosis for most of these diseases is very poor and treatment options are limited. what happens when the immune system is complete but malfunctions, or when its normal functioning has undesirable effects? there are several instances in which inappropriate actions of the immune system can result in pathologic consequences that tip the balance from health to disease ( fig. - ) . firstly, the normal attack of a healthy immune system on a foreign tissue transplant meant to preserve life results in transplant rejection and deleterious consequences for the transplant patient. secondly, when the tolerance of an individual's adaptive immune response fails, self tissues are attacked in a way that can lead to autoimmune disease. thirdly, an immune response that is too strong or long can result in hypersensitivity to an entity. in this situation, the normal inflammation that accompanies the response gets out of control and causes significant collateral tissue damage. when an individual's normal adaptive immune system responds inappropriately to an antigen that is generally harmless, the immune response is manifested clinically as a form of hypersensitivity called allergy. lastly, if normal regulation of the inflammatory response is lost for an extended period for any reason, chronic inflammation ensues that can set up conditions allowing the development of tumors, heart disease or autoimmune disorders. the remainder of this book will take the reader on a logical tour of the immune response. chapter continues our basic immunology section, providing detailed descriptions of the various cells, tissues and cytokines involved in mediating immune responses. chapters - cover all aspects of the innate and adaptive immune responses in depth. the chapters in our clinical immunology section, chapters - , address immunological principles not only of practical value to the more medically oriented reader but also of interest to all who seek to understand the pivotal role of the immune system in health and disease. it is our hope that the reader will come away with a solid understanding of the cellular and molecular mechanisms underlying immunity and how these mechanisms can go awry to cause illness. we also provide a glimpse of how researchers seek to manipulate these mechanisms with the goal of ensuring good health for all. the pathological consequences of immune system irregularities include immunodeficiency (ch. ), cancer (ch. and ), transplant rejection (ch. ), hypersensitivity (ch. ) and autoimmune disease (ch. ). the way in which the immune system responds can maintain a healthy body or lead to disease. in a healthy individual, the immune system mounts an appropriate response that clears damaged cells and kills infectious organisms or cancerous cells. however, the immune system is a powerful and multi-faceted weapon that requires strict controls to remain in balance. when the immune system fails to respond adequately, or mounts an overzealous or inappropriate attack, or ignores normal control mechanisms, a wide variety of disease states can result. immunodeficiency autoimmune disease hypersensitivity and allergy destruction of cancer cells • the immune system is a central player in the maintenance of human health. • immune responses depend on the coordinated action of leukocytes that travel throughout the body to recognize and eliminate threats posed by trauma as well as by extracellular and intracellular pathogens, toxins and cancerous cells. • cytokines are secreted intercellular messenger proteins that mediate complex interactions among leukocytes. • complete protection from and clearance of unwanted entities often involve both innate and adaptive responses. • some innate mechanisms require no induction and are completely non-specific, whereas others are inducible and involve broad recognition of a limited number of molecular patterns (pamps and damps) by prms. • adaptive responses are mounted after innate responses have failed to remove an unwanted entity, and are dependent on cytokines produced during the innate response. • adaptive immunity involves the selective activation of lymphocytes by the engagement of their antigen receptors by antigens derived from the particular inciting entity. • the three subsets of lymphocytes are th, tc and b cells, which use distinct mechanisms to recognize antigen and carry out effector functions to eliminate specific entities. • elements of innate immunity influence adaptive immunity and vice versa. • malfunctions of the immune system are related to autoimmune disorders, allergy and other hypersensitivities, transplantation rejection, primary and acquired immunodeficiencies, chronic inflammation, and cancer development. section a ) can you define these terms? immunology, immune system, immunity, pathogen ) what was koch's theory and why was it important? ) who coined the term "vaccination" and on what basis? ) can you define these terms? homeostasis, mucosae, leukocyte, cytokine, immunopathic ) what is the difference between infection and disease? ) what is the major difference between extracellular and intracellular pathogens? ) distinguish between cell-mediated and humoral immunity. ) can you define these terms? senescent, phagocytosis, inflammation, prm, prr, mhc, lymphocyte, tcr, bcr, antibody, clonal selection, tolerance, self antigen, immunological memory ) what are three major differences between innate and adaptive immune responses? ) distinguish between damps and pamps. patient # is having surgery in the course of an elective hip replacement in a modern hospital. patient # is not so lucky and has stepped on a rusty nail in her garden. what sort of damps and/or pamps might be present in each case, and what sorts of immune responses might you expect to find in these patients? ) if a person's immune system was unable to produce memory lymphocytes why are b lymphocyte responses usually associated with defense against extracellular rather than intracellular pathogens? how might they also help protect a host against intracellular pathogens? can you extrapolate? some conceptual questions would you like to read more? overview of the immune response regulation of adaptive immunity by the innate immune system friendly and dangerous signals: is the tissue in control? key concepts in immunology key: cord- -gevrlcvy authors: shewen, p.e.; carrasco-medina, l.; mcbey, b.a.; hodgins, d.c. title: challenges in mucosal vaccination of cattle date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: gevrlcvy recognition of the mucosal portal of entry for many infectious diseases and of the relevance of mucosal immune response to protection has encouraged the development of vaccines administered by mucosal routes, principally oral and intranasal, for stimulation of intestinal and nasopharyngeal lymphoid tissues respectively. the oral route is problematic in cattle and other ruminants where antigen degradation in the rumen is likely, prior to transit to the intestine. on the other hand, rumination can be exploited for exposure of nasopharyngeal tissues during cudding if vaccine antigen is expressed by a fibrous feed like alfalfa. an increase in anti-leukotoxin (lkt) iga was demonstrated in nasal secretions of calves following feeding of alfalfa expressing a truncated lkt from mannheimia haemolytica, and there is evidence suggesting that such vaccination may protect against experimentally induced pneumonia. intranasal vaccination is an alternative approach for use in pre-ruminating calves. intranasal administration of iscoms carrying soluble antigens of m. haemolytica, including native lkt, induced lkt specific iga in nasal secretions after vaccination at and weeks of age. subcutaneous (s.c.) administration of the same vaccine induced lkt specific igg in both serum and nasal secretions, whereas s.c. administration of a commercial m. haemolytica vaccine did not. regardless of the vaccination strategy employed it is difficult to assess the immunogenicity of mucosally administered vaccines because production of secreted antibodies tends to be transient, and they do not persist on the mucosal surface in the absence of ongoing antigenic stimulation. an additional challenge is demonstration of vaccine efficacy in response to experimental infection. protection of the mucosally vaccinated animal will most probably result from recall response, which may not amplify sufficiently to counter the effects of experimental pulmonary delivery of a large bolus of virulent bacteria, even though the response would suffice over the more prolonged and gradual infection that occurs in natural induction of pneumonia. the vast majority of infectious diseases in all species are initiated by colonization of, or entry across, mucosal surfaces of the respiratory, intestinal or urogenital tracts. there has, therefore, been a great deal of interest in immune response at these sites and in development of vaccines that target these portals of entry (hodgins et al., ) . the reality is that most current vaccines for such infections are delivered parenterally and act thorough induction of systemic rather than mucosal immunity. protection is typically mediated by ''spill-over'' of mediators onto mucosal surfaces or by blocking of infection once the mucosal surface is breached; examples include vaccines for influenza viruses, vibrio, salmonella, and veterinary immunology and immunopathology ( ) - a r t i c l e i n f o mucosal immunity vaccination mannheimia haemolytica cattle a b s t r a c t recognition of the mucosal portal of entry for many infectious diseases and of the relevance of mucosal immune response to protection has encouraged the development of vaccines administered by mucosal routes, principally oral and intranasal, for stimulation of intestinal and nasopharyngeal lymphoid tissues respectively. the oral route is problematic in cattle and other ruminants where antigen degradation in the rumen is likely, prior to transit to the intestine. on the other hand, rumination can be exploited for exposure of nasopharyngeal tissues during cudding if vaccine antigen is expressed by a fibrous feed like alfalfa. an increase in anti-leukotoxin (lkt) iga was demonstrated in nasal secretions of calves following feeding of alfalfa expressing a truncated lkt from mannheimia haemolytica, and there is evidence suggesting that such vaccination may protect against experimentally induced pneumonia. intranasal vaccination is an alternative approach for use in pre-ruminating calves. intranasal administration of iscoms carrying soluble antigens of m. haemolytica, including native lkt, induced lkt specific iga in nasal secretions after vaccination at and weeks of age. subcutaneous (s.c.) administration of the same vaccine induced lkt specific igg in both serum and nasal secretions, whereas s.c. administration of a commercial m. haemolytica vaccine did not. regardless of the vaccination strategy employed it is difficult to assess the immunogenicity of mucosally administered vaccines because production of secreted antibodies tends to be transient, and they do not persist on the mucosal surface in the absence of ongoing antigenic stimulation. an additional challenge is demonstration of vaccine efficacy in response to experimental infection. protection of the mucosally vaccinated animal will most probably result from recall response, which may not amplify sufficiently to counter the effects of experimental pulmonary delivery of a large bolus of virulent bacteria, even though the response would suffice over the more prolonged and gradual infection that occurs in natural induction of pneumonia. ß elsevier b.v. all rights reserved. (corbeil et al., ; wilkie and markham, ) . despite these successes, development of mucosally delivered vaccines remains an area of active investigation in many laboratories, for both human and veterinary pathogens. why vaccinate mucosally? immune mediators, both immunoglobulins and effector t cells generated by mucosal exposure to antigens differ from those generated by systemic immunization (boyaka et al., ) . certainly, where the goal is prevention of infection, the presence of mediators on the mucosal surface is needed. memory cells generated at mucosal sites and in draining lymph nodes, home preferentially to other mucosal locations providing a primed response at all potential portals of exposure (youngman et al., ) . there are also non-immunologlical reasons for seeking vaccines that are delivered without injection, including ease of delivery and the absence of injection site reactions. vaccination of food producing animals would be facilitated by mass delivery of vaccine in feed, water or by aerosol, meaning less labor cost for producers and reduced stress on the animals. additionally, carcass condemnation due to needle breakage or injection site reactions would be avoided (roeber et al., ) . increasing consumer pressure for organically produced food and a natural approach to disease management is more compatible with disease prevention using noninvasive methods of vaccine delivery. mucosal delivery of antigens triggers immune response in mucosa-associated lymphoid tissues including the peyer's patches of the small intestine, the tonsil and associated pharyngeal lymphoid tissues, the bronchus associated lymphoid tissues of the lung and diffuse lymphoid aggregates lining the urogential tract. induction sites for mucosal immunity have been most thoroughly described for the intestinal tract where antigens are delivered to underlying peyer's patches by specialized membranous non-ciliated epithelial cells, m cells, located in villous crypts, and by dendritic cells (dcs) that send processes to the surface between ciliated columnar epithelial cells of the villi. these dcs deliver antigen to peyer's patches and to draining mesenteric nodes (meeusen et al., ) . similar mechanisms for antigen acquisition exist at other mucosal sites, including in tonsilar crypts and in balt where both m cells and epithelial dcs have been identified (gebert and pabst, ; stanley et al., ) , and presumably also at genital sites (hodgins et al., ) . experimentally, calves have been immunized by the vulvovaginal and rectal routes (loehr et al., (loehr et al., , , but most mucosal vaccines deliver antigens by oral administration, which targets intestinal induction, or intranasally which targets pharyngeal and, depending on particle size, deeper respiratory tissues. there are many challenges inherent in mucosal antigen delivery. intranasal vaccines must be delivered in dosages sufficient to overcome innate clearance mechanisms and facilitate uptake in the pharynx. oral vaccines must be protected against degradation by digestive processes, mechanisms are needed to facilitate antigen adherence to the mucosal epithelium and avoid clearance with the mucociliary blanket coating the gut, and the potential for induction of tolerance rather than active immunity must be considered . in mouse models, oral delivery of auto-antigens may lead to oral tolerance and reversal or reduction of autoimmunity. however, oral tolerance has not yet been reported in studies where plant expressed antigens have been delivered as vaccines in mice or other monogastric animals (arntzen et al., ; rice et al., ) . successful oral vaccines exist; vaccines for polio and rabies are excellent examples. vaccines using avirulent live bacteria (e.g. salmonella, bordetella) or viruses (e.g. adenovirus, coronavirus, reovirus) have been shown to be quite effective in stimulating mucosal immune responses and in cases where the vaccine organism is at least minimally invasive, systemic responses as well. perhaps the greatest challenge for mucosal immunization lies in vaccination with non-replicating antigens. such preparations are difficult to protect against digestion, tend not to adhere to mucosal surfaces, and generally fail to trigger the danger signals needed to initiate appropriate cellular stimulation for active immune response. experimentally several strategies have been employed to overcome these inherent challenges. vaccine antigens have been enclosed within microspheres, linked to bacterial toxin subunits such as ct or lt, or administered with molecules that induce danger signals, like cpg motifs, or with cytokines to stimulate lymphocyte activation (holmgren et al., ) . ruminating animals pose a particular challenge for development of orally administered vaccines. encapsulation of antigens in polymer microparticles or microspheres that resist digestion in the rumen is one approach that shows promise for stimulation of mucosal immune response in peyer's patches by oral delivery (bowersock et al., ) . an alternative approach is to exploit the process of rumination for exposure of the pharyngeal lymphoid tissues including the tonsil during cudding. this could be a particularly valuable approach for vaccination against both respiratory and intestinal diseases, since it is recognized that memory cells produced in tonsilar lymphoid tissue migrate preferentially to the lung and intestine, priming these sites for subsequent natural exposure (brandtzaeg and johansen, ) . with this in mind, we hypothesized that delivery of protective antigens in a palatable fibrous feed, such as alfalfa, could lead to repeated exposure of pharyngeal lymphoid tissue by cudding during rumination. because of its relevance as an economically important disease of cattle, we selected bovine pneumonic pasteurellosis as the target disease for study. our extensive experience with immunity to the principal causative bacterium mannheimia haemolytica and its protective antigens made this both an appropriate and convenient model system to test both the immunogenicity and efficacy of edible vaccines comprised of transgenic alfalfa. the first antigen targeted in this research was the leukotoxin (lkt). many studies have demonstrated lkt's importance in pathogenesis and the correlation between the presence of anti-lkt antibodies in serum and protection against pneumonia; however, most suggest that an anti-lkt response is essential but not sufficient alone to provide immunity (shewen and wilkie, ; jeyaseelan et al., ) . the transgenic alfalfa used in these experiments expressed a truncated form of leukotoxin (lkt ) that contained the neutralizing epitope (lee et al., ) . the concentration of lkt was at least mg/g of dried plant material, estimated as a percentage of total soluble protein. posttranslational modification of proteins occurs in plants ( gomord and faye, ) , including both n-linked and olinked glycosylation, but plant glycosylation patterns can differ from those found in bacteria. since the extent of glycosylation and type of glycan added to proteins can alter their immunogenicity, an early step in this work was demonstration of the immunogenicity of plant expressed lkt following intramuscular inoculation of rabbits, and verification that antisera from immunized rabbits recognized both recombinant and native lkt (lee et al., ) . use of transgenic alfalfa as a vehicle provides an efficient means for delivery of antigen that also furnishes protection against immediate dilution and destruction in the rumen. the natural process of cudding means that the fibrous feed is regurgitated, chewed slowly and held as a cud in the posterior oral cavity. typically this activity occurs - times over a period of several days, spraying the pharyngeal lymphoid tissues with antigen during each cycle, before it is finally digested and passed on. with this system, concerns about delivery, avoidance of innate clearance mechanisms and antigen destruction are addressed, but at least two major challenges remain as barriers to vaccine development. the first is demonstration of immunogenicity when the anticipated response is predominately mucosal and therefore inherently both difficult to sample and transient in the absence of continual antigen stimulation. it is also quite possible that pharyngeal exposure will merely prime the lung for an anamnestic response on infection, rather than lead to production of mucosal antibodies in response to the levels of antigen delivered by vaccination. the second related challenge is demonstration of efficacy, given that protection is most likely derived from a recall response. such response should suffice during natural exposure since this is gradual and continual over a period of hours or days, but can easily be overwhelmed in experimental challenge where the successful challenge model uses intrabronchial delivery of a large number of organisms, sufficient to cause pneumonia, as a single bolus. to determine the extent of these challenges and to address related questions of dose and duration of feeding needed for immunization, we have conducted a series of pilot studies feeding transgenic alfalfa to small groups of calves, typically two vaccinates and two controls that receive an equal amount of wild type alfalfa in lieu of the transgenic feed. for initial studies we opted to use colostrum deprived animals, reared in an isolation facility, to avoid interference by passive maternal antibodies and to minimize commensal colonization by m. haemolytica that might confound demonstration of response to vaccination. the animals were not germfree, nor were they caesarian derived, thus there was a low level of colonization and a low baseline antibody titer in serum at the time of first feeding, typically about months of age. all experiments were conducted under approval of the university of guelph animal care committee and adhered to the canadian council of animal care guidelines for use of animals in research. growth, processing, storage and feeding of the transgenic alfalfa as well as disposal of animals and animal waste were as specified in letters of permission from the canadian food inspection agency, which is responsible for regulation of gmos and use of experimental vaccines. in early trials, calves were fed in two rounds, g of dried alfalfa, each day for days, at a -week interval. they were then challenged approximately weeks after the second feeding by intrabronchial administration of ml of m. haemolytica (atcc ) at approximately cfu/ ml. this dose was estimated to cause infection sufficient to elicit recall response, but not produce pneumonia in controls. calves were euthanized or days postchallenge and mononuclear cells were harvested from blood, tonsil, spleen, and retropharyngeal, bronchial and mesenteric lymph nodes. serum and nasal swabs were collected at various points throughout the trial, and the presence of antibodies was determined by an lkt specific elisa using either alfalfa expressed lkt or native leukotoxin, in log phase serum-free culture supernatant, as the antigen. the latter elisa was further adapted as an elispot assay for detection of antibody secreting cells in mononuclear cell preparations following incubation with native leukotoxin. mononuclear cell culture supernatants were also assayed for production of interferon gamma using a commercial kit (bovigam, pfizer). during these studies it was noted that feeding of transgenic lkt alfalfa led to an increase in lkt specific iga in nasal secretions week following the second feeding ( fig. ) . this increase was transient and by the time of challenge levels of specific nasal iga were similar in vaccinates and controls. no changes in serum antibodies were observed prior to challenge. low level intrabronchial challenge with m. haemolytica, < cfu/ml, resulted in an increase in both serum and nasal antibodies in all calves at days post-challenge. these observations illustrate the difficulty in demonstrating humoral immune response to mucosal vaccination. the response is likely to be local, not systemic, and transient. thus sampling site and timing become critical. a similar response to vaccination was recently observed in a larger scale feeding trial in colostrum sufficient calves ( vaccinates and controls) (fig. ) . baseline serum antibodies to lkt were higher in all calves than previously observed in colostrum deprived animals, and feeding did not result in a noticeable change in serum antibodies. several calves fed recombinant alfalfa maintained elevated nasal iga to lkt from week after the second feeding up to the day of challenge. that the response was sustained in these animals may be a characteristic of conventional calves, since continuous antigen stimulation due to natural exposure could help maintain the response that was enhanced by vaccination. in this experiment where the challenge, ml of m. haemolytica at . cfu/ml, was intended to induce pneumonia, a correlation between the level of anti-lkt nasal iga and protection against pneumonia was observed. however, there was also a correlation between the level of anti-lkt igg in serum and protection, even though we could not demonstrate an effect of vaccination on the circulating level of those antibodies. thus, despite the encouraging nasal antibody results, we cannot confidently associate protection with vaccination. during one pilot study the challenge dose, at cfu/ ml, was sufficient to induce pneumonia in the two control calves (lesion scores / for both, % and % pneumonic tissue). both calves fed alfalfa expressing lkt had no clinical signs of pneumonia and no lesions at necropsy. although caution must be exercised in interpreting this as evidence of vaccine efficacy, since calf numbers are small, several interesting observations were made with respect to the response of mononuclear cells in susceptible versus resistant calves. vaccinated calves had lkt specific antibody producing cells in and day old cultures of cells derived from blood and bronchial nodes harvested at necropsy and there was evidence of class switching since igg and iga producing cells were present as well as cells producing igm. each of the control calves had only a single blood cell ( in ) producing antibodies, one igm and one iga. there was no interferon gamma production by blood mononuclear cells of any calves, response in tonsil cells was very weak, and there was no differential between vaccinated and control calves in ifng response in bronchial node cells. spleen and retropharyngeal node cells from vaccinates produced ifng rapidly within h of incubation, whereas cells from control calves responded later or not at all. the most striking difference was observed on culture of cells from mesenteric nodes with lkt. cells from vaccinates responded by producing ifng, controls did not (fig. ) . this was especially interesting since the challenge was pulmonary, not intestinal; thus, one would not expect activation of lymphocytes in these nodes. does this confirm trafficking of antigen-specific memory cells following exposure of the nasopharynx to transgenic alfalfa or does this suggest that vaccine antigen survived rumination and sensitized galt by transport across the intestinal epithelium? these questions remain to be addressed but at least we have identified a tissue to target in ongoing investigations. is the edible format the future for veterinary vaccines in ruminanting animals? perhaps, but even if that approach succeeds it will not address the question of pneumonia in younger calves that are not yet ruminating. bacteria in the pasteurellaceae family are major contributors to enzootic pneumonia that occurs at - weeks of age in both veal calves and replacement dairy heifers (van donkersgoed et al., ) . mucosal delivery of vaccine is also a relevant goal in these neonates, in particular intranasal delivery has appeal for both logistical (ease of delivery) and immunological (targets nasopharyngeal lymphoid tissues) per- fig. . iga antibodies to leukotoxin (lkt) in nasal secretions, determined in an elisa assay using alfalfa expressed lkt as antigen. od = optical density against lkt alfalfa minus od using a mock antigen preparation derived from alfalfa expressing gfp. grids span feeding days, solid vertical bar indicates day of challenge, oval indicates increase in specific iga days after feeding in vaccinated calves ( vaccinates, control). group means for iga antibodies to leukotoxin in nasal swabs obtained from calves fed alfalfa expressing lkt and control calves fed wild-type. median sample to positive (s/p) ratios determined in an elisa assay using native lkt as antigen. grids span feeding days, solid vertical bar indicates day of challenge. spectives. the added difficulty in calves of this age is the potential for interference in active immune response by passive maternal antibodies. in fact, pneumonia, at - weeks of age, occurs precisely during the period when passive immunity has waned to the extent that it is no longer protective, but the effects of interference with active immune response have delayed generation of protective immunity (prado et al., ) . previously we demonstrated that calves do not produce leukotoxin neutralizing antibodies in response to parenteral vaccination with a commercial m. haemolytica culture supernatant vaccine (presponse, wyeth/fort dodge) prior to weeks of age (hodgins and shewen, ) . earlier induction of active immunity may be possible through selection of an appropriate adjuvant or delivery by a mucosal immune route. immune stimulating complexes (iscoms) are an antigen delivery and adjuvant system (morein et al., ) wherein many antigens can be incorporated within or on the surface of small ( - nm) cage like structures formed of cholesterol, saponin and phosphatidylcholine. antigens contained in multivalent subunit iscom vaccines are found both within the cytosol and endosomic vesicles of antigen presenting cells (villacres et al., ) . iscom vaccines have been shown to stimulate both humoral and cell mediated immune responses and are claimed to override the down-regulatory effects of passively acquired maternal antibodies (nordengrahn et al., ; hagglund et al., ) . iscoms, prepared using supernatants from log-phase cultures, were shown to contain native lkt, as the target antigen, as well as other soluble antigens of m. haemolytica. iscom vaccines were used to vaccinate groups (n = or per group) of colostrum fed dairy heifers at and weeks of age, by either the intranasal or subcutaneous routes. response to vaccination was compared to that in unvaccinated controls and a group of calves that received a commercial vaccine, presponse sq (wyeth/fort dodge) subcutaneously at and weeks. all vaccinated calves were challenged to assess recall response by subcutaneous vaccination with the commercial vaccine at weeks of age, an age where response to vaccination might normally be expected. all three vaccines were standardized by antigen capture elisa to contain concentrations of lkt equivalent to that present in the commercial vaccine. sera and nasal swabs were collected weekly from to weeks of age and antibody responses were determined by direct and indirect agglutination, for antibodies to bacterial surface antigens, and by elisa for isotypic response to lkt. antibody responses were expressed as the change in titer from that at week , the time of initial vaccination, to adjust for antibodies present due to passive transfer. subcutaneous vaccination with iscoms induced an increase in the direct agglutination titer in serum at week , one week after the second vaccination and the titer remained elevated for the duration of the study (data not shown). vaccination with the commercial s.c. vaccine had no effect on titer by any assay before weeks of age. the earlier i.m. formulation of the same vaccine had been shown previously to induce agglutinating antibodies and igm to capsular polysaccharide, but not lkt neutralizing responses, in calves vaccinated at and weeks of age (hodgins and shewen, ) . both iscom vaccines induced an increase in serum igg to lkt week following the second dose (fig. , top panel) . the rate of decline in titer, from week to , was less in calves receiving presponse compared to controls. titers in control calves rose by week , consistent with a naturally induced active immune response to commensal colonization. as hoped, intranasal vaccination induced a significant change in lkt specific iga in nasal secretions week following first vaccination at week , the earliest age at which we have succeeded in inducing active immunity to lkt to date. this may be particularly important given the correlation between nasal iga to lkt and protection, recently demonstrated in our edible vaccine trial (above). by weeks of age nasal iga titers had begun to increase in all groups (fig. , middle panel) . interestingly, subcutaneous vaccination with iscom vaccine induced an increase in lkt specific igg in nasal secretion, which could relate to spillover from systemic response, but may reflect homing of memory cells to the nasopharynx permitting enhanced response to natural exposure locally (fig. , lower panel) . thus iscom vaccines, but not the commercial vaccine, induced immune response to lkt in serum and nasal secretions following vaccination at and weeks of age. this preceded responses arising from natural exposure that were evident at weeks of age. the hope would be that this earlier response would protect should the calves receive a challenge sufficient to induce pneumonia in the - week old period, but that was not assessed in this trial. these studies demonstrate that it is possible to detect immune response to mucosal vaccination targeted at the nasopharyngeal lymphoid tissue by examining antibodies in nasal secretions. we have also collected saliva and feces from many of these animals and will analyze those to determine their utility as alternative indicators of mucosal response. we could demonstrate enhanced response, compared to controls, in sera from some calves vaccinated mucosally using an edible vaccine, but only post-challenge. this would be important as a protective response to natural exposure, but is not useful for demonstration of immunogenicity in response to vaccination per se. additionally, though it may be adequate to stave off pneumonia during the course of natural infections, the recall response was not adequate to provide protection against an experimental challenge sufficient to induce pneumonia using a single bolus exposure. therefore, it may be necessary to further refine challenge protocol to enable differentiation, for example by adjusting challenge dose to animal weight, prechallenge serum titer or other criteria. additionally it is important to continue to examine immune response to mucosal antigen exposure, to improve our understanding of factors that lead to its stimulation and those parameters that reflect stimulation. this will assist in finding new and innovative means to enhance responsiveness, including adjuvants and delivery systems, and improved methods for detection of immunogenicity. author p. shewen is co-author on patents that protect the commercial vaccine presponse and receives loyalty revenue from wyet/fort dodge related to this vaccine. she currently holds a contract with dow agrosciences that funds an unrelated project. with these exceptions, she and her co-authors have no financial or personal relationship with other people or organizations that could inappropriately influence or bias the paper entitled ''challenges in mucosal vaccination of cattle''. isolation unit for excellent care of calves. the natural sciences and engineering research council of canada; the ontario ministry of agriculture, food and rural affairs; the ontario cattlemen's association and the agricultural adaptation council, and dow agrosciences funded various portions of this research. plant-derived vaccines and antibodies: potential and limitations oral vaccination of animals with antigens encapsulated in alginate microspheres mucosal vaccines: an overview mucosal b cells: phenotypic characteristics, transcriptional regulation, and homing properties immunity in the female bovine reproductive tract based on response to campylobacter fetus m cells at locations outside the gut posttranslational modification of therapeutic proteins in plants bovine respiratory syncytial virus iscoms-protection in the presence of maternal antibodies mucosal veterinary vaccines: comparative vaccinology serologic responses of young colostrum fed dairy calves to antigens of pasteurella haemolytica a mucosal immunization and adjuvants: a brief overview of recent advances and challenges role of mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis towards development of an edible vaccine against bovine pneumonic pasteurellosis using transgenic white clover expressing a mannheimia haemolytica a leukotoxin fusion protein gene gun-mediated dna immunization primes development of mucosal immunity against bovine herpesvirus- in cattle suppository-mediated dna immunization induces mucosal immunity against bovine herpesvirus- in cattle advances in mucosal vaccination immune response versus mucosal tolerance to mucosally administered antigens a novel structure for antigenic presentation of membrane protein from enveloped viruses equine herpesvirus type (ehv- ) as a predisposing factor for rhodococcus equi pneumonia in foals: prevention of the bifactorial disease with ehv- immunostimulating complexes maternally and naturally acquired antibodies to mannheimia haemolytica and pasteurella multocida in beef calves plant-made vaccines: biotechnology and immunology in animal health frequencies of injection-site lesions in muscles from rounds of dairy and beef cow carcasses vaccination of calves with leukotoxic culture supernatant from pasteurella haemolytica characterization of ovine nasal associated lymphoid tissue and identification of m cells in the overlying follicle-associated epithelium epidemiological study of enzootic pneumonia in dairy calves in saskatchewan internalization of iscom-borne antigens and presentation under mhc class i or class ii restriction bronchoalveolar washing cells and immunoglobulins of clinically normal calves lymphocyte homing: chemokines and adhesion molecules in t cell and iga plasma cell localization in the mucosal immune system key: cord- -aaokc authors: stanberry, lawrence r.; strugnell, richard title: vaccines of the future date: - - journal: perspectives in vaccinology doi: . /j.pervac. . . sha: doc_id: cord_uid: aaokc nan the advances made in vaccine technology since edward jenner vaccinated the young james phipps against smallpox have had a spectacular impact on human health over the last two centuries (see chapter e vaccine evolution). vaccines have been fundamental in the control and elimination of many debilitating and lethal diseases, and more diseases are currently targeted for eradication by vaccination. recent major breakthroughs in immunology, molecular biology, genomics, proteomics, biochemistry and computing sciences have driven vaccine technology forward, and will continue to do so. many challenges remain, however, including persistent or latent infections, pathogens with complex life cycles, antigenic drift and shift in pathogens subject to selective pressures, challenging populations and emerging infections. to address these challenges researchers are exploring many avenues: novel adjuvants are being developed that enhance the immune response elicited by a vaccine while maintaining high levels of tolerability; methods of protective antigen identification are iterated with every success; vaccine storage and transport systems are improving (including optimising the cold chain and developing temperature-stable vaccines); and new and potentially more convenient methods of vaccine administration are being pursued. high priority targets include life-threatening diseases, such as malaria, tuberculosis (tb) and human immunodeficiency virus (hiv), as well as problematic infections caused by ubiquitous agents, such as respiratory syncytial virus (rsv), cytomegalovirus (cmv) and staphylococcus aureus. non-traditional vaccines are also likely to become available for the management of addiction, and the prevention, treatment and cure of malignancies. this chapter is not meant as a compendium of all new-generation vaccines, but rather as an outline of the modern principles that will likely facilitate the development of future vaccines. as shown in figure . , there are several key elements that are likely to be understanding modern vaccines the foundation for the development of future vaccines. this chapter will illustrate these elements and provide examples that show promise. since the first use of an adjuvant in a human vaccine over years ago, adjuvant technology has improved significantly with respect to improving vaccine immunogenicity and efficacy. over the advances in adjuvant design have been driven by parallel advances in vaccine technology as many modern vaccines consist of highly purified antigens e with low non-specific reactogenicity which require combination with adjuvants to enhance the immune response. future developments in adjuvant technology are expected to provide stronger immune priming, enhance immune responses in specific populations, and lead to antigen sparing. adjuvants to date have demonstrated an ability new adjuvants must aim to drive the immune response that is associated with lifelong protection. new adjuvants and adjuvant combinations will play many roles in future vaccines as illustrated in figure . . adjuvants will need to be individually selected for specific vaccine targets in order to achieve the desired goal (ie enhanced immunogenicity, induction of specific immune profile etc). to deliver this aim, some adjuvants will be mixed with free antigens, while others will need to be covalently linked to the antigenic moiety as part of a complex molecule. some examples of new adjuvants that have been evaluated in humans or that are in clinical trials are listed in table . (also see chapter e vaccine adjuvants). modern approaches to antigen design tend to eschew classical trial and error techniques in favour of identifying the type of pathogenic structures (ie antigens) that are most likely to be important immunogens based on their structural signature or physical location within the pathogen (table . ) (see chapter e vaccine antigens). the t or b cell immune responses to an antigen are targeted to precise regions of the antigen (ie epitopes e either linear or three-dimensional conformational structures; in the case of protein antigens these are specific peptide epitopes). historically, simple, linear, synthetic peptide epitope vaccines have been poorly immunogenic because they lack a specific conformation and are easily degraded by a variety of extracellular and cell-surface proteases that serve to limit epitope presentation to t cells and/or result in destruction of the b-cell epitope. peptide vaccines need to union. every effort has been made to verify the information in the above table. the information included is not meant to be exhaustive but is intended to provide an overview of the subject matter. subunit and individual epitope vaccines need to be optimised to ensure adequate immunogenicity. novel strategies are being developed and exploited in order to identify antigens recognised by t and b cells, thus facilitating a more knowledge-based vaccine design. one of the most common ways to identify these antigens is to measure cellular proliferation (t or b cells) upon in vitro stimulation with antigen. high-throughput screening assays of candidate synthetic peptides that drive cellular proliferation help speed the rate of antigen discovery. reverse vaccinology combines knowledge of the pathogen's genome sequence with known protein sequences via computer analysis, to predict protein expression and post-translational modifications and identify likely vaccine candidates (see chapter e vaccine antigens; figure . ). the development of epitope-based vaccines is one example of reverse vaccinology where computer software combines prediction algorithms to suggest sequences similar to those for pathogenic components. epitope mapping, combined with the creation of more stable poly-epitope vaccines, may lead to the successful translation of this technology into products. mhc molecules exhibit widely varying binding specificities; a vaccine expressing a single peptide antigen would therefore only target a few mhc molecules and thus only be recognised by the t cells of individuals carrying a specific mhc phenotype. poly-epitope technology could be used to generate a synthetic protein carrying antigenic epitopes from multiple strains or pathogens. this would overcome the mhc restriction and afford protection in individuals carrying different mhc types. the screening of pathogen peptide libraries is another example of new approaches to antigen discovery. screening methods are used to identify antigens that can stimulate cd þ or cd þ t cells, or which bind to antibodies from humans known to have been infected with the relevant pathogen. where peptide screening uses antibodies, an additional consideration is the synthesis of antigens that contain the tertiary (folding/three-dimensional) structure of the native immunogen, since vaccine efficacy can be impacted by infidelities in the structure of the final product. incorrect protein folding may result in a less immunogenic antigen or an antigen that induces an immune response that differs from that of the native immunogen. the mimicking of the three-dimensional structure of the native immunogen is important during the synthesis of antigens that are being used to target b-cell responses. conversely, the requirement for folding is reduced for t cells since t cells bind only processed peptides, from degraded proteins. likewise, dna expression libraries using the pathogen genomic dna have been screened using animal model systems to identify genes encoding proteins that afford protection against infection or disease caused by the pathogen. one example is genocea's vaccine development programmes that are built around a broad platform for the rapid discovery of t-cell antigens. the process is explained in figure . . t-cell antigens, specifically antigens that stimulate cd þ and cd þ t cells, are critical to generating disease-specific cellular immune responses and long-term t-cell memory. stability of the final product is another important consideration. adverse environmental conditions can result in degradation of the vaccine, rendering it non-immunogenic. in order to maintain product integrity many vaccines (particularly live vaccines) must be stored at cold temperatures ( c). the maintenance of the vaccine at this temperature from production site to distribution site, and medical office or clinic, is referred to as the 'cold chain'. maintaining the cold chain is much less of a challenge in resource-rich countries, but can be a major barrier to vaccine implementation in resource-limited areas. ongoing research designed to increase our understanding of vaccine degradation may address the problems associated with cold chain management and lead to the development of thermostable vaccines. the level of antigen presentation which occurs with some current vaccines may sometimes be insufficient to drive long-lasting immune responses of high quality (see chapter e vaccine antigens). this may be due to inadequate exposure of the antigen to immature antigen-presenting cells (apcs) rapid or subimmunogenic degradation or sequestration of antigens, or lack of immunogenicity due to the physical presentation of the antigen. the discovery and modifying vaccine formulations to increase tolerance to temperature fluctuations is likely to increase the shelf-life of the product and reduce transport and wastage issues. understanding modern vaccines refinement of new and varied options for antigen presentation is expected to allow the design of vaccines to produce specific immune profiles. some of these technologies have been shown to facilitate oral delivery to target mucosal immune responses and also trigger both innate and adaptive immune systems, including t-and b-cell effector and memory responses. candidate viral vector vaccines utilise a non-pathogenic virus to carry and subsequently induce expression of genes that produce immunogenic foreign proteins at high levels in the host. these are taken up by immature apcs, and have been shown to lead to a robust, long-lasting immune response to the target antigen ( figure . ). viral vector vaccines, eg recombinant poxvirus vaccines, can be administered mucosally to stimulate mucosal immune responses. the attenuated modified vaccinia virus ankara (rmva) vectors are showing promise as mucosal delivery vectors. pre-existing immunity to the viral vaccine vector is an impediment to successful use of this approach. as ways to avoid anti-vector immunity, viruses can be attenuated or inactivated, by deleting or replacing pathogenic genes. figure . demonstrates how viral vaccine vectors are made. dna expressing an immunogenic transgene (the vaccine antigen) is inserted into the viral vector genome for expression following administration into the recipient; expression of the vaccine antigen can be boosted by using a variety of dna promoters. if the viral vector is no longer able to grow and replicate, the virus is grown using a cell line (a so-called complementing cell line) that has been engineered to produce the missing viral product. often, viral genes are removed in an effort to reduce or eliminate the pathogenicity of the vector and in some cases viral genes are removed to make the vector itself less immunogenic; an anti-vector immune response would greatly reduce the ability of the vector to induce an antigen-specific response. examples of viral vector candidate vaccines in clinical development are listed in table . . non-pathogenic bacterial vectors have many features that make them an attractive vaccine platform. bacterial vectors can be engineered for maximum safety (eg deletion of two or more genes from the same metabolic pathway), and to express large numbers of foreign antigens ( figure . ). two key issues affecting bacterial vaccine vectors are: a) to decide whether the optimal platform should be a bacterial vaccine in its figure . viral vectors for vaccines. viral vector vaccines exploit the natural ability of viruses to infect or otherwise enter (in the case of disabled viral vectors) host cells, and then deliver pathogen-specific antigens. antigen-encoding genes are isolated from the pathogen and inserted into the viral vector genome. the viral vector can then be used as a factory for production of large quantities of pathogen antigen in vivo, following introduction of the vector into the vaccine recipient, with the pathogen antigen then expressed on the surface of the infected/transduced host cells or exported out of the producer cell. mhc, major histocompatibility complex. own right or a bacterial vector system to deliver exogenous antigens; and b) to determine whether re-administration of the vector, either with the same or different target antigens, will fail because of the immune response to the bacterial vector vaccine at the time of its initial administration. initial assessments of the feasibility of using attenuated bacterial vectors for the delivery of foreign antigens have focused on salmonella species. bacterial vaccine vectors for humans, however, have been disappointing so far. it may be necessary to develop unique bacterial vaccine vectors for delivering exogenous antigens, in which case the vectors can be modified to allow for re-use. for example, if immunity against the vector, which is a major impediment to vaccine re-use, is determined by antibodies against the surface structures of the bacterium (such as lipopolysaccharide [lps]), the dedicated vaccine vector could be developed to lack expression of lps or to express truncated/ different forms of lps to the target, thereby avoiding priming of the immune response and allowing for re-use of the vector and/or vaccine. some potential options for live, attenuated bacterial vectors are shown in table . . dna vaccines are the result of the discovery in the early s that the gene, rather than the encoded protein, if delivered in an 'expressible' form, could induce an immune response (see chapter e vaccine evolution). the principle behind dna vaccines is that the antigenic molecule is produced within the host from the dna or rna that is injected, in contrast to more traditional vaccination where the antigen is supplied in the vaccine formulation. the gene(s) for target antigen(s) is/are usually encoded in a circular plasmid expression vector under the control of promoter sequences that direct gene expression in mammalian cells, which is achieved after injection into mammals. the dna vaccine process can circumvent some of the major issues resulting from recombinant protein administration. the construction and production of the plasmids carrying the gene of interest together with the promoter sequences is relatively simple; antigens expressed from plasmids retain their native conformation, the gene can be readily modified to produce tailored antigens, and bacterial plasmid dna is intrinsically immunogenic (subsequently shown to result from the pathogen-associated molecular patterns [pamps] it carries). additional desirable features include the ability to engineer and deliver genetic adjuvants in tandem or parallel with the antigen, the potential to deliver multiple antigen genes in one construct or within other constructs that encode adjuvanting protein(s), and the ability to induce both cellular and humoral immune responses. despite promising data in pre-clinical testing, dna vaccine candidates have shown only limited success in clinical settings so far. one of the current drawbacks of dna vaccines is the inefficiency of conventional delivery methods for the plasmid dna; however, understanding modern vaccines emerging proprietary particle-mediated delivery technology or electroporation technology seeks to improve this situation. with the electroporation method, brief electrical pulses are applied at the site of immunisation which causes a transient disruption of cell membranes. this results in an enhancement in uptake of the dna vaccine between e -fold. examples of dna candidate vaccines in clinical development are presented in table . . dendritic cell (dc) vaccines typically use monocytes harvested from the blood (in most cases from the individual who will receive the vaccine) to produce immature dcs in vitro. the monocytes are antigen-loaded and treated to induce their maturation into apcs and infused back into the patient. the first food and drug administration (fda)-approved dc vaccine, designed for the treatment of prostate cancer, was licensed in (sipuleucel-t); examples of other targets for dc vaccine therapy are presented in table . . dc vaccines offer an individualised approach to therapeutic vaccine development, but represent a specialised method of vaccination that is currently limited to aggressive cancers, and the treatment of serious, intractable infections. a comparison between the strengths and weaknesses of selected new vaccine platforms is presented in table . . developing administration techniques that place the vaccine directly at the site(s) where pathogens are most likely to initiate an infection (eg mucosal or respiratory sites) is likely to improve vaccine efficacy and safety. traditional methods of vaccine administration can potentially pose a number of limitations with respect to reactogenicity, immunogenicity, convenience, efficacy, safety and cost-effectiveness. the information included is not meant to be exhaustive but is intended to provide an overview of the subject matter. ongoing research on alternative experimental administration strategies includes ballistic delivery to skin (the gene gun), the transdermal patch and other intradermal methods, plus sublingual, aerosol, rectal and vaginal mucosal vaccines. the main advantages of alternative delivery strategies are the potential to induce immune responses at the common portals of pathogen entry (eg oral polio vaccine replicating in the gut), potential convenience (eg ease of use of the transdermal patch), potential combination of vaccines to reduce or simplify the vaccination schedule, and reduction or elimination of administration via standard hypodermic needle injection. despite the intuitive value of these approaches, few vaccines today are administered via non-im routes. this is for several reasons including feasibility, lack of proven efficacy and limited safety data. some problems have been observed with new routes of delivery, for example, after the launch of an inactivated intranasal influenza vaccine (a virosome formulation adjuvanted by heat labile enterotoxoid of escherichia coli), post-licensure data indicated a significantly increased risk of bell's palsy in vaccinees and forced its withdrawal from the market. this experience led to a higher level of caution in the development of intranasal vaccines. today, the only example of a licensed vaccine against a latent infection is the zoster vaccine; the vaccine formulation is the high potency (about -fold) version of the live, attenuated varicella zoster virus (vzv) vaccine. this vaccine has been used to boost the anti-vzv cell-mediated immune response in older subjects and has been shown to reduce the overall incidence of zoster by % in subjects aged years or older (oxman et al., ) . future vaccines may control persistent infections either by preventing the initial infection or disease (ie prophylactic vaccines) aerosol delivery: 'mass immunization of almost all susceptible children in a short period of time, has the potential of rapidly eliminating measles as a public health problem. immunization by inhalation of aerosolised measles vaccine provides a procedure that could make such a mass programme possible, especially in parts of the world where measles continues to be a serious problem.' (sabin et al., ) . administering the measles vaccine as an aerosol, either as nebulised vaccine or as an increased understanding of human immunology and of hostepathogen interactions should enable the identification of the type(s) of immunity required to effectively prevent or control persistent infections (see chapter e vaccine immunology). some examples of persistent infections are shown in table . . mycobacterium tuberculosis can persist in a latent state within the human host for years without causing disease (latent tb). protection against miliary (disseminated) tb in children is provided by the bacille calmetteeguérin (bcg) vaccine, developed through culture attenuation of mycobacterium bovis early in the th century, which is routinely given in many countries. the vaccine, however, provides only modest and often temporary protection against pulmonary tb, and provides lower efficacy in resource-limited regions closer to the equator. in addition, vaccination with live, attenuated mycobacterium bovis is a particular concern in hiv-positive individuals, especially those with advanced immune suppression; this population would particularly benefit from tb vaccination as tb is a leading cause of death worldwide for people with hiv/acquired immunodeficiency syndrome (aids). however, a recent phase iii trial demonstrated that protection against tb can be provided to individuals with hiv by using an inactivated whole-cell mycobacterial vaccine (von reyn et al., ) . the current state of tb vaccine development has been summarised in reviews by walker et al. ( ) and lambert et al. ( ) and examples of vaccines in development are shown in table . . cytomegalovirus, a herpes virus, establishes latent infection in cells in the bone marrow and peripheral blood. primary infection during pregnancy is associated with congenital infection that frequently causes a well-characterised spectrum of abnormalities and disabilities, which may be severe or fatal. reactivation in pregnancy is common, but is unlikely to cause severe congenital infection, although some manifestations, especially hearing loss, remain common. reactivation of cmv is of special concern in immunocompromised individuals, where severe and fatal pulmonary, hepatic and central nervous system infections are common. gastrointestinal disease and retinitis are common in association with hiv. a successful cmv vaccine has proved elusive for more than years. based upon the observation that antibodies to the cmv envelope glycoprotein b (gb) could pass et al., ) . a recent phase ii clinical trial in cmv-seronegative women year post-partum has shown the potential of gb/mf in decreasing incident cases of maternal and congenital cmv infection (pass et al., ) . this is the first evidence that a cmv vaccine can protect against infection. an alternative approach to the development of a cmv vaccine has been to utilise dna vaccination to induce host responses to cmv gb and phosphoprotein (pp is another viral target). recent studies have shown that injection of combinations of plasmids, formulated with an adjuvant, can induce vaccine-specific immune responses, and can prime for effective memory responses. the hallmark of herpes simplex virus types and (hsv- and hsv- ) is their ability to establish and maintain latent infection in to be exhaustive but is intended to provide an overview of the subject matter. sensory ganglion neurons. periodic reactivation of the latent infection results in recurrent infections. both hsv- and hsv- can cause myriad diseases but the greatest public health problem is genital herpes. genital hsv- infection increases the risk of hiv acquisition and transmission, and control of genital herpes has been predicted to significantly impact the hiv epidemic. given the complex natural history of hsv infections, vaccines could have a variety of possible risks and benefits (table . ). an effective hsv vaccine has been sought for more than years. recently, an hsv- glycoprotein d (gd ) candidate vaccine containing the as adjuvant (see chapter e vaccine adjuvants), was tested in three large, double-blind, phase iii controlled trials. the first two studies recruited volunteers with a partner with genital herpes disease and found the candidate vaccine was % effective against genital herpes disease in women seronegative for both hsv- and hsv- (stanberry et al., ) . trends towards protection against infection were also observed, but were not statistically significant. the candidate vaccine was not effective in hsv- seropositive women; or in understanding modern vaccines men, regardless of their hsv seropositivity status. these were the first studies to report a significant difference in vaccine efficacy between men and women. this finding could have important implications for other vaccines targeting sexually transmitted diseases. the basis for this difference could relate to differences in how men and women respond to novel adjuvants or may reflect differences in the acquisition and natural history of genital herpes in men and women. a third phase iii efficacy trial of the gd candidate vaccine in hsv- and hsv- negative women who thought themselves possibly at risk of acquiring genital herpes (a different risk population than in the original two trials) has been completed and is being analysed. an initial assessment of the results of the third trial showed that the vaccine had an acceptable safety profile but the primary trial endpoint, prevention of genital herpes disease, was not met (niaid, ) . although the development of the vaccine has been stopped, further analyses and comparison of the trials may guide researchers as they continue seeking vaccines to control hsv infections. as discussed in chapter e vaccine immunology, some pathogens have complex life cycles. one specific example is parasites, sometimes using more than a single host, where each development phase is marked by differential expression of major proteins, meaning that possible antigen targets are host-and development-phase specific. taenid worms aside, vaccines against parasites have been extremely difficult to develop and only a limited number have performed well in later-stage clinical trials. the protozoan parasite plasmodium falciparum, the most common cause of malaria, has a complex life cycle, as shown in figure . . the plasmodium parasite has a genome encoding more than proteins, and presents different allelic and immunogenic table ) . one of the furthest advanced of these new candidate vaccines is rts,s/as . the vaccine targets the pre-erythrocytic stage of the parasite (figure . ) . to be protective, a vaccine targeted at this phase needs to induce humoral immunity, to prevent parasites from invading the liver, and cell-mediated immunity to destroy hepatocytes that become infected in the face of the humoral immune response. the rts,s antigen, produced in saccharomyces cerevisiae, contains sequences of the p. falciparum circumsporozoite protein, linked to the hepatitis b surface antigen (hbsag). this chimeric protein spontaneously assembles into mixed polymeric particulate structures. in phase ii studies, the rts,s/as candidate vaccine induced a strong neutralising antibody response and cell-mediated immunity, and afforded protection against malaria (bejon et al., ; abdulla et al., ) . rts,s/as has been selected to proceed to phase iii clinical testing due to its higher efficacy compared with alternative formulations. if successful, the rts,s/as candidate vaccine could be the first licensed human vaccine against a parasite. other malaria candidate vaccines in development are shown in appendices, supplementary table . pathogens may mutate or recombine to change their antigenic profile. antigenic drift refers to a gradual process whereby point mutations in genes encoding antigenic proteins change the antigen sufficiently so that over time previously effective antibodies and vaccines no longer effectively control the pathogen and hence new vaccines need to be created. antigenic shift is a more dramatic event where there is a recombination of genes between different pathogen strains that gives rise to a new strain with a unique antigenic profile. in theory, pathogens are susceptible to selective pressure and an immunological environment that provides strong selective pressures should provide the 'bottleneck' that drives selection. this occurs with influenza viruses, where the high mutation frequency allows for the selection of mutants that are not neutralised. the risk of vaccine-mediated immune selection of pathogens, though certainly present, is difficult to demonstrate. moreover, peptide vaccines only use the antigenic epitope so the risk of pathogen evolution is theoretically increased. however, this phenomenon has not been regularly observed in experimental studies and may reflect the complex nature of most vaccine antigens and the presence of immune responses against multiple antigens and multiple epitopes within antigens. serotype replacement, where the distribution of specific microbial serotypes within communities changes after the introduction of vaccines, has occurred for some bacterial pathogens and may be a consequence of the use of capsular vaccines that address only a limited number of serotypes. similarly, since their introduction in the s, the use of antibiotics has exerted a selective pressure on bacterial strains leading to selection for common resistance alleles (eg the extended-spectrum beta-lactamase [esbl] resistance of enteric bacteria and beta-lactamase resistance in gonococci). to date, there has been no requirement to remodel a vaccine because of vaccine-mediated immune escape; however, new vaccines against the pneumococcus have been licensed, including additional capsular types, to expand the geographical coverage of most frequent types and, in part, to counter the observed phenomenon of serotype replacement. annual seasonal influenza infections are subject to natural antigenic drift which requires the reformulation of the vaccine when drifts occur, but there is no evidence that the deployment of the vaccine accelerates this drift. antigenic shift, while not the result of selective pressure, gives rise to viral strains containing a mixture of the surface antigens from the parent strains. pathogens that can undergo antigenic shift, including influenza viruses (figure . ), present major challenges for vaccine developers. chapter e vaccine adjuvants, there has been progress in the another approach to the problem of influenza genome shifts has been to target weakly immunogenic conserved antigens such as the influenza m e protein. one approach to addressing the weak immunogenicity of the antigen has been to link it to a potent toll-like receptor adjuvant such as flagellin, an approach developed by vaxinnate inc. during primary infection of a single individual with hiv, mutations in surface proteins of the virus lead to selection of a 'cloud' of antigenic variants that can evade the cell-mediated immune responses complicating the development of broadly effective vaccines. this propensity for mutation has given rise to many strains of hiv (figure . ). two types of hiv, hiv- and hiv- , have been identified, with hiv- being the most common. on a global scale, hiv- strains are differentiated according to their respective group and subtypes (or 'clades') within groups. the amino acid sequence of the viral envelope glycoprotein gp shows e % divergence between clades and up to % divergence within any given clade, which constitutes a formidable hurdle to vaccine development. this is made worse by recombination between clades of hiv- , which has produced circulating recombinant forms (crfs) which differ in antigenicity depending on the geographical region. since the initiation of hiv vaccine programmes, more than candidate vaccines have been tested in over phase i/ii clinical trials involving more than , healthy human volunteers. regrettably, all attempts to date have failed to yield a licensed hiv vaccine. questions remain concerning the immune mechanisms behind vaccines that achieve partial protection. regardless of the unknowns, the ability to prevent infection in at least some individuals still offers real hope that a globally effective hiv vaccine might be possible. current research is comparing the immune responses of subjects who are naturally protected against hiv with those who were infected, seeking to find the elusive immunological mechanisms of protection to help guide the design of future t-cell vaccines against the virus. infections of group a streptococcal serotypes (ie streptococcus pyogenes) account for approximately % of cases of uncomplicated bacterial pharyngitis and streptococcal invasive infections in north america. the m protein of group a streptococci is a major virulence determinant of these organisms and also functions as a major target for protective antibodies. one of several strategies for vaccine prevention of these infections is based on type-specific m protein epitopes. however, group a streptococcal vaccine development faces many obstacles: i) the widespread diversity of circulating m protein types; ii) immunological cross-reactivity between epitopes in the m protein and several human tissues introducing an autoimmune risk; and iii) animal models are of limited value because humans are the only hosts for group a streptococci. in an attempt to partially overcome some of these obstacles, a design strategy akin to that of the pneumococcal polysaccharide vaccines has been employed to generate a group a streptococci multivalent m protein-based vaccine containing type-specific determinants from different m serotypes. this multivalent vaccine is currently in clinical development. the 'prime-boost' approach the term 'prime boost' (or heterologous boosting) describes an approach to vaccination where one type of vaccine, such as a live-vector vaccine, is administered followed by a second type of vaccine, such as a recombinant subunit vaccine. this is in contrast with the traditional method of homologous boosting in which two or more doses of the same vaccine are given successively. the intent of prime-boost vaccination is to induce different types of immune responses and enhance the overall immune response, a result that may not occur if only one type of vaccine were to be given for all doses. this approach has been employed in trials with, for example, tb, cmv, malaria and hiv candidate vaccines. for example, in studies on new tb vaccines, subjects already primed with the live, attenuated bcg vaccine have been boosted with a subunit adjuvanted vaccine (see tuberculosis). respiratory syncytial virus is a common cause of bronchiolitis and pneumonia in infants, and exacerbations of chronic obstructive pulmonary disease in the elderly. the development of an effective vaccine has been challenging; natural immunity to rsv infection is incomplete and re-infections occur in all age groups. moreover, the primary target population for vaccination is newborns and young infants, and they are a challenging population as they have relatively immature immune systems and the presence of maternal antibodies may interfere with vaccination of the young infant (see chapter e vaccine immunology). the initial efforts to develop a formalin-inactivated cell culture-derived rsv vaccine resulted in an unanticipated enhancement of natural rsv disease in some of the rsv-naïve infants who received the vaccine in a clinical trial and subsequently were exposed to rsv. the exacerbated disease is thought to be due to an exaggerated t helper type cell immune response (see chapter e vaccine immunology). safety concerns regarding the potential of vaccines to trigger or prime for immunopathological responses has resulted in a cautious approach to the development of rsv vaccines. the vaccine candidates most advanced in clinical development use two different approaches e one uses a live, attenuated virus with a gene deletion deliberately targeted to minimise immunopathological responses. the other approach uses a live viral vector to deliver only a key rsv surface antigen, thereby avoiding the risk of an immunopathological response arising from exposure to the rsv virus itself. infectious illnesses exert a major burden of disease in developing countries. the greatest burden is caused by diseases for which we currently have no vaccines, eg taeniid cestode parasites are associated with high human morbidity and losses in livestock. global efforts to reduce these infections in humans are ongoing through the use of antihelminthics and the implementation of lifestyle changes, but this is having little effect. however, substantial progress has been made towards developing veterinary vaccines which encourages investigation of the potential use of similar vaccines in humans to prevent, for example, hydatid disease (arising from infection with echinococcus granulosus) and cysticercosis (from infection with taenia solium). relative to their burden on society, such diseases have a low priority for funding. unless comprehensive measures are taken to address the gaps in funding, research and global immunisation coverage, developing countries will continue to be overwhelmed by some of the most devastating diseases. in order to improve the situation, collaborative schemes are underway that bring together academic institutions, industry and public/charitable financing organisations. microbiome project is a national institutes of health initiative that seeks to determine the relationship between human health and changes in the human microbiome. by using revolutionary sequencing technologies to characterise the microbiology of five body sites e oral cavity, skin, vagina, gut and nasal tract/lung e an association may be made between the microbiomes associated with either the healthy body state or disease. characterising microbes associated with disease-related pathogens may allow for the development of new vaccines that preserve or protect the healthy microbiome and hence could protect human health. some of the areas of current research are outlined in the box, right. some conditions traditionally thought of as non-infectious may in fact have infectious origins (table . ); therefore, vaccination could be a strategy to prevent these diseases. other diseases may result from an interaction between the host's genetic background and a particular microbe (a so-called gene-environment interaction). some diseases have an established link with an identified infectious agent. for example, primary cmv infection is a known cause of congenital mental retardation; similarly the link between bacterial vaginosis and foetal prematurity is widely accepted. while some links have been established, others remain speculative (table . ). candidate vaccines are in development for the prevention and treatment of various types of addiction. the basic concept is to induce the production of antibodies which will bind the drug and impede its crossing the bloodebrain barrier to exert its psychoactive effects. several nicotine candidate vaccines have now entered clinical trials. a cocaine candidate vaccine has also shown some benefit in a phase iib clinical trial. the key issue to date for both nicotine and cocaine candidate vaccines has been to induce continued on next page high immunoglobulin (ig)g anti-drug antibody levels, which appear to be critical in achieving some degree of efficacy. candidate vaccines against methamphetamine addiction are also in early development. to date, the approach to developing prophylactic cancer vaccines has been to target infectious diseases that cause or contribute to the development of cancer such as hpv (cervical cancer) and hbv (hepatocellular carcinoma). examples of infectious diseases associated with cancer are shown in table . . the successful development of a nicotine vaccine would be expected to reduce cigarette smoking-related lung cancer. some cancers express tissue-specific antigens that can be targeted by the immune system. therapeutic cancer vaccines aim to target tumour-associated antigens (taa) with t-cell mediated immune responses. taa can be related to the genetic changes that drive the cancer (eg ras oncogene), or inappropriate up-regulation/ expression of genes (eg carcinoembryonic antigen). with such taa targets, vaccines aim to maximally stimulate a cytotoxic t-cell response and their design often includes adjuvants to enhance antigen presentation. tumours develop in a multistep process in the face of the host immune response and frequently evolve to escape immune control. mechanisms of evasion include genetic changes (loss of human leukocyte antigen/taa expression) and induction of immune regulatory systems (t-cell anergy due to the activity of t reg cells) which limit anti-tumour immunity. the key approach for therapeutic cancer vaccines is resetting the immune response to deliver anti-tumour immunity that alters or destroys cancer cells and hence eliminates or reduces the tumour. one strategy uses the patient's own tumour as the immunogen, thereby providing all the potential idiotypic changes that might act as taa, in conjunction with antigen-presenting dcs harvested from the same patient and activated in vitro (see dendritic cell vaccines). there are different types of therapeutic candidate vaccines currently undergoing clinical trials for numerous types of cancer (table . ). the most advanced candidates currently in phase iii are described in chapter e vaccine adjuvants. there has been some success in the development of therapeutic cancer vaccines, with the fda approval of the first dc vaccine oxford biomedica muc , mucin cell surface; hil , human cytokine interleukin- ; asci, antigen-specific cancer immunotherapeutics; mage, melanomaassociated antigen; ctl, cytotoxic t lymphocyte. every effort has been made to verify the information in this table. the information included is not meant to be exhaustive but is intended to provide an overview of the subject matter. vaccine approaches. however, this presents opportunities for the application of novel technologies and adjuvants. some of the considerations for vaccines designed for use in special populations include: immunosenescence in the elderly; the poor immunological response to traditional vaccines seen in immunocompromised individuals (patients with hiv, transplant recipients); the crossing of vaccine components into the foetal bloodstream when vaccines are administered to pregnant women; and the safety and immunogenicity concerns surrounding vaccines for neonates due to their naïve and immature immune system. cell-mediated immunity is depressed in pregnant women, leaving them at high risk of infection from pathogens, including those harmful to the foetus. most live, attenuated vaccines are contraindicated during pregnancy because of the theoretical risk of foetal infection from the vaccine. however, inactivated viral or bacterial vaccines can be administered. pregnant women can, therefore, be vaccinated against some infections, including several that pass from mother to foetus (such as hepatitis a and b), and against infections acquired by the infant in the first few months of life (often from close contact with the mother). in the latter case, the infant can be protected by transfer of maternal antibodies during late gestation. examples of diseases that can be prevented in pregnant women include influenza, tetanus, diphtheria and probably pertussis. other diseases, such as those caused by the so-called torch pathogens (toxoplasma, others including syphilis, cmv and hsv), are not yet preventable through vaccination though encouraging phase ii results have been presented for a vaccine to prevent group b streptococcus carriage in pregnant women (hillier et al., ; smith, ). the h n pandemic influenza outbreak posed an increased risk to pregnant women and vaccination was specifically recommended in pregnant women as one of the high-risk groups. the pandemic example has emphasised once more the importance of protecting pregnant women against influenza. seasonal influenza vaccination in pregnancy is well tolerated and the benefiterisk profile when administered to pregnant women supports its use during pregnancy. many public health authorities worldwide recommend seasonal influenza vaccination in pregnant women and this recommendation is motivated not only by the potentially severe course of influenza during pregnancy, but also by the need to protect vulnerable infants against influenza during their first months of life. boosting rsv immunity in pregnant women through vaccination may be another approach to protecting the newborn against rsv infection during the most vulnerable early months after birth. neonatal immunisation is a strategy to protect infants against infections during a particularly vulnerable period. a recent study showed that immunisation with an acellular pertussis vaccine at birth and month of age induces high igg anti-pertussis antibody titres by months of age (wood et al., ) . it is hoped that this approach may reduce death and morbidity from bordetella pertussis infection in the first months of life. the elderly respond poorly to vaccination as the immune system becomes more senescent with increasing age and, therefore, new vaccine technologies are needed to improve the response to vaccination in this population. in the late s, an influenza vaccine adjuvanted with the oil-in-water emulsion, mf Ô, was shown to be more effective at inducing high immune responses in the elderly (minutello et al., ) . alternative vaccine administration techniques have also been studied in the elderly. research showed that in subjects years of age or older, an influenza vaccine administered with an intradermal microinjection system induced significantly higher antibody titres compared with im vaccination (arnou et al., ). subsequently, a microinjection system influenza vaccine was licensed for use in europe, and a high antigen dose formulation has been licensed for the elderly in the usa. individuals with cancer, hiv infection or who are asplenic can be immunocompromised as a result of their condition. patients can also be immunocompromised as a result of therapy, eg when receiving an organ transplant, radiation therapy or immunosuppressive medication. such patients are therefore at an elevated risk of infection from pathogens such as herpesviruses (particularly cmv and epsteinebarr virus), hbv, hcv, pneumocystis and coinfections and represent a special population regarding immunisation. despite a likely reduction in the efficacy of vaccinations in immunocompromised individuals, immunisation remains a frequent recommendation in the hope that at least partial immunity will be achieved. eliciting a response from vaccination in immunocompromised patients may require an increase in the dose and/or number of doses; altering the dosing interval; selecting a different vaccine formulation; or administration via an alternative route. evidence in this patient population is lacking and guidelines are often based on theoretical assumptions. live vaccines are generally contraindicated in immunocompromised or immunosuppressed individuals due to the risk of an active and symptomatic infection resulting from the vaccine itself (non-controlled replication process). encouragingly, vaccine formulations with highly purified antigens and novel adjuvants or alternative deliveries have been shown to induce more effective immune responses than the classical inactivated vaccines in immunocompromised hosts, including patients with end-stage renal diseases in pre-haemodialysis and haemodialysis (see chapter e vaccine adjuvants), patients with hiv and those who have received haematological stem cell transplants. the future of vaccine development can build on the knowledge and experience gained over the last years, and at the same time can take advantage of the most cutting-edge technologies and understanding modern vaccines research. new approaches to antigen selection and production, antigen delivery, adjuvantation and vaccine administration will allow us to target established and emerging diseases, and populations with complex needs. vaccination has been one of the most successful and cost-effective health interventions ever conceived and is now expanding further into cancer and chronic diseases. this expansion of scope and the subsequent impact on human disease is likely to continue into the future in currently unforeseen ways, further increasing the importance of vaccine science and engineering in improving human health. safety and immunogenicity of rts,s/as d malaria vaccine in infants intradermal influenza vaccine for older adults: a randomized controlled multicenter phase iii study efficacy of rts,s/as e vaccine against malaria in children to months of age women receiving group b streptococcus serotype iii tetanus toxoid (gbs iii-tt) vaccine have reduced vaginal and rectal acquisition of gbs type iii new vaccines against tuberculosis safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with mf adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults a subunit cytomegalovirus vaccine based on recombinant envelope glycoprotein b and a new adjuvant vaccine prevention of maternal cytomegalovirus infection vaccination with alvac and aidsvax to prevent hiv- infection in thailand successful immunization of children with and without maternal antibody by aerosolized measles vaccine. i. different results with undiluted human diploid cell and chick embryo fibroblast vaccines associate clinical professor of medicine glaxosmithkline herpes vaccine efficacy study group. glycoprotein-d-adjuvant vaccine to prevent genital herpes prevention of tuberculosis in bacille calmette-guérin-primed, hiv-infected adults boosted with an inactivated whole-cell mycobacterial vaccine the second geneva consensus: recommendations for novel live tb vaccines acellular pertussis vaccine at birth and one month induces antibody responses by two months of age genocea technology developing cell culture-derived pandemic vaccines seeking new pathways for hiv vaccine discovery infectious diseases and global cancer control intrauterine infection and preterm delivery the nih human microbiome project alliance for case studies for global health. case studies for global health: building relationships alliance for case studies international aids vaccine initiative vaccines of the future national institute of allergy and infectious diseases (niaid). statement: study finds genital herpes vaccine ineffective in women world health organization's special programme for research and training in tropical diseases aidsvaxÔ is a trademark of global solutions for infectious diseases corporation; alvacÔ is a trademark of connaught technology corporation mf Ô is a trademark of novartis; montanideÔ is a trademark of seppic; nanostatÔ is a trademark of nanobio corporation. for details of trademarks not listed above please see the manufacturer's website (see section internet resources) vaccines of the future key: cord- -vlklgd x authors: kim, yushim; kim, jihong; oh, seong soo; kim, sang-wook; ku, minyoung; cha, jaehyuk title: community analysis of a crisis response network date: - - journal: soc sci comput rev doi: . / sha: doc_id: cord_uid: vlklgd x this article distinguishes between clique family subgroups and communities in a crisis response network. then, we examine the way organizations interacted to achieve a common goal by employing community analysis of an epidemic response network in korea in . the results indicate that the network split into two groups: core response communities in one group and supportive functional communities in the other. the core response communities include organizations across government jurisdictions, sectors, and geographic locations. other communities are confined geographically, homogenous functionally, or both. we also find that whenever intergovernmental relations were present in communities, the member connectivity was low, even if intersectoral relations appeared together within them. other or are friends, know each other, etc." which generally refers to a social circle (mokken, , p. ) , while a community is formed through concrete social relationships (e.g., high school friends) or sets of people perceived to be similar, such as the italian community and twitter community (gruzd, wellman, & takhteyev, ; hagen, keller, neely, depaula, & robert-cooperman, ) . in social network analysis, a clique is operationalized as " . . . a subset of actors in which every actor is adjacent to every other actor in the subset (borgatti, everett, & johnson, , p. ) , while communities refer to " . . . groups within which the network connections are dense, but between which they are sparser" (newman & girvan, , p. ) . the clique and its variant definitions (e.g., n-cliques and k-cores) focus on internal edges, while the community is a concept based on the distinction between internal edges and the outside. we argue that community analysis can provide useful insights about the interrelations among diverse organizations in the ern. we have not yet found any studies that have investigated cohesive subgroups in large multilevel, multisectoral erns through a community lens. with limited guidance from the literature on erns, we lack specific expectations or hypotheses about what the community structure in the network may look like. therefore, our study focuses on identifying and analyzing communities in the middle east respiratory syndrome coronavirus (mers) response in south korea as a case study. we address the following research questions: ( ) in what way were distinctive communities divided in the ern? and ( ) how did the interorganizational relations relate to the internal characteristics of the communities? by detecting and analyzing the community structure in an ern, we offer insights for future empirical studies on erns. the interrelations in erns have been examined occasionally by analyzing the entire network's structure. for example, the katrina case exhibited a large and sparse network, in which a small number of nodes had a large number of edges and a large number of nodes had a small number of edges (butts, acton, & marcum, ) . the katrina response network can be thought of as " . . . a loosely connected set of highly cohesive clusters, surrounded by an extensive 'halo' of pendant trees, small independent components, and isolates" (butts et al., , p. ) . the network was sparse and showed a tree-like structure but also included cohesive substructures. other studies on the katrina response network have largely concurred with these observations (comfort & haase, ; kapucu, arslan, & collins, ) . in identifying cohesive subgroups in the katrina response network, these studies rely on the analysis of cliques: "a maximal complete subgraph of three or more nodes" (wasserman & faust, , p. ) or clique-like (n-cliques or k-cores). the n-cliques can include nodes that are not in the clique but are accessible. similarly, k-cores refer to maximal subgraphs with a minimum degree of at least k. many cliques were identified in the katrina response network, in which federal and state agencies appeared frequently (comfort & haase, ; kapucu, ) . using k-cores analysis, butts, acton, and marcum ( ) suggest that the katrina response network's inner structure was built around a small set of cohesive subgroups that was divided along institutional lines corresponding to five state clusters (alabama, colorado, florida, georgia, and virginia), a cluster of u.s. federal organizations, and one of nongovernmental organizations. while these studies suggest the presence of cohesive subgroups in erns, we have not found any research that thoroughly discussed subsets of organizations' significance in erns. from the limited literature, we identify two different, albeit related, reasons that cohesive subgroups have interested ern researchers. in their analysis of cohesive subgroups using cliques, comfort and haase ( ) assume that a cohesive subgroup can facilitate achieving shared tasks as a group, but it can be less adept at managing the full flow of information and resources across groups and thus decreasing the entire network's coherence. kapucu and colleagues ( ) indicate that the recurrent patterns of interaction among the sets of selected organizations may be the result of excluding other organizations in decision-making, which may be a deterrent to all organizations' harmonious concerted efforts in disaster responses. comfort and haase ( ) view cliques as an indicator of " . . . the difficulty of enabling collective action across the network" (p. ), and others have adhered closely to this perspective (celik & corbacioglu, ; hossain & kuti, ; kapucu, ) . cohesive subgroups such as cliques are assumed to be a potential hindrance to the entire network's performance. the problem with this perspective is that one set of eyes can perceive cohesive subgroups in erns as a barrier, while another can regard them as a facilitator of an effective response. while disaster and emergency response plans are inherently limited and not implemented in practice as intended (clarke, ) , stakeholder organizations' responses may be performed together with presumed structures, particularly in a setting in which government entities are predominant. for example, the incident command system (ics) was designed to improve response work's efficiency by constructing a standard operating procedure (moynihan, ). structurally, one person serves as the incident commander who is responsible for directing all other responders (kapucu & garayev, ) . ics is a somewhat hierarchical command-and-control system with functional arrangements in five key resources and capabilities-that is, command, operations, planning, logistics, and finance (kapucu & garayev, ) . in an environment in which such an emergency response model is implemented, it is realistic to expect clusters and subgroups to reflect the model's structural designs and arrangements, and they may be intentionally designed to facilitate coordination, communication, and collaboration with other parts or subgroups efficiently in a large response network. others are interested in identifying cohesive subgroups because they may indicate a lack of cross-jurisdictional and cross-sectoral collaboration in erns. during these responses, public organizations in different jurisdictions participate, and a sizable number of organizations from nongovernmental sectors also become involved (celik & corbacioglu, ; comfort & haase, ; kapucu et al., ; spiro, acton, & butts, ) . organizational participation by multiple government levels and sectors is often necessary because knowledge, expertise, and resources are distributed in society. participating organizations must collaborate and coordinate their efforts. however, studies have suggested that interactions in erns are limited and primarily occur among similar organizations, particularly within the same jurisdiction. that is, public organizations tend to interact more frequently with other public organizations in specific geographic locations (butts et al., ; hossain & kuti, ; kapucu, ; tang, deng, shao, & shen, ) . these studies indicate that organizations have been insufficiently integrated across government jurisdictions (tang et al., ) or sectors (butts et al., ; hossain & kuti, ) , and the identification of cliques composed of similar organizations reinforces such a concern. in our view, there is a greater, or perhaps more interesting, question related to the crossjurisdictional and cross-sectoral integration in interorganizational response networks: how are intergovernmental relations mixed with intersectoral relations in erns? here, we use the term interorganizational relations to refer to both intergovernmental and intersectoral relations. intergovernmental relations refer to the interaction among organizations across different government levels (local, provincial, and national) , and intersectoral relations involve the interaction among organizations across different sectors (public, private, nonprofit, and civic sectors). recent studies have suggested that both intergovernmental and intersectoral relations shape erns (kapucu et al., ; kapucu & garayev, ; tang et al., ) , but few have analyzed the way the two interorganizational relations intertwine. if the relation interdependencies in the entire network are of interest to ern researchers, as is the case in this article, focusing on cliques may not necessarily be the best approach to the question because clique analysis may continue to find sets of selected organizations that are tightly linked for various reasons. the analysis of cliques is a very strict way of operationalizing cohesive subgroups from a social network perspective (moody & coleman, ) , and there are two issues with using it to identify cohesive subgroups in erns. first, clique analysis assumes complete connections of three or more subgroup members, while real-world networks tend to have many small overlapping cliques that do not represent distinct groups (moody & coleman, ) . even if substantively meaningful cliques appear, they may not necessarily imply a lack of information flow across subgroups or other organizations' exclusion, as previous ern studies have assumed (comfort & haase, ; kapucu et al., ) . second, clique analysis assumes no internal differentiation in members' structural position within the subgroup (wasserman & faust, ) . in a task-oriented network such as an ern, organizations within a subgroup may look similar (e.g., all fire organizations). however, this does not imply that they are identical in their structural positions. when these assumptions in clique analysis do not hold, identifying cohesive subgroups as cliques is inappropriate (wasserman & faust, ) . similarly, other clique-like approaches (n-cliques and k-cores) demand an answer to the question: "what is the n-or k-?" the clique and clique-like approaches have a limited ability to define and identify cohesive subgroups in a task-oriented network because they do not clearly explain why the subgroups need to be defined and identified in such a manner. we proposed a different way of thinking about and finding subsets of organizations in erns: community. when a network consists of subsets of nodes with many edges that connect nodes of the same subset, but few that lay between subsets, the network is said to have a community structure (wilkinson & huberman, ) . network researchers have developed methods with which to detect communities (fortunato, latora, & marchiori, ; latora & marchiori, ; lim, kim, & lee, ; newman & girvan, ; yang & leskovec, ) . optimization approaches, such as the louvain and leiden methods, which we use in this article, sort nodes into communities by maximizing a clustering objective function (e.g., modularity). beginning with each node in its own group, the algorithm joins groups together in pairs, choosing the pairs that maximize the increase in modularity (moody & coleman, ) . this method performs an iterative process of node assignments until modularity is maximized and leads to a hierarchical nesting of nodes (blondel, guillaume, lambiotte, & lefebvre, ) . recently, the louvain algorithm was upgraded and improved as the leiden algorithm that addresses some issues in the louvain algorithm (traag, waltman, & van eck, ) . modularity (q), which shows the quality of partitions, is measured and assessed quantitatively: in which e ii is the fraction of the intra-edges of community i over all edges, and e ij is the fraction of the inter-edges between community i and community j over all edges. modularity scores are used to compare assignments of nodes into different communities and also the final partitions. it is calculated as a normalized index value: if there is only one group in a network, q takes the value of zero; if all ties are within separate groups, q takes the maximum value of one. thus, a higher q indicates a greater portion of intra-than inter-edges, implying a network with a strong community structure (fortunato et al., ) . currently, there are two challenges in community detection studies. first, the modular structure in complex networks usually is not known beforehand (traag et al., ) . we know the community structure only after it is identified. second, there is no formal definition of community in a graph (reichardt & bornholdt, ; wilkinson & huberman, ) , it simply is a concept of relative density (moody & coleman, ) . a high modularity score ensures only that " . . . the groups as observed are distinct, not that they are internally cohesive" (moody & coleman, , p. ) and does not guarantee any formal limit on the subgroup's internal structure. thus, internal structure must be examined, especially in such situations as erns. despite these limitations, efforts to reveal underlying community structures have been undertaken with a wide range of systems, including online and off-line social systems, such as an e-mail corpus of a million messages in organizations (tyler, wilkinson, & huberman, ) , zika virus conversation communities on twitter (hagen et al., ) , and jazz musician networks (gleiser & danon, ) . further, one can exploit complex networks by identifying their community structure. for example, salathé and jones ( ) showed that community structures in human contact networks significantly influence infectious disease dynamics. their findings suggest that, in a network with a community structure, targeting individuals who bridge communities for immunization is better than intervening with highly connected individuals. we exploit the community detection and analysis to understand an ern's substructure in the context of an infectious disease outbreak. it is difficult to know the way communities in erns will form beforehand without examining clusters and their compositions and connectivity in the network. we may expect to observe communities that consist of diverse organizations because organizations' shared goal in erns is to respond to a crisis by performing necessary tasks (e.g., providing mortuary and medical services as well as delivering materials) through concerted efforts on the part of those with different capabilities (moynihan, ; waugh, ) . organizations that have different information, skills, and resources may frequently interact in a disruptive situation because one type alone, such as the government or organizations in an affected area, cannot cope effectively with the event (waugh, ) . on the other hand, we also cannot rule out the possibility shown in previous studies (butts et al., ; comfort & haase, ; kapucu, ) . organizations that work closely in normal situations because of their task similarity, geographic locations, or jurisdictions may interact more frequently and easily, even in disruptive situations (hossain & kuti, ) , and communities may be identified that correspond to those factors. a case could be made that communities in erns consist of heterogeneous organizations, but a case could also be made that communities are made up of homogeneous organizations with certain characteristics. it is equally difficult to set expectations about communities' internal structure in erns. we can expect that, regardless of their types, sectors, and locations, some organizations work and interact closely-perhaps even more so in such a disruptive situation. emergent needs for coordination, communication, and collaboration also can trigger organizational interactions that extend beyond the usual or planned structure. thus, the relations among organizations become dense and evolve into the community in which every member is connected. on the other hand, a community in the task network may not require all of the organizations within it to interact. for example, if a presumed structure is strongly established, organizations are more likely to interact with others within the planned structure following the chain of command and control. even without such a structure, government organizations may coordinate their responses following the existing chain of command and control in their routine. we may expect to observe communities with a sparse connection among organizations. thus, the way communities emerge in erns is an open empirical question that can be answered by examining the entire network. several countries have experienced novel infectious disease outbreaks over the past decade (silk, ; swaan et al., ; williams et al., ) and efforts to control such events have been more or less successful, depending upon the instances and countries. in low probability, high-consequence infectious diseases such as the mers outbreak in south korea, a concerted response among individuals and organizations is virtually the only way to respond because countermeasures-such as vaccines-are not readily available. thus, to achieve an effective response, it is imperative to understand the way individuals and organizations mobilize and respond in public health emergencies. however, the response system for a national or global epidemic is highly complex (hodge, ; sell et al., ; williams et al., ) because of several factors: ( ) the large number of organizations across multiple government levels and sectors, ( ) the diversity of and interactions among organizations for the necessary (e.g., laboratory testing) or emergent (e.g., hospital closure) tasks, and ( ) concurrent outbreaks or treatments at multiple locations attributable to the virus's rapid spread. all of these factors create challenges when responding to public health emergencies. we broadly define a response network as the relations among organizations that can act as critical channels for information, resources, and support. when two organizations engage in any mers-specific response interactions, they are considered to be related in the response. examples of interactions include taking joint actions, communicating with each other, or sharing crucial information and resources (i.e., exchanging patient information, workforce, equipment, or financial support) related to performing the mers tasks, as well as having meetings among organizations to establish a collaborative network. we collected response network data from the following two archival sources: ( ) news articles from south korea's four major newspapers published between may , , and december , (the outbreak period), and ( ) a postevent white paper that the ministry of health and welfare published in december . in august , hanyang university's research center in south korea provided an online tagging tool for every news article in the country's news articles database that included the term "mers (http://naver.com)." a group of researchers at the korea institute for health and social affairs wrote the white paper ( pages, plus appendices) based on their comprehensive research using multiple data sources and collection methods. the authors of this article and graduate research assistants, all of whom are fluent in korean, were involved in the data collection process from august to september . because of the literature's lack of specific guidance on the data to collect from archival materials to construct interorganizational network data, we collected the data through trial and error. we collected data from news articles through two separate trials (a total of , articles from the four newspapers). the authors and a graduate assistant then ran a test trial between august and april . in july , the authors developed a data collection protocol based on the test trial experience collecting the data from the news articles and white paper. then, we recollected the data from the news articles between august and september using the protocol. when we collected data by reviewing archival sources, we first tagged all apparent references within the source text to organizations' relational activities. organizations are defined as "any named entity that represents (directly or indirectly) multiple persons or other entities, and that acts as a de facto decision making unit within the context of the response" (butts et al., , p. ) . if we found an individual's name on behalf of the individual's organization (e.g., the secretary of the ministry of health and welfare), we coded the individual as the organization's representative. these organizational interactions were coded for a direct relation based on "whom" to "whom" and for "what purpose." then, these relational activity tags were rechecked. all explicit mentions of relations among organizations referred to in the tagged text were extracted into a sociomatrix of organizations. we also categorized individual organizations into different "groups" using the following criteria. first, we distinguished the entities in south korea from those outside the country (e.g., world health organization [who], centers for disease control and prevention [cdc] ). second, we sorted governmental entities by jurisdiction (e.g., local, provincial/metropolitan, or national) and then also by the functions that each organization performs (e.g., health care, police, fire). for example, we categorized local fire stations differently from provincial fire headquarters because these organizations' scope and role differ within the governmental structure. we categorized nongovernmental entities in the private, nonprofit, or civil society sectors that provide primary services in different service areas (e.g., hospitals, medical waste treatment companies, professional associations). at the end of the data collection process, organizational groups from , organizations were identified (see appendix). we employed the leiden algorithm using python (traag et al., ) , which we discussed in the previous section. the leiden algorithm is also available for gephi as a plugin (https://gephi.org/). after identifying communities, the network can be reduced to these communities. in generating the reduced graph, each community appears within a circle, the size of which varies according to the number of organizations in the community. the links between communities indicate the connections among community members. the thickness of the lines varies in proportion to the number of pairs of connected organizations. this process improves the ability to understand the network structure drastically and provides an opportunity to analyze the individual communities' internal characteristics such as the organizations' diversity and their connectivity for each community. shannon's diversity index (h) is used as a measure of diversity because uncertainty increases as species' diversity in a community increases (dejong, ) . the h index accounts for both species' richness and evenness in a community (organizational groups in a community in our case). s indicates the total number of species. the fraction of the population that constitutes a species, i, is represented by p i below and then multiplied by the natural logarithm of the proportion (lnp i ). the resulting product is then summed across species and multiplied by À : high h values represent more diverse communities. shannon's e is calculated by e ¼ h=ln s, which indicates various species' equality in a community. when all of the species are equally abundant, maximum evenness (i.e., ) is obtained. while limited, density and the average clustering coefficient can capture the basic idea of a subgraph's structural cohesion or "cliquishness" (moody & coleman, ) . a graph's density (d) is the proportion of possible edges presented in the graph, which is the ratio between the number of edges present and the maximum possible. it ranges from (no edges) to (if all possible lines are present). a graph's clustering coefficient (c) is the probability that two neighbors of a node are neighbors themselves. it essentially measures the way a node's neighbors form a -clique. c is in a graph connected fully. the mers response network in the data set consists of , organizations and , edges. table shows that most of the organizations were government organizations (approximately %) and % were nongovernmental organizations from different sectors. local government organizations constituted the largest proportion of organizations ( %). further, one international organization (i.e., who) and foreign government agencies or foreign medical centers (i.e., cdc, erasmus university medical center) appeared in the response network. organizations coordinated with approximately three other organizations (average degree: . ). however, six organizations coordinated with more than others. the country's health authorities, such as the ministry of health and welfare (mohw: edges), central mers management headquarters (cmmh: edges), and korea centers for disease control and prevention (kcdc: edges), were found to have a large number of edges. the ministry of environment ( edges) also coordinated with many other organizations in the response. the national medical center had edges, and the seoul metropolitan city government had . the leiden algorithm detected communities in the network, labeled as through in figures - and tables and . the final modularity score (q) was . , showing that the community detection algorithm partitioned and identified the communities in the network reasonably well. in real-world networks, modularity scores " . . . typically fall in the range from about . to . . high values are rare" (newman & girvan, , p. ) . the number of communities was also consistent in the leiden and louvain algorithms ( communities in the louvain algorithm). the modularity score was slightly higher in the leiden algorithm than the q ¼ . in the louvain. figure presents the mers response network with communities in different colors to show the organizations' clustering using forceatlas layout in gephi. in figure , the network's community structure is clear to the human eye. from the figures (and the community analysis in table ), we find that the mers response network was divided into two sets of communities according to which communities were at the center of the network and their nature of activity in the response, core response communities in one group and supportive functional communities in the other. the two core communities ( and ) at the center of the response network included a large number of organizations, with a knot of intergroup coordination among the groups surrounding those two. these communities included organizations across government jurisdictions, sectors, and geographic locations ( table , description) and were actively involved in the response during the mers outbreak. while not absolute, we observe that the network of a dominating organization had a "mushroom" shape of interactions with other organizations within the communities (also see figure a ). the dominant organizations were the central government authorities such as the mohw, the cmmh, and kcdc. the national health authorities led the mers response. other remaining communities were ( ) confined geographically, ( ) oriented functionally, or ( ) both. first, some communities consisted of diverse organizations in the areas where two mers hospitals are located-seoul metropolitan city and gyeonggi province (communities and ). organizations in these communities span government levels and sectors within the areas affected. second, two communities consisted of organizations with different functions and performed supportive activities (community , also see figure b ). other supportive functional communities that focus on health (community , see figure c ) or foreign affairs (community ) had a "spiderweb" shape of interactions among organizations within the communities. third, several communities consisted of a relatively small number of organizations connected to one in the center (communities , , , and ) . these consisted of local fire organizations in separate jurisdictions (see figure d ) that were both confined geographically and oriented functionally. table summarizes the characteristics of the communities in the response network. in table , we also note distinct interorganizational relations present within the communities. the two core response communities include both intergovernmental and intersectoral relations. that is, organizations across government jurisdictions or sectors were actively involved in response to the epidemic in the communities. while diverse organizations participated in these core communities, the central government agencies led and directed other organizations, which reduced member connectivity. among the supportive functional communities, those that are confined geographically showed relatively high diversity but low connectivity (communities , , and through ). these communities included intergovernmental relations within geographic locations. secondly, communities of organizations with a specialized function showed relatively high diversity or connectivity. these included organizations from governmental and nongovernmental sectors and had no leading or dominating organizations. for example, communities and had intersectoral relations but no intergovernmental relations. thirdly, within each community of fire organizations in different geographic locations, one provincial or metropolitan fire headquarters was linked to multiple local fire stations in a star network. these communities, labeled igf, had low member diversity and member connectivity, while they were organizationally and functionally coherent. table summarizes the results elaborated above. in addition to the division of communities along the lines of the nature of their response activities, we observe that the structural characteristics of communities with only intersectional or international relations showed high diversity and high connectivity. whenever intergovernmental relations were present in communities, however, the member connectivity was low, even if intersectoral relations appeared together within them. we use the community detection method to gain a better understanding of the patterns of associations among diverse response organizations in an epidemic response network. the large data sets available and increased computational power significantly transform the study of social networks and can shed light on topics such as cohesive subgroups in large networks. network studies today involve mining enormous digital data sets such as collective behavior online (hagen et al., ) , an e-mail corpus of a million messages (tyler, wilkinson, & buberman, ) , or scholars' massive citation data (kim & zhang, ) . the scale of erns in large disasters and emergencies is noteworthy (moynihan, ; waugh, ) , and over , organizations appeared in butts et al. ( ) study as well as in this research. their connections reflect both existing structural forms by design and by emergent needs. the computational power needed to analyze such large relational data is ever higher and the methods simpler now, which allows us to learn about the entire network. we find two important results. first, the national public health ern in korea split largely into two groups. the core response communities' characteristics were that ( ) they were not confined geographically, ( ) organizations were heterogeneous across jurisdictional lines as well as sectors, and ( ) the community's internal structure was sparse even if intersectoral relations were present. on the other hand, supportive functional communities' characteristics were that ( ) they were communities of heterogeneous organizations in the areas affected that were confined geographically; ( ) the communities of intersectoral, professional organizations were heterogeneous, densely connected, and not confined geographically; and ( ) the communities of traditional emergency response organizations (e.g., fire) were confined geographically, homogeneous, and connected sparsely in a centralized fashion. these findings show distinct features of the response to emerging infectious diseases. the core response communities suggest that diverse organizations across jurisdictions, sectors, and functions actually performed active and crucial mers response activities. however, these organizations' interaction and coordination inside the communities were found to be top down from the key national health authorities to all other organizations. this observation does not speak to the quality of interactions in the centralized top-down structure, but one can also ask how effective such a structure can be in a setting where diverse organizations must share authority, responsibilities, and resources. second, infectious diseases spread rapidly and can break out in multiple locations simultaneously. the subgroup patterns in response networks to infectious diseases can differ from those of location-bound natural disasters such as hurricanes and earthquakes. while some organizations may not be actively or directly involved in the response, communities of these organizations can be formed to prepare for potential outbreaks or provide support to the core response communities during the event. second, we also find that the communities' internal characteristics (diversity and connectivity) differed depending upon the types of interorganizational relations that appeared within the communities. based on these analytical results, two propositions about the community structure in the ern can be developed: ( ) if intergovernmental relations operate in a community, the community's member connectivity may be low, regardless of member diversity. ( ) if community members are functionally similar, (a) professional organization communities' (e.g., health or foreign affairs) member connectivity may be dense and (b) emergency response organization communities' (e.g., fire) member connectivity may be sparse. the results suggest that the presence of intergovernmental relations within the communities in erns may be associated with low member connectivity. however, this finding does not imply that those communities with intergovernmental relations are not organizationally or functionally cohesive. instead, we may expect a different correlation between members' functional similarity and their member connectivity depending upon the types of professions, as seen in (a) and (b). organizations' concerted efforts during a response to an epidemic is a prevalent issue in many countries (go & park, ; hodge, gostin, & vernick, ; seo, lee, kim, & lee, ; swaan et al., ) . the mers outbreak in south korea led to , suspected cases, infected cases, and deaths in the country (korea centers for disease control and prevention, ) . the south korean government's response to it was severely criticized for communication breakdowns, lack of leadership, and information secrecy (korea ministry of health and welfare, ). the findings of this study offer a practical implication for public health emergency preparedness and response in the country studied. erns' effective structure has been a fundamental question and a source of continued debate (kapucu et al., ; nowell, steelman, velez, & yang, ). the answer remains unclear, but the recent opinion leans toward a less centralized and hierarchical structure, given the complexity of making decisions in disruptive situations (brooks, bodeau, & fedorowicz, ; comfort, ; hart, rosenthal, & kouzmin, ) . our analysis shows clearly that the community structure and structures within communities in the network were highly centralized (several mushrooms) and led by central government organizations. given that the response to the outbreak was severely criticized for its poor communication and lack of coordination, it might be beneficial to include more flexibility and openness in the response system in future events. we suggest taking advice from the literature above conservatively because of the contextual differences in the event and setting. this study's limitations also deserve mention. several community detection methods have been developed with different assumptions for network partition. some algorithms take deterministic group finding approaches that partition the network based on betweenness centrality edges (girvan & newman, ) or information centrality edges (fortunato et al., ) . other algorithms take the optimization approaches we use in this article. in our side analyses, we tested three algorithms with the same data set: g-n, louvain, and leiden. the modularity scores were consistent, as reported in this article, but the number of communities in g-n and the other two algorithms differed. the deterministic group finding approach (g-n) found a substantively high number of communities. the modularity score can help make sense of the partition initially, but the approach is limited (reichardt & bornholdt, ) . thus, two questions remain: which algorithm do we choose and how do we know whether the community structure is robust (karrer, levina, & newman, ) ? in their nature, these questions do not differ from which statistical model to use given the assumptions and types of data in hand. the algorithms also require further examination and tests. while we reviewed the data sources carefully multiple times to capture the response coordination, communication, and collaboration, the process of collecting and cleaning data can never be free from human error. it was a time-consuming, labor-intensive process that required trial and error. further, the original written materials can have their own biases that reflect the source's perspective. government documents may provide richer information about the government's actions but less so about other social endeavors. media data, such as newspapers, also have their limitations as information sources to capture rich social networks. accordingly, our results must be interpreted in the context of these limitations. in conclusion, this article examines the community structure in a large ern, which is a quite new, but potentially fruitful, approach to the field. we tested a rapidly developing analytical approach to the ern to generate theoretical insights and find paths to exploit such insights for better public health emergency preparedness and response in the future. much work remains to build and refine the theoretical propositions on crisis response networks drawn from this rich case study. the katrina response network consisted of , organizations and connections with a mean degree except for the quote, comfort and haase ( ) do not provide further explanation incident command system was established originally for the response to fire and has been expanded to other disaster areas in the end, we found that the process was not helpful because of the volume and redundancy of content in news articles different newspapers published, which is not an issue in analysis because it can be filtered and handled easily using network analysis tool. because we had not confronted previous disaster response studies that collected network data from text materials, such as news articles and situation reports, and reported their reliability we also classified organizations based on specialty, such as quarantine, economy, police, tourism, and so on regardless of jurisdictions. twenty-seven specialty areas were classified. we note that the result of diversity analysis using the specialty areas did not differ from that using the organizational groups. the correlation of the diversity indices based on the two different classification criteria was r ¼ . . we report the result based on organization groups 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ebola response in the united states epidemics crisis management systems in south korea infectious disease threats and opportunities for prevention extended structures of mediation: re-examining brokerage in dynamic networks ebola preparedness in the netherlands: the need for coordination between the public health and the curative sector leveraging intergovernmental and cross-sectoral networks to manage nuclear power plant accidents: a case study from from louvain to leiden: guaranteeing well-connected communities e-mail as spectroscopy: automated discovery of community structure within organizations social network analysis: methods and applications terrorism, homeland security and the national emergency management network a method for finding communities of related genes cdc's early response to a novel viral disease, middle east respiratory syndrome coronavirus structure and overlaps of communities in networks author biographies yushim kim is an associate professor at the school of public affairs at arizona state university and a coeditor of journal of policy analysis and management. her research examines environmental and urban policy issues and public health emergencies from a systems perspective jihong kim is a graduate student at the department of seong soo oh is an associate professor of public administration at hanyang university, korea. his research interests include public management and public sector human resource management he is an associate editor of information sciences and comsis journal. his research interests include data mining and databases her research focuses on information and knowledge management in the public sector and its impact on society, including organizational learning, the adoption of technology in the public sector, public sector data management, and data-driven decision-making in government jaehyuk cha is a professor at the department of computer and software, hanyang university, korea. his research interests include dbms, flash storage system the authors appreciate research assistance from jihyun byeon and useful comments from chan wang, haneul choi, and young jae won. the early idea of this article using partial data from news articles was presented at the dg.o research conference and published as conference proceeding (kim, kim, oh, kim, & ku, ) . data are available from the author at ykim@asu.edu upon request. we used python to employ the leiden community detection algorithm (see the source code: https://github.com/ vtraag/leidenalg). network measures, such as density and clustering coefficient, as well as the diversity index were calculated using python libraries (networkx, math, pandas, nump). we used gephi . . for figures and mendeley for references. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported by the national research foundation of korea grant funded by the korean government (ministry of science and ict; no. r a a ). supplemental material for this article is available online. key: cord- -kgpvdb authors: sa ribero, margarida; jouvenet, nolwenn; dreux, marlène; nisole, sébastien title: interplay between sars-cov- and the type i interferon response date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: kgpvdb the severe acute respiratory syndrome coronavirus- (sars-cov- ) is responsible for the current covid- pandemic. an unbalanced immune response, characterized by a weak production of type i interferons (ifn-is) and an exacerbated release of proinflammatory cytokines, contributes to the severe forms of the disease. sars-cov- is genetically related to sars-cov and middle east respiratory syndrome-related coronavirus (mers-cov), which caused outbreaks in and , respectively. although ifn treatment gave some encouraging results against sars-cov and mers-cov in animal models, its potential as a therapeutic against covid- awaits validation. here, we describe our current knowledge of the complex interplay between sars-cov- infection and the ifn system, highlighting some of the gaps that need to be filled for a better understanding of the underlying molecular mechanisms. in addition to the conserved ifn evasion strategies that are likely shared with sars-cov and mers-cov, novel counteraction mechanisms are being discovered in sars-cov- –infected cells. since the last coronavirus epidemic, we have made considerable progress in understanding the ifn-i response, including its spatiotemporal regulation and the prominent role of plasmacytoid dendritic cells (pdcs), which are the main ifn-i–producing cells. while awaiting the results of the many clinical trials that are evaluating the efficacy of ifn-i alone or in combination with antiviral molecules, we discuss the potential benefits of a well-timed ifn-i treatment and propose strategies to boost pdc-mediated ifn responses during the early stages of viral infection. the severe acute respiratory syndrome coronavirus (sars-cov- ) is a beta-coronavirus that emerged at the end of in china and rapidly spread around the world, causing a pandemic [ , ] . sars-cov- infection is responsible for covid- , a disease associated with mild symptoms in the majority of cases but that can progress to an acute respiratory distress syndrome [ , ] . so far (july th, ), the virus has infected more than million people and caused more than , deaths worldwide. sars-cov- is genetically related to other betacoronaviruses that have caused epidemics: sars-cov and mers-cov (for middle east respiratory syndrome-related coronavirus), in phosphorylation, irf and/or irf dimerize and translocate into the nucleus, where they induce the expression of ifn-i and a subset of isgs referred to as early isgs (reviewed in [ ] ). secreted ifn-i then bind to the interferon alpha and beta receptor (ifnar, composed of the ifnar and ifnar subunits), leading to the activation of the jak tyrosine kinases tyrosine kinase (tyk ) and janus kinase (jak ), which in turn phosphorylate the signal transducer and activator of transcription (stat) and stat [ , ] . phosphorylated stats heterodimerize and associate with the dna binding protein irf to form a complex known as ifn-stimulated growth factor (isgf ). the isgf complex translocates into the nucleus and binds to interferon-stimulated response elements (isres) in isg promoters, thus inducing the expression of hundreds of isg products that establish the antiviral state at the site of viral infection [ ] . the antiviral response is intensified by various signaling factors, including sensors and transcriptional regulators, which are themselves isgs induced by isgf and/ or directly by the irf /irf transcriptional activators. aside from the ifn-i response, the recognition of double-stranded viral rna elements by the protein kinase receptor (pkr) triggers a translational arrest in infected cells (reviewed in [ , , ] ). this host response is highly connected to the ifn-i response because pkr is also an isg (reviewed in [ , ] ). ifn-i response requires fine-tuning because its overactivation is deleterious to the host. notably, some isgs are involved in the regulation of cell metabolism, intracellular rna degradation, translation arrest, and cell death, for which changes can be potentially detrimental to the host. ifn-i also potentiates the recruitment and activation of various immune cells. thus, although a robust ifn-i response is required as a first line of defense against viral infections, systemic/uncontrolled or prolonged ifn-i production can lead to inflammatory diseases. for example, an exacerbated ifn-i response contributes to the development of autoimmune diseases [ ] . covid- is no exception to the rule, and it is therefore critical to understand the regulation of the ifn-i response upon infection. sars-cov- is a poor inducer of ifn-i response in vitro and in animal models as compared with other respiratory rna viruses [ , ] . ifn-i levels in the serum of infected patients are below the detection levels of commonly used assays, yet isg expression is detected [ , ] , thus suggesting that a limited ifn-i production could be sufficient to induce isgs. alternatively, ifn-i production could be restricted to specific immune cells, such as plasmacytoid dendritic cells (pdcs). despite a more efficient replication in human lung tissues, sars-cov- induced even less ifn-i than sars-cov [ ] , which is itself a weak inducer in human cells [ ] [ ] [ ] ]. an ineffective ifn-i response seems to be a hallmark of other coronavirus infections, as observed with mers-cov in ex vivo respiratory tissue cultures [ ] and with animal coronaviruses such as the porcine epidemic diarrhea virus (pedv) or the mouse hepatitis virus (mhv), which are alpha-and beta-coronaviruses, respectively [ , ] . indeed, coronaviruses have developed multiple strategies to escape and counteract innate sensing and ifn-i production. sars-cov encodes at least proteins that allow the virus to either escape or counteract the induction and antiviral action of ifn (fig and table ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . initial observations already suggest that the sars-cov- anti-ifn arsenal is at least as efficient as that of sars-cov [ , , ] , although detailed mechanistic studies are required to determine whether the ifn antagonists identified in other coronaviruses have equivalently competent counterparts in sars-cov- . a virus-cell protein interaction map performed with of the sars-cov- proteins expressed in human embryonic kidney (hek) t identified several innate immune signaling proteins as partners of viral proteins cells (fig ) [ ] . sars-cov- orf b, like sars-cov orf b, interacts with mavs through its association with tom , thus suggesting a conserved mechanism of ifn-i evasion [ , ] (fig ) . furthermore, sars-cov- nsp and nsp were found to interact with tbk and the tbk activator ring finger protein (rnf )/nrdp , respectively [ ] (fig ) . nsp , which is a highly conserved endoribonuclease encoded by various coronaviruses, including sars-cov [ , , ], antagonizes the induction of ifn-i by cleaving the -polyuridines of the negative-sense viral rna, as demonstrated for mhv and pedv in various cellular models [ , , ] (table and fig ) . if further validated, the interaction between sars-cov- nsp and tbk may reveal that the viral endoribonuclease antagonizes ifn induction via at least mechanisms. sars-cov orf b was reported to inhibit ifn induction and to act either on irf or possibly on mavs because it translocates to mitochondria when overexpressed in vero cells [ , ] . despite the fact it encodes a shorter protein than sars-cov, sars-cov- orf b was recently found to suppress ifn induction even more efficiently [ ] . by screening , sars-cov- sequences, the authors identified a natural variant encoding a longer orf b and displaying an even greater inhibitory activity [ ] . finally, sars-cov- nsp was also recently found to bind s ribosomal subunits (fig ) , thus inhibiting host mrna translation, including that of ifn-i on this cartoon are schematically represented the signaling pathways triggered by sars-cov rna recognition by the cytoplasmic rna sensors rig-i and mda , which leads to ifn induction (a) and subsequent ifn signaling in surrounding cells, resulting in the expression of isgs (b). sars-cov proteins that have been reported to interfere with these pathways are indicated. ifn, interferon; ifnar, interferon alpha and beta receptor; iκb, inhibitor of nuclear factor κb; ikkε, iκb kinase-ε; irf, ifn regulatory factor; isg, ifn-stimulated gene; jak, janus kinase; m, membrane; mavs, mitochondrial antiviral signaling protein; mda , melanoma differentiation-associated gene ; n, nucleocapsid; nsp, nonstructural protein; orf, open reading frame; p, phosphate; plp, papain-like protease; rig-i, retinoic acid-inducible gene ; sars-cov, severe acute respiratory syndrome coronavirus; stat, signal transducer and activator of transcription; tank, traf family member associated nf-κb activator; tbk , tank-binding kinase ; traf , tumor necrosis factor receptor-associated factor ; tyk , tyrosine kinase . https://doi.org/ . /journal.ppat. .g [ ], a feature that was previously demonstrated for other coronavirus-encoded nsp , including sars-cov [ , ] (fig and table ) . another viral strategy to inhibit ifn-i signaling is to enhance the host retrocontrol of this pathway. several isgs are themselves repressors of the ifn-i response, and their regulatory functions operate at the viral and host mrna transcription and translation steps, acting via a wide-range of mechanisms (reviewed in [ , ] ). for example, the inducible negative regulators such as the suppressor of cytokine signaling (socs and socs ) act at various levels of the jak-stat pathway or by targeting irf for degradation [ ] . in the context of sars-cov, the s protein induces the expression of socs expression in b cells [ ] . induction of socs / expression is also detected in sars-cov-infected cells, albeit to a lower extent as compared with other respiratory viruses [ ] . recent genomic screen approaches identified a set of repressors of the ifn-i response depending on the cell type and activation pathway involved [ ] [ ] [ ] . hence, one might anticipate that distinct repressors of the ifn-i response are induced depending on the cell type targeted by sars-cov- , the level of replication, and the microenvironment. for example, in the context of coronaviruses, an inefficient detection of mhv infection likely results from an inhibition of the basal levels of sensors mrna expression in several cell types [ ] . it is conceivable that this inhibition might involve negative regulators such as the ifn-inducible rnf , which targets signaling components such as rig-i, mda , and mavs for degradation [ ] . inhibition of protein synthesis is a conserved host response to prevent viral infections. the host translation is dynamically regulated by pkr, activated via recognition of viral rna (reviewed by [ ] ). activated pkr inhibits the eukaryotic initiation factor (eif α), a major regulator of the initiation phase of mrna translation, by phosphorylating its α subunit. the pkr-induced translational arrest shuts down the negative feedback on the ifn-i response, which can thus result in a prolonged and/or amplified ifn-i response [ ] . because pkr is an isg, the translational arrest is, in turn, potentiated by the ifn-i response (reviewed in [ ] ). this highlights a paradoxical situation in which translation arrest prevents viral replication but also set a threshold of viral detection to commensurate the host transcriptional antiviral response to the level of infection [ ] . whether the pkr pathway is modulated by sars-cov is unknown, yet different coronaviruses regulate pkr-eif α axis and host translation. for example, the mers-cov protein a (p a) accessory protein impedes pkr activation [ , ] . future studies are needed to further uncover the relationship between ifn-i response and host translation and their dynamics in the context of sars-cov infection. the ifn-i response varies among different cell types and within different microenvironments. studies at the single-cell level suggest that the amplitude and kinetic of the response is also heterogeneous for a given cell type. mathematic modeling revealed that ifn-i response is, at least in part, stochastic because only a fraction of cells are able to produce ifn-i upon activation by agonists of the sensors and are sensitive to the paracrine stimulation by ifn-i [ ] [ ] [ ] [ ] [ ] . the heterogeneity of the ifn-i response can be imprinted by the state of the cell at the activation time, including its global translation activity, metabolism, expression levels of signaling molecules (sensors, adaptors, and receptors) [ ] [ ] [ ] [ ] . additionally, the distinct onsets of the ifn-i induction depend on the rapidity and amplitude of viral replication. this heterogeneous responsiveness at the individual cell level consequently shapes the dynamics of the host antiviral response at the whole population level [ ] [ ] [ ] . this model of the ifn-i response dynamics yielded in the context of various rna viruses provides a framework likely at play for coronavirus infections. a delayed induction of the isg expression via virus-induced modulation of the basal activity of transcriptional activity of stat and pkr pathways leads to a peak of coronavirus replication preceding the isg response [ ] . additionally, in vivo study of the dynamic of mhv infection showed that a fast and robust ifn-i production by pdcs down-regulate the ifn-i response by other cells [ ] . this suggests that the ifn-i response at play in different cell types might drive the control of coronavirus infection and potentially contribute to the progression of the disease. as mentioned above, coronaviruses possess various mechanisms to defeat the ifn-i response within infected cells, and this inhibition ability is associated with clinical severity (reviewed in [ ] ). clinical studies showed that coronaviruses evade innate immunity during the first days of infection, which corresponds to a period of widespread inflammation and steadily increasing viral load [ , ] . elevated virus replication eventually leads to inflammation and hypercytokinemia, referred to as a "cytokine storm" [ ] [ ] [ ] [ ] (fig ) . the delayed ifn-i response indeed promotes the accumulation of pathogenic monocyte-macrophages [ , ] . this cell infiltrate results in lung immunopathology, vascular leakage, and suboptimal t cell response [ , ] . immune phenotypic profiling in peripheral blood mononuclear cells (pbmcs) of covid- patients similarly revealed that high viremia is associated with an exacerbated ifn-i response, an aggravated cytokine secretion, and inflammation, driving clinical severity [ ] . although the ifnar signaling pathway was up-regulated at an earlier disease stage, down-regulation of isgs, together with exacerbated nf-κb activation, promotes a cytokine storm and hyperinflammation, found in critically ill patients [ ] . collectively, these findings highlight the negative impact of a delayed ifn-i response on viral control and disease severity. however, the underlining mechanisms that drive the temporal control of the ifn-i response in patients are still elusive. in particular, the host and viral determinants driving the on/off switch of the ifn-i response in infected cells, noninfected cells, and/or stimulated immune cells need to be investigated. such studies will certainly benefit from longitudinal studies of immune profiling in sars-cov infected patients at the single-cell level and in combination with the clinical data. by producing , -fold more ifn-i than any other cell types, pdcs are at the heart of the antiviral ifn-i response [ , ] . they also produce other proinflammatory cytokines, which contribute to modulate the function of several immune cells, such as the mobilization of natural killer (nk) cells or the licensing of virus-specific t cell responses [ ] [ ] [ ] [ ] . because pdcs are refractory to most viral replication, their antiviral response cannot be inhibited by viral proteins [ , ] . this unopposed response likely contributes to the exceptional magnitude of pdc ifn-i production [ , [ ] [ ] [ ] . pdcs are circulating immune cells; nonetheless, their response is mostly localized at the infection site because their activation requires physical contact with infected cells [ , , ] . the contact site between pdcs and infected cells, which we named the interferogenic synapse, is a specialized platform for pamp transfer from infected cell to the toll-like receptor (tlr ) sensor in pdc, leading to an antiviral response [ ] . previous studies on sars-and mers-cov demonstrated that the rapid production of ifn-i by pdcs is essential for the control of potentially lethal coronavirus infections in mouse models [ , ] . pdcs migrate into the lungs at the early phase of infection (i.e., pdc number peaks at day ), temporally coinciding with the peak of ifnα production [ , ] . the pdc number was found to be reduced in blood of covid- patients as compared with control patients [ ] , potentially resulting from a prior response followed by a vanished number of circulating pdcs and/or their mobilization to the infected site. future studies are needed to address how pdc responsiveness evolves in the course of sars-cov- infection and how pdcs respond to contact with coronavirus-infected cells. despite the abovementioned viral inhibitory mechanisms of ifn-i response (table ) , exogenous ifn-i in cell cultures efficiently inhibit sars-cov, sars-cov- , and mers-cov spread [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . consistently, ifn-i was shown to have a protective effect against sars-cov and mers-cov, alone or in combination with other antivirals, in various animal models including mice, marmosets, and macaques [ , , ] . ifn-i and iii interfere with virus infection by inducing the expression of several hundred isgs [ ] . numerous welldescribed isgs exhibit direct antiviral activities by targeting specific stages of the viral life cycle, including entry into host cells, replication, protein translation, and assembly of new virus particles. as mentioned above, many signaling regulators are themselves isgs, thus leading to amplification of the antiviral ifn-i pathway. as a first step towards identifying isgs able to restrict sars-cov- replication, transcriptomic responses to infection have been analyzed in different cellular models, including primary cells, organoids, and clinical samples [ , [ ] [ ] [ ] , as summarized in table . these studies demonstrate that, despite triggering very little to no ifn expression (table ) , sars-cov- replication induces moderate levels of a limited number of isgs. a small subset of infected cells may be refractory to the antagonistic mechanisms of sars-cov- , producing minute but sufficient amounts of ifns to trigger isg induction in larger population of cells. alternatively, isgs may be up-regulated in noninfected cells, which were analyzed together with infected ones. indeed, interpretation of genome-wide investigations of virus-pathogen interactions are often obscured by analyses of mixed populations of infected and uninfected cells [ ] . of note, by contrast to low-multiplicity of infection (moi) infection of a cells expressing angiotensin i converting enzyme (ace ), normal human bronchial epithelial (nhbe) cells, and patient samples, high-moi infections of a -ace and calu- cells led to the high induction of ifns and isgs, including isgs with broad antiviral activities [ ] ( table ). this discrepancy of ifn production/signaling between the levels of viral replication and/or proportion of infected cells might reflect that the counteraction measures employed by sars-cov- are less potent at high moi. alternatively, as suggested by blanco-melo and colleagues, high-moi infections in cell culture may generate more pamps, such as defective noninfectious viral particles, than low-moi infections [ ] . despite being expressed at moderate levels in vitro and in vivo, several up-regulated isgs identified by these transcriptomic studies ( table ) exhibit well-characterized broad-spectrum antiviral activities and could thus have additive restrictive effects on sars-cov- replication. for instance, the members of the interferon-induced protein with tetratricopeptide repeats (ifitm) family, known to inhibit entry of numerous enveloped rna viruses [ ] , similarly restrict entry of sars-cov, mers-cov, and the globally circulating human coronaviruses e and nl in t and a cell lines [ , ] . oas and mycovirus resistance protein (mx)a could also contribute to the ifn-i-mediated inhibitory effect on sars-cov- because a clinical study revealed that single nucleic polymorphisms in the oas -utr and mxa promoter region appear associated with host susceptibility to sars-cov in the chinese han population [ ] . moreover, the fact that mers-cov nonstructural protein b (ns b) is a - - oligoadenylate synthetase (oas)-rnase l antagonist [ ] suggests that the oas pathway contributes to the antiviral effects of ifns on coronavirus replication. isgs positively potentiating ifn signaling, such as ifih /mda , tank, irf , and stat , were also increased in the bronchoalveolar lavage fluid (balf) of covid- patients as compared with healthy controls [ ] and could potentially contribute to the amplification of ifn-i response against sars-cov- replication. zinc finger antiviral protein (zap), which is encoded by an isg, contributes to the anti-sars-cov- effect of ifns in human lung calu- cells [ ] . zap is known for restricting the replication of numerous viruses such as retroviruses and filoviruses [ ] . the protein recruits the cellular mrna degradation machinery to viral rna via -c-phosphate-g- (cpg) dinucleotide recognition [ ] . to further determine which individual isg or combination of isgs mainly restricts sars--cov- replication in vitro, several previously established approaches could be used, such as, for example, screening for single or combined isg activity using a lentiviral vector-based library, as successfully performed by schoggins and colleagues for other viral infections [ ] [ ] [ ] [ ] . indeed, this library of around human isgs was recently screened in human hepatoma cells for antiviral activity against hcov- e [ ] . the screen identified ifn-inducible lymphocyte antigen complex, locus e (ly e) as a potent inhibitor of the replication of multiple coronaviruses, including sars-cov, sars-cov- , and mers-cov, by blocking fusion of viral and cellular membranes [ ] . mice studies revealed that ly e directly protects primary b cells and dendritic cells from murine coronavirus infection [ ] . pursuing the identification and characterization of ifn effectors with potent anti-sars-cov- activities will reveal weakness points in the life cycle of sars-cov- and may lead to the design of drugs that activate antiviral isgs or either mimic or amplify their action. recent advances in systematic screening strategies have revealed the existence of a small subset of isgs exhibiting proviral activities [ , ] . these proviral isgs act either by exhibiting direct proviral activities such as facilitating viral entry [ ] or via their abilities to negatively regulate ifn signaling and facilitate the return to cellular homeostasis. the receptor tyrosine kinase axl is a well-characterized example of an isg that is used by enveloped virus for cellular internalization [ ] [ ] [ ] . alternatively, isgs that possess antiviral activities against a viral family can be hijacked by unrelated viruses to favor infection. this is the case for ifitm and ifitm , which potently block entry of a broad range of enveloped viruses [ ] while promoting entry step of human coronavirus oc (hcov-oc ) in human cells [ ] . sars-cov- uses ace and transmembrane serine protease (tmprss ) to enter cells [ ] . viral tropism is thus largely dictated by ace and tmprss coexpression. analysis of human, nonhuman primate, and mouse single-cell rna-sequencing (scrna-seq) data sets generated from healthy or diseased individuals revealed that expression of ace is primarily restricted to type ii pneumocytes in the lung, absorptive enterocytes within the gut, and goblet secretory cells of the nasal mucosa [ ] . interestingly, this meta-analysis identified an association between ace expression and canonical isgs or components of the ifn-signaling pathway in different tissues. independent analyzes of publicly available data sets concluded that ace expression pattern is similar to isgs [ ] . in vitro validations were performed by treating primary human upper airway cells with numerous inflammatory cytokines. ifnα , and to some extent ifny, led to greater and more significant ace up-regulation compared with all other tested cytokines [ ] . substantial up-regulation of ace was also observed in primary skin and primary bronchial cells treated with either ifn-i or ifn-ii. moreover, ace expression was also up-regulated upon ex vivo influenza a infection in human lung explants isolated following surgical resection [ ] . because the majority of cells robustly up-regulating ace were epithelial, this observation potentially explains why previous analyses to define canonical isgs within immune populations did not identify ace as an induced gene [ ] . finally, stat , stat , irf , and irf binding sites were identified within − , to + bp of the transcription start site of ace [ ] . despite need for direct evidence that ifns up-regulate ace in target cells in vivo, altogether these studies suggest that ace could be an isg that enhances sars-cov- internalization in human epithelial cells [ , ] . elucidating tissue and cell type specificity of isgs, as well as their mechanisms of action, is essential for understanding the potential dual role of ifns during human sars-cov- infection. it may also guide the use of ifns in clinical trials. although ifn-i treatment gave some encouraging results against sars-cov and mers-cov in vitro and in animal models, including mice, marmosets, and macaques [ , , , , ] , additional knowledge to optimize its therapeutic efficiency in humans is required [ ] [ ] [ ] [ ] [ ] . previous information yielded from these animal studies provided guidance for treating the current pandemic virus. first, it became clear from these former studies that ifnβ is a more potent inhibitor than ifnα as shown both in vitro and in patients [ , ] . second, the timing of ifn-i treatment seems determinant for infection outcomes. indeed, as shown in mice and in macaques, ifn-i is protective when administered prior to sars-cov or mers-cov infection or early in the course of infection, whereas late administration could be either ineffective or detrimental [ , ] . in humans as well, ifn-i-based therapies were not beneficial to critically ill patients with multiple comorbidities and who were diagnosed late with mers-cov, thus pointing out that ifn-i has to be administered early after infection [ , ] . the first clinical trials using ifn-i alone or in combination with other antivirals are currently carried out in covid- patients in several countries. for instance, the multicenter, adaptive, randomized, open clinical trial discovery evaluates, among other treatment, the efficacy of ifnβ as a treatment for covid- in hospitalized adults in europe. a recent openlabel, randomized, phase trial performed in adults with covid- in hong kong showed that the triple combination of ifnβ- b, lopinavir-ritonavir, and ribavirin was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospital stay in patients with mild to moderate covid- [ ] . it has to be noted that the patients were treated in the early stages of the disease because the median number of days from symptom onset to start of study treatment was days, further reinforcing the fact that the timing of ifn-i treatment is key [ ] . other therapeutic approaches are under investigation to avoid the adverse effects of ifn-i therapy and/or its potential inefficacity when administrated too late postinfection. one strategy is to use aerosol formulations of recombinant ifn to deliver the cytokine directly inside the lung [ , ] . this approach has several benefits because it is a noninvasive route of administration, and the local concentration reached in the tissue can be higher than through systemic injection and is thus expected to minimize the adverse effects of ifn. nebulized ifnα- b was used on covid- patients in wuhan, alone or in combination with arbidol [ ] . the study, performed on adults, showed a significant reduction of the duration of detectable virus in the upper respiratory tract in ifnα- b-treated patients, with or without arbidol [ ] . another study currently ongoing in beijing aims at evaluating the efficacy and safety of recombinant human ifnα spray in preventing sars-cov- infection in highly exposed medical staffs (chictr ). type iii ifns (ifnλs or ifn-iii) are gaining an increased interest in antiviral therapies [ ] [ ] [ ] . like ifn-i, they activate the jak-stat signaling pathway. they do so via a receptor that is largely restricted to cells of epithelial origin, including respiratory epithelial cells (reviewed in [ ] ). ifn-iiis are induced upon viral infections, and they are growing evidence that they provide important first-line defense against viral infections of the respiratory and gastrointestinal tracts [ ] [ ] [ ] . in mice, ifn-iii was shown to protect epithelial cells of the respiratory and tract from infections with several respiratory viruses, including mers-cov [ ] . a study investigating sars-cov- infection of intestinal epithelial cells, using both colon-derived cell lines and primary colon organoids, showed that ifn-iii response was more efficient than ifn-i at controlling viral replication [ ] . however, ifn-iiis produced by dendritic cells in the lung were recently shown to cause barrier damage and to compromise host tissue tolerance and predispose to lethal bacterial superinfections [ ] . therefore, although the antiviral properties are promising, the benefit of ifn-iii to treat covid- patients awaits careful evaluation. the first clinical trials using ifn-iii are ongoing, including one launched at the massachusetts general hospital to evaluate the safety and efficacy of pegylated ifnλ on a small number of covid- patients (nct ). besides the use of recombinant ifn as a therapeutic treatment, one interesting alternative strategy would be to boost the natural innate immune defenses of covd- patients at early stages of the disease. because pdcs are seemingly crucial to control coronavirus infections [ , ] , a possibility would be to either amplify or prolong their activation to make them produce more ifn-i and ifn-iii. a number of negative feedback loops prevent an exacerbated activation of pdcs, which can be deleterious for the organism in the long term. thus, transitorily inhibiting these negative retrocontrols may increase the antiviral activity of pdcs. for instance, the bone marrow stromal cell antigen (bst ) is an isg that activates the immunoglobulin-like transcript (ilt ) inhibitory receptor expressed by pdcs to interrupt the ifn-i response [ ] . the blockade of this interaction using either antibodies or inhibitory molecules should thus increase the duration of pdc activation. one could also envisage to take advantage of viral proteins that counteract the antiviral activity of bst , such as hiv- viral protein u (vpu) [ ] . other pdc inhibitory molecules include natural monamines such as histamine, dopamine, or serotonin, which bind to the c-x-c motif chemokine receptor (cxcr ) at the surface of pdcs [ ] . because the cxcr antagonist amd (also known as plerixafor) blocks the binding of monoamines to pdcs, it can prevent the amine-dependent inhibition of pdc activation [ ] . amd is already used in clinics as an immunostimulatory molecule able to mobilize hematopoietic stem cells in cancer patients [ ] . finally, we recently reported that the peptidyl-prolyl isomerase peptidyl-prolyl cis-trans isomerase nimainteracting (pin ) switches off the ifn-i expression by pdcs by inducing irf degradation [ ] . a number of pin inhibitors have been developed and could be tested for their potential activity on human pdcs [ ] and could represent another possible therapeutic strategy to boost pdc-mediated ifn-i production. sars-cov- emerged in the human population around months ago, yet it seems well adapted to avoid and inhibit the ifn-i response in its new host. such efficient strategies allow the virus to replicate and disseminate in infected individuals without encountering the initial host defense. this modest ifn response could explain why viremia peaks at early stages of the disease, at the time of symptoms appearance, and not around to days following symptoms, like during sars-cov and mers-cov infections. ifnβ treatment would be expected to improve the antiviral response of patients at the early stage of covid- and, if possible, at plos pathogens the site of infection. indeed, ifnβ appeared to be pivotal to improve patient states in a combined therapy regiment of ifnβ, lopinavir-ritonavir, and ribavirin [ ] . nonetheless, ifnresistant viral mutants may arise and be able to control ifn even more efficiently than parental viruses. the exacerbated production of proinflammatory cytokines observed at later stage of covid- might challenge the efficiency of an ifnβ treatment administrated after appearance of symptoms. 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influenza virus infection targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals we thank nathalie j. arhel for helpful discussion. key: cord- -on zu w authors: falcinelli, shane d.; chertow, daniel s.; kindrachuk, jason title: integration of global analyses of host molecular responses with clinical data to evaluate pathogenesis and advance therapies for emerging and re-emerging viral infections date: - - journal: acs infectious diseases doi: . /acsinfecdis. b sha: doc_id: cord_uid: on zu w [image: see text] outbreaks associated with emerging and re-emerging viral pathogens continue to increase in frequency and are associated with an increasing burden to global health. in light of this, there is a need to integrate basic and clinical research for investigating the connections between molecular and clinical pathogenesis and for therapeutic development strategies. here, we will discuss this approach with a focus on the emerging viral pathogens middle east respiratory syndrome coronavirus (mers-cov), ebola virus (ebov), and monkeypox virus (mpxv) from the context of clinical presentation, immunological and molecular features of the diseases, and omics-based analyses of pathogenesis. furthermore, we will highlight the role of global investigations of host kinases, the kinome, for investigating emerging and re-emerging viral pathogens from the context of characterizing cellular responses and identifying novel therapeutic targets. lastly, we will address how increased integration of clinical and basic research will assist treatment and prevention efforts for emerging pathogens. e merging and re-emerging viruses pose a significant threat to public health and global economies. moreover, outbreaks caused by emerging and re-emerging viruses continue to increase in frequency as a result of changing socio-economic, environmental, and ecological factors. notably, the zoonotic viral pathogens, severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), ebola virus, chikungunya virus, and zika virus, have emerged on a global scale in recent years; although less widely publicized, other emerging viral pathogens such as monkeypox virus and andes virus have led to smaller recurrent outbreaks. a critical challenge for combating these outbreaks is often the discordant relationship between the economic status of outbreak "hotspots" and resource distribution or control capacity within these regions. in addition, the development and delivery of therapeutics for combating such outbreaks have been complicated by both the associated costs in design and development for novel antiinfective therapeutics and the requirements for regulatory approval and licensure. importantly, emerging infectious diseases present the additional inherent challenge that they are only "emerging", and thus limited resources are made available for research until they present a significant risk. for many emerging viral pathogens, the requirement for highcontainment facilities has further impeded widespread research. from the perspective of drug development, the limited knowledge and understanding of molecular pathogenesis for these agents is a daunting challenge to overcome when outbreaks emerge. in the face of an increasing burden of emerging and reemerging pathogens, it is necessary to overcome the barriers imposed by a paucity of information regarding molecular pathogenesis for these agents. omics-based approaches present a mechanism to rapidly generate large amounts of data in regard to host responses and assist in target identification for drug development efforts. in addition, omics-based analyses allow for the characterization of molecular events that mitigate cellular responses to viral pathogens from a global perspective across multiple levels of cellular complexity (individual cell types < tissues < organs). high-throughput global analyses of host gene expression, including microarrays and rna-seq, provide important information regarding transcriptional responses during infection. although these validated approaches are among the most widespread of the omics-based technologies for infectious disease investigations, they do not provide a direct measure of the activation status of the cell signaling pathways that regulate underlying cellular responses. in contrast, global investigations of cellular kinase activities (the kinome) are able to provide insight into the activation status of cell signaling networks (including those that mediate pathogen recognition and innate immune activation, cell cycle activities, metabolic status, wound healing and repair, and cell death) at the level of individual kinase-mediated phosphorylation events. in addition, kinome investigations allow for potential identification of kinase drug targets. kinases are currently one of the top targets for drug design and development, and there is potential for repurposing of kinase inhibitors with existing regulatory approval. as emerging viral infections often result in severe illness including respiratory failure [severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and influenza] and multiorgan failure [ebola virus disease (evd)], understanding complex pathogenesis of these infections is required for effective vaccine and therapeutic design and for improved patient care. healthcare providers caring for patients with severe emerging viral infections are generally focused on clinical care and biosafety as compared to the complex molecular events that underlie pathogenesis. in contrast, basic researchers typically focus on discrete aspects of pathogenesis through a variety of in vitro and in vivo analyses rather than the complex interplay between these events and the clinical, physiologic, and pathologic abnormalities observed by the clinician. integrating basic and clinical research is needed to accelerate the translation of knowledge for emerging infections toward vaccine development and therapeutic discovery. specifically, detailed natural history studies merging multiple data streams including omics approaches (high-throughput gene expression and kinomics) and focused translational investigations utilizing relevant models that can be validated to human disease are needed to clarify disease pathogenesis, advance therapeutic discovery, and facilitate regulatory approval. although an integrated approach between basic and clinical research is ideal for investigating the connections between molecular and clinical pathogenesis, there has been a paucity of investigations for which this has been undertaken. here, we will discuss emerging pathogens for which there is available information regarding the clinical course of disease, host immune responses during natural infection, and molecular information regarding the global cellular responses to infection, with particular attention on host kinome investigations. in this regard we will focus on the emerging viral pathogens mers-cov, ebola virus (ebov), and monkeypox virus (mpxv) (figure ). we will review the clinical presentation and immunological and molecular features of the diseases and summarize available omics data informing pathogenesis of these pathogens. lastly, we will discuss the benefit of improved integration of available clinical knowledge or data regarding the pathologic manifestations of disease with basic research investigations to advance treatment and prevention of severe emerging viral infections. global gene expression investigations have provided information regarding host response to emerging and re-emerging review pathogens at the level of individual genes or gene clusters. however, there is a paucity of information regarding the relationship of cell signaling networks, and in particular their activation status, with the biological/pathological events that occur throughout infection. it has been well established that many biological processes can be regulated independent of transcriptional or translational changes through post-translational modification (ptm) events. indeed, kinase-mediated phosphorylation of proteins, in which kinases catalyze the transfer of the γ phosphate group from atp to the hydroxyl group of a specific ser, thr, tyr residue, is figure . generation of kinome peptide array targets and kinome peptide arrays: ( ) species-specific proteomic or genomic information from a diverse range of species can be used to identify kinase recognition motifs that are composed of a central phosphorylation target and the surrounding amino acids (normally + and − amino acids from the central phosphorylated residue); ( ) peptides that comprise the kinase recognition motifs identified in ( ) are synthesized and covalently linked to a glass surface. peptide targets are spotted in replicates of three to nine spots on each array to account for intra-array variability. individual amino acids of the peptides are represented by orange, red, purple, and green spheres. ( ) biological samples are processed to generate cell lysates that are activated with atp and applied to the kinome peptide array. ( ) following the application of the cell lysate, activated kinases in the cell lysate will recognize their respective kinase recognition motifs and phosphorylate the central phosphorylated residue of the peptide. ( ) kinome peptide arrays are subsequently stained with a phospho-specific fluorescent stain and imaged followed by comparative bioinformatics analyses. tissue and cell images were derived and/or modified from servier medical arts under a creative commons attribution . unported license. among the most thoroughly characterized ptm. virtually all cell signal transduction events are regulated by kinases independent of biological complexity of the host (i.e., prokaryotes and eukaryotes). lending further credence to the biological importance of kinases, > kinases have been identified in the human genome, and ∼ % of the human proteome is modulated by kinase-mediated phosphorylation events. , thus, considering the central role of kinases in a broad range of cellular processes (including growth and development, metabolism, and immune responses), it has been postulated that the activities of individual kinases may represent more reliable predictors of cellular phenotypes than transcriptional or translational changes. indeed, transcriptional or translational-based omics approaches are often unable to account for regulatory events including gene silencing, mrna stability, translational efficiencies, protein turnover, enzyme/ substrate subcellular sequestration, or protein activation/ repression ptms. given the central role of kinases in the regulation of biological processes, kinases are a logical drug target. as a testament to this, kinase inhibitors have been granted licensure by the u.s. food and drug administration (fda) for a broad range of malignancies, and there are a continually increasing number of kinase inhibitors that are in various stages of preclinical trials. furthermore, kinases are the second most frequently targeted gene class in cancer therapy after the g protein coupled receptors. , the recent prioritization for the repurposing of approved therapeutics for alternative malignancies by the national institutes of health center for advancing translational sciences (ncats) , also provides considerable impetus for the investigation of licensed kinase inhibitors as infectious disease therapeutics. concerns exist regarding the therapeutic application of kinase inhibitors as novel therapeutics for infectious disease, in particular to the potential immunosuppressive effects following prolonged treatment. however, it should be appreciated that the application of kinase inhibitors in such cases would need to be targeted in terms of timing and dose, with appropriate molecular biomarkers guiding initiation and cessation. it should also be appreciated that the clinical symptoms associated with many emerging and re-emerging pathogens have been associated with dysregulated host immune responses (in particular pro-inflammatory responses). thus, the global analysis of the activation state of host kinases (the kinome) can provide critical insight into the specific activation state of individual kinases, cell signaling pathways, or larger biological networks. in addition, kinome investigations may offer important, and predictive, insight into the cellular mechanisms that regulate phenotypic changes within cells. as many kinases recognize a particular phosphorylation motif composed of the central phosphoacceptor site and the amino acids + and − residues from the central phosphorylation site, peptides representing this kinase target motif can be synthesized with relatively high efficiency and low expense. indeed, kinase target motif peptides have been shown to be appropriate substrates for their respective kinases with v max and k m values approaching those of the fulllength protein. thus, peptide kinome arrays can be constructed in an analogous manner to traditional dna microarrays where kinase target motif peptides are spotted onto a glass slide representing hundreds to thousands of unique peptide targets for kinases ( figure ). following this, samples in the form of cellular lysates from whole organs, tissues, or individual cell types can be applied to the kinome peptide arrays, allowing for the phosphorylation of specific peptide targets by kinases within the lysate (figure ). the development of kinome-specific bioinformatics analysis software, including the platform for intelligent, integrated kinome analysis (piika), has provided a mechanism to identify the complex patterns of kinase-mediated phosphorylation events and quantitate the differences between compared conditions. clinical findings during mers-cov infection. syndromic case-definition for mers infection requires a compatible clinical syndrome and an epidemiologic risk factor including travel to an affected region or contact with a known or suspected case. initial symptoms of mers-cov infection include fever, chills, cough, shortness of breath, myalgia, and malaise following a mean incubation period of days, which can range from to days. mildly symptomatic or possibly asymptomatic infections have been reported, and progression to severe disease is associated with pre-existing medical conditions including cardiopulmonary disease, obesity, and diabetes. most ( %) of reported mers cases are among adults, with a median age of years. in severe cases respiratory failure requiring mechanical ventilation typically occurs within days of symptom onset. laboratory abnormalities include lymphopenia, leukopenia, thrombocytopenia, elevated serum creatinine levels consistent with acute kidney injury, and elevated liver enzymes. − high lactate levels and consumptive coagulopathy have also been reported. , chest radiographic abnormalities are observed in most cases consistent with viral pneumonitis, secondary bacterial pneumonia, or acute respiratory distress syndrome. cytokine levels in serum and bronchoalveolar lavage (bal) from two mers patients, one with fatal disease and one who survived. higher levels of retinoic acid-inducible gene (rig- ), melanoma differentiation-associated protein (mda ), interferon regulatory factor (irf)- and - , interleukin (il) a, and il- and lower levels of il- and ifnγ were observed in the fatal case compared with the survivor. more recently, min et al. performed a temporal analysis of cytokine, chemokine, and growth factor blood levels from patients during the recent outbreak of mers in south korea. the patients were subcategorized into four groups on the basis of disease severity: group i patients developed fever and recovered. group ii patients developed mild pneumonia without hypoxemia. group iii patients had prolonged and severe pneumonia. group iv patients had severe pneumonia and acute respiratory distress syndrome. group iv patients included five fatal cases of mers; all patients in groups i−iii fully recovered from illness. ifnα was elevated in all groups and largely peaked during the second week of illness. granulocyte-colony stimulating factor (g-csf) and granulocyte macrophage (gm)-csf were similarly elevated across all patient groups; however, patients with fatal disease had reduced gm-csf responses following antiviral treatment as compared to patients that recovered. patients with pneumonia had relative elevations of il- , tumor necrosis factor (tnf)-α, il- , and il- during the second and third weeks of illness. elevated il- and il- appeared to trend positively with the severity of illness. a robust induction of multiple chemokines was found in most patients. notably, eotaxin and regulated on activation, normal t expressed and secreted (rantes) was elevated in all patients. in contrast, il- , monocyte chemotactic protein (mcp)- and macrophage inflammatory protein (mip)- β were more prominent in groups ii and iii as compared to groups i and iv. furthermore, elevated interferon gamma induced protein (ip)- correlated with the development of pneumonia (groups i−iii). multiple growth factors, including epidermal growth factor (egf), fibroblast growth factor (fgf)- , vascular endothelial growth factor (vegf), and tgf-α, were significantly elevated across all patients; however, egf was significantly higher in patients that recovered from disease as compared to the fatal cases. further evaluations are needed to characterize the natural history of immune response during acute mers-cov infection and recovery. transcriptome analyses of mers-cov. in an effort to better characterize mers-cov pathogenesis in the absence of available samples from human patients and, in particular, address pathologic changes associated with infection, multiple animal species have been employed in mers-cov investigations. while multiple small animals are not susceptible to mers-cov infection, rhesus macaques and marmosets develop mild to severe lung pathology following experimental infection. transcriptome analysis in mers-cov-infected rhesus macaques revealed that genes related to antiviral immunity, chemotaxis, and inflammation were overexpressed in lesional versus grossly normal lung tissue at days postinfection. a significantly smaller number of differentially expressed genes was found on day postinfection with no obvious trends following pathway enrichment analysis. significant changes in the transcriptome profiles of peripheral blood mononuclear cells (pbmcs) were observed at only day postinfection. this global analysis suggests a key role for an initial rapid innate immune and inflammatory response (through pattern recognition receptors) followed by rapid resolution. a study of mers-cov-infected marmosets evaluated lung lesions by rnaseq at days − postinfection. pathway analyses demonstrated that chemotaxis and cell migration, cell cycle progression, and cell proliferation and fibrogenesis were highly over-represented relative to uninfected controls. to a lesser degree, pathways associated with inflammation, vascularization, endothelial activation, proliferation of smooth muscle, and tissue repair were also overrepresented in infected animals. differences were most significant on days and postinfection during illness progression relative to day . recently, menachery and colleagues examined the interaction between mers-cov emc/ and the host ifnstimulated gene (isg) response by transcriptomics. isg responses in mers-cov infected calu cells, a lung adenocarcinoma cell line, had no discernible induction initially upon infection but were up-regulated by h postinfection. down-regulation of a subset of isgs resulted in altered histone modifications, a potential epigenetic contributor to early impairment of antiviral cellular defenses. in a separate analysis genetically distinct mers-cov strains, mers-cov sa and mers-cov eng , produced distinct gene expression profiles in calu- cells. these analyses may better inform early host-cell antiviral responses and the impact of viral evolution on these and other complex biological responses. proteomics analysis corroborated these transcriptional data with induction of isgs observed h postinfection. significantly reduced levels of stat and pkr compared with uninfected controls were also noted. differential host transcriptome responses to mers-cov sa and mers-cov eng highlight both the propensity of emerging viral pathogens to evolve rapidly and the importance of additional host response analyses for augmenting and clarifying such complex biological responses. kinome analyses of mers-cov infection. host responses to mers-cov infection through kinome analysis were recently assessed using huh- cells, an immortalized human hepatocyte cell line, that are highly permissive to mers-cov infection. temporal analysis of kinome responses by peptide arrays revealed selective modulation of extracellular signal-regulated kinases (erk)/mitogen-activated protein kinases (mapk) and phosphatidylinositol- -kinases (pi k)/akt (also known as protein kinase b)/mechanistic target of rapamycin (mtor) signaling responses. over-representation analysis (ora) revealed erk/mapk and pi k/akt/mtor signaling responses were consistently up-regulated during infection. multiple erk/mapk family members formed central components of functional networks and signaling pathways throughout infection. similar results were observed for intermediates of the pi k/akt/mtor signaling pathway at and h postinfection, suggesting that modulation of erk/ mapk and pi k/akt/mtor signaling may be important for productive mers-cov infection. downstream analysis of the phosphorylation patterns of pathway intermediates from the erk/mapk and pi k/akt/ mtor signaling supported observations from the kinome analysis. both investigations demonstrated that nuclear factor kappa-light-chain-enhancer of activated b cells (nfκb)regulated family members were important mediators of mers-cov infection. il -and ifn-mediated signalings were also modulated during mers-cov infection, consistent with prior analyses. , these results were also in agreement with in vitro transcriptional analysis of mers-cov infection. prophylactic or therapeutic addition of fda-licensed kinase review inhibitors targeting activated kinases in mers-cov infection impaired viral replication. these hypothesis-generating data may inform directed investigations into mers-cov pathogenesis and, importantly, demonstrate the potential to identify novel host-centric therapeutic targets. ebolaviruses. the filoviridae family of viruses consists of three genera: ebolavirus, marburgvirus, and the newly identified cuevavirus. structurally, filoviruses have a pleomorphic enveloped, filamentous virion particle that encapsulates a negative-sense single-stranded rna genome. ebolaviruses were first described in following disease outbreaks in the democratic republic of congo and sudan and are composed of five viral species, including ebola virus (ebov), sudan virus (sudv), bundibugyo virus (bdbv), taı̈forest virus (tafv), and reston virus (restv). sporadic outbreaks of ebov, sudv, bdbv, and tafv have occurred throughout central africa for more than three decades, resulting in thousands of infections. case fatality rates during these outbreaks have routinely exceeded %. isolated outbreaks of restv have occurred outside africa in nonhuman primate facilities in the united states, italy, and the phillipines, and infection results in high morbidity and mortality in nonhuman primates; however, restv has only been associated with asymptomatic infections in humans. although ebolaviruses have been historically associated with isolated outbreaks involving small cohorts of infected patients (< ), an outbreak of evd in west africa beginning in has resulted in , cases and , deaths ( % cfr) as of june (http://www.who.int/csr/ disease/ebola/en/). although virus transmission has greatly decreased in west africa, surveillance for sporadic infections continues. clinical findings in evd. ebov transmission occurs through exposure of infected body fluids or tissues to mucous membranes or nonintact skin. the mean incubation period is − days, ranging from to days. initial signs and symptoms are nonspecific including fever, myalgia, and malaise and cannot be reliably distinguished from other endemic illnesses in africa including malaria and enteric infections. whereas mild illness has been described, most patients develop severe disease within days of symptom onset. massive gastrointestinal fluid losses of up to − l per day due to vomiting and watery diarrhea may result in progressive dehydration and hypovolemic shock. even in the setting of adequate fluid and electrolyte replacement, sequential multiorgan failure may occur. ebov infects multiple organs and cell types throughout the body with the notable exception of lymphocytes that are indirectly depleted early during infection. organ injury due to direct viral or indirect host-mediated responses results in severe complications including meningoencephalitis, uveitis, respiratory failure, secretory diarrhea, disordered coagulation, renal failure, hepatic necrosis, and myositis. the clinical presentation, laboratory values, viral kinetics, and clinical management of evd patients in west africa, europe, and the united states during the − outbreak have been recently well-characterized. soluble immune mediators associated with ebov infections. there is a paucity of information regarding ebov pathogenesis in humans primarily due to the limited frequency of evd outbreaks prior to and limitations presented by sample acquisition from infected patients in the field as well as the overall size of patient cohorts. largely contradictory findings regarding the immune responses in those who survive or succumb to evd have further confounded the under-standing of ebov pathogenesis in human patients. for example, villinger et al. reported that serum cytokine concentrations (including ifnα, ifnγ, tnf-α, il- , and il- ) were elevated in patients with fatal infections in comparison to survivors. in contrast, additional studies have suggested that fatal infections were instead related to general immunosuppression including ifnγ, il- , and il- . − an investigation of sudv infection in humans by sanchez et al. demonstrated limited changes in the expression levels of cytokines, fas antigen, and fas ligand in pbmcs from infected patients relative to those found for uninfected patients. furthermore, an investigation of fatally infected evd patients by wauquier et al. has further confounded the role of host immune responses in fatal evd as hypersecretion of multiple cytokines and growth factors and decreased secretion of t lymphocyte-derived cytokines were associated with fatal disease. transcriptome analyses of ebola virus infection. to date, no investigations of host gene expression in ebov-infected patients have been reported, although limited data are available from animal models of infection or from in vitro investigations. in a study of pbmcs from ebov-infected crab-eating macaques, rubins et al. found few notable changes in the early stages of infection ( − days); however, broad changes were observed over days − post-infection. pro-inflammatory cytokines (il- β, il- , il- , and tnf-α) and chemokines (mip- α and mcp − ) were up-regulated at days − postinfection relative to healthy controls. multiple genes related to apoptosis including bcl- family members, multiple caspases, fas-associated death domain protein, and tnf superfamily member were also up-regulated at late time points. ifn-regulated genes were up-regulated by day postinfection and remained so through study day . yan et al. investigated pbmc gene expression in ebovinfected rhesus macaques with or without anticoagulant administration. untreated animals displayed up-regulation of immune response genes, b cell receptor signaling intermediates, nk cell mediated cytotoxicity, leukocyte activation, and lymphocyte activation compared with anticoagulant-treated animals during the early stages of infection. the expression levels of these gene clusters fell to pre-infection levels at the late-stage of infection. in contrast, genes related to defense responses, apoptosis, wounding, inflammation, coagulation, and leukocyte activation remained elevated during early-and latestage infection. following the isolation of restv from pigs, subsequent investigations have demonstrated that pigs were susceptible to both restv and ebov infection with preferential targeting of macrophages in the lungs. recently, nfon et al. demonstrated that ebov infection in pigs resulted in up-regulation of chemokine expression beginning on day postinfection as compared to mock-infected pigs. the most pronounced changes in gene expression were found on days and postinfection and included the up-regulation of a broad set of cytokines (il- , il- , il- , il- , il- , il- , il- , resistin), chemokines (ccl , ccl , ccl , ccl , amcf-ii, ccl l , ccl ), cell adhesion protein (selectin), antimicrobial protein, palate, lung, and nasal epithelium clone proteins, and pro-apoptotic molecules (multiple caspases, caspase recruitment domain-containing protein (card), apoptosisassociated tyrosine kinase (aatk), fas, fas-associated protein with death domain (fadd), tnf receptor-associated factor (traf ), tnfα-induced protein -interacting protein review (tnip )). in addition, expression of multiple genes related to microbial sensing (pattern recognition receptors) or antiviral responses (isgs) was up-regulated in the lungs of infected animals. although the localization of the cytokine response of pigs and humans or nhps differs during the course of ebov infection (localized responses in the lungs of pigs versus a predominantly systemic response in humans and nhps), the cytokine profiles of pigs, humans, and nhps were quite similar. for example, comparison of nhp and porcine responses during ebov infection demonstrated multiple gene expression similarities between the two species (i.e., il- , il- , caspase family members). it is also likely that direct comparison of both data sets would likely yield many common gene signatures that are conserved in their identity as well as their directionality (upregulation vs down-regulation). macrophages are an early target of ebov infection and support high-level viral replication. ebov attachment and entry into human macrophages in vitro induces pro-inflammatory mediators including il- , il- , and tnf-α as early as h postinfection. noncardiogenic pulmonary edema is a recognized complication of evd, and human autopsy data support that alveolar macrophages are a target of ebov infection. ebov infection of alveolar macrophages in vitro resulted in an early, transient increase in cytokine and chemokine expression, supporting that paracrine-soluble mediators of inflammation may contribute to vascular leakage in the lungs. gene expression responses of ebov-and marvinfected huh cells resulted in the global suppression of antiviral responses, including toll-like receptor (tlr), irf , and protein kinase r (pkr)-mediated pathways. however, signal transducers and activators of transcription (stat) phosphorylation in ebov-and marv-infected cells were differentially modulated. ebov-mediated ifn inhibition has been well characterized and is thought to be attributable to ebov proteins vp and vp . interestingly, restv infection, which does not induce clinical illness in humans, resulted in the activation of > % of the ifn-stimulated genes (isgs). kinome analysis of ebola virus. hepatocytes are an early target of ebov infection, directly contributing to diffuse hepatic necrosis observed in fatal cases. ebov infection of huh cells has been evaluated by kinome analyses, shedding light on liver pathogenesis in evd. ebov infection of huh- cells resulted in temporal modulation of the tgf-β signaling pathway as compared to mock-infected cells. pathway ora demonstrated that multiple tgf-β-mediated signaling pathways were up-regulated at and h post ebov infection. furthermore, these responses were associated with changes in the expression patterns of multiple cellular proteins associated with a mesenchyme-like transition. these included the upregulation of matrix metalloproteinase , n-cadherin, and fibronectin and down-regulation of e-cadhering and claudin . in this process cells lose polarity and cell-to-cell adhesion transforming into mesenchymal stem cells that contribute to wound healing or organ fibrosis; however, the role of these events in ebov infection remains to be elucidated. additional analysis demonstrated that inhibition of pi k/akt, erk/ mapk, or pkc pathways with kinase inhibitors reduced ebov replication when administered prophylactically or therapeutically. supporting this observation, a subset of kinase inhibitors administered to ebov-infected mice reduced lethality. defining mechanisms by which kinase inhibitors show benefit in these models will better clarify their role as potential therapeutics. monkeypox virus. mpxv, a member of the genus orthopoxvirus, causes zoonotic infections with a case fatality rate of ∼ %. mpxv, vaccinia virus (vacv), cowpox virus (cpxv), ectromelia virus, and variola virus (varv), the etiologic agent of human smallpox, comprise the orthopoxviridae family of viruses. mpxv was first isolated in from cynomolgus macaques in denmark; however, human mpxv infections were not recognized until following the isolation of the virus from a suspected case of smallpox infection in the democratic republic of congo. mpxv is composed of two distinct clades that are genetically, clinically, and geographically distinct. the congo basin mpxv (central african mpxv) clade is considered to have both higher lethality and morbidity than the west african mpxv clade as demonstrated from comparative infection models in various animal species (including nonhuman primates, mice, prairie dogs, and ground squirrels) and as well natural infection in humans. although human mpxv infections have been recorded in west africa, the majority of human mpxv infections have occurred in the congo basin region of central africa, largely in the democratic republic of congo. clinical findings in mpxv infections. clinical and epidemiological information regarding human mpxv disease has been derived from enhanced surveillance campaigns in the congo basin. from this work, it has been demonstrated that human mpxv infection and illness largely mirror those of discrete, ordinary smallpox. the incubation period for both viruses (varv and mpxv) is − days with an initial febrile prodromal period of − days. this prodromal period is normally accompanied by fever, headache, backache, malaise, and prostration. the rash period for both smallpox and mpxv (including lesion appearance and desquamation) normally occurs − days postinfection with highly similar appearance, distribution, and progression of lesions. , as with smallpox, mpxv-associated rash progresses through macular, papular, vesicular, and pustular phases. a second febrile period occurs when the lesions become pustular and is often associated with deteriorating conditions in the patient. lymphadenopathy (maxillay, cervical, or inguinal) is often associated with mpxv infections prior to, or concomitant with, rash development but is absent in varv infections. it has been postulated that this reflects the effective generation of host immune responses during mpxv infection as compared to varv; however, this has yet to be validated. , severe complications have been noted late in the course of mpxv infection, including pulmonary distress or bronchopneumonia, corneal scarring and permanent vision loss, and encephalitis. severe dehydration due to excessive vomiting or diarrhea may also occur. long-term sequelae in survivors are most commonly associated with pitted scarring. soluble immune mediators associated with mpxv infections. although mpxv infections in humans have been recorded for over four decades, there has been little information review regarding host immune responses during the course of natural infection. as disease presentation is highly similar during mpxv and varv infections, it has been postulated that immune responses would likely be highly conserved. recently, johnston et al. provided the first empirical evidence for a relationship between cytokine responses and disease severity during mpxv infection. serum cytokines were analyzed from patients with confirmed mpxv infections ranging from mild to severe as assessed by the who smallpox lesion scoring system. , serum concentrations of il- β, il- ra, il- r, il- , il- , il- , il- , il- , il- , il- , mcp- , and rantes were elevated in all disease groups (mild to severe) as compared to normal serum concentrations. il- concentrations were also elevated in all disease groups and were proportional to disease severity. however, patients with serious mpxv disease had significantly higher concentrations of il- compared to all other disease groups. mpxv infection resulted in elevated mip- α and mip- β; mild cases had significantly elevated levels above the moderate or severe disease groups. serum concentrations of il- r were elevated across all disease groups; however, patients with serious disease had significantly higher il- r serum levels than those with mild to severe mpxv disease. gm-csf levels were significantly elevated only in those with serious mpxv disease as compared to normal serum ranges. on the basis of these observations, mpxv infection resulted in prominent t helper (th ) and dampened th responses. transciptome analyses in mpxv infection. transcriptome analyses have largely been employed for the in vitro investigation of the molecular pathogenesis of mpxv infection. alkhalil et al. investigated the host transcriptome responses to mpxv infection during the first cycle of viral replication ( and h postinfection) in rhesus macaque kidney epithelial cells. interestingly, mpxv infection resulted in a strong downregulation of host transcriptional responses. of the transcripts that met the authors' criteria for significance, % of the transcripts were found to be down-regulated at both postinfection time points. comparative functional analysis from both time points suggested that the primary biological functions associated with these down-regulated transcriptional responses were largely related to cell morphology, cell development, metabolic responses, and post-translational modifications. canonical pathway analysis demonstrated a general conservation in the identities of over-represented pathways at both time points including multiple growth factor signaling pathways, p signaling, and cell cycle-related pathways. more recently, bourquain et al. investigated host transcriptome responses in mpxv-infected hela cells, a cervical epithelial cell line. at h post-mpxv infection, only . % of the transcripts analyzed were found to have > fold changes in gene expression. in contrast to alkhalil et al., the majority of these transcripts (∼ %) were found to be upregulated as compared to mock-infected controls. functional analysis of all transcripts with > -fold changes in gene expression demonstrated a strong over-representation of genes involved in the negative regulation of mapk signaling and the intracellular protein cascade. positive regulation of pathways related to toll-like receptor signaling, chemotaxis, and regulation of leukocyte migration was also predicted from the data. an investigation by rubins et al. compared the temporal host transcriptome response to mpxv in multiple human cells targeted by mpxv including primary macrophages, primary fibroblasts, and hela cells. the trasncriptome of mpxv-infected fibroblasts was found to have the most significant changes where mpxv infection resulted in the depletion of ∼ genes by a factor of ≥ . interestingly, mpxv infection resulted in the broad repression of many transcripts related to innate immune responses in all cell types tested. in contrast, inactivated mpxv resulted in strong upregulation of innate immune responses in all of the cell types. it was also noted that mpxv infection resulted in strong cytopathic effects across all of the cell types in contrast to an almost universal repression of innate immune responses. kinome analyses in mpxv infection. human mpxv infections and infection models of mpxv in various animal species have demonstrated that the congo basin mpxv clade is more virulent than the west african mpxv clade. however, there has been a paucity of information regarding the underlying molecular mechanisms mitigating these virulence differences. furthermore, previous investigations focusing on gene expression or proteomic changes during mpxv infection have focused solely on congo basin mpxv. to address this, host kinome analysis was performed on congo basin and west african mpxv-infected human monocytes, a host cell targeted by orthopoxviruses. as the genomes of both mpxv clades demonstrate considerable diversity in the regions coding host response modifier proteins, and in particular in genes associated with anti-apoptotic activities, it was postulated that the virulence differences of the two mpxv clades may be related to differential modulation of host cellular responses. hierarchical clustering of the kinome data sets suggested limited similarities at the level of host kinase modulation between the two mpxv clades. the congo basin mpxv kinome data set clustered most strongly with the kinome data set from cpxv-infected monocytes and moderately with the vacv-infected monocyte data set. both cpxv and vacv can cause serious disease in humans. the pathway ora of the kinome data demonstrated that congo basin mpxv infection resulted in strong down-regulation of a large proportion of host cell responses, most notably apoptosis, in comparison to west african mpxv. biological validation through fluorescenceactivated cell sorting (facs) and caspase activity analyses confirmed this phenomenon. from the perspective of individual phosphorylation events, the kinome data also suggested that akt phosphorylation at ser was increased in congo basin mpxv-infected cells as compared to west african mpxv-infected cells. pharmacologic inhibition of akt phosphorylation at ser resulted in a > -fold inhibition of congo basin mpxv virus yields, whereas those for west african mpxv were unaffected. prior investigations with cpxv and vacv demonstrated that pharmacological inhibition of akt resulted in decreased viral yields for both viruses. overall, this investigation provided significant insight into the host cellular response differences between the two mpxv clades. emerging and re-emerging pathogens are a continual threat to global health. in recent years, disease outbreaks associated with sars and the influenza pandemic have also demonstrated that these pathogens can have considerable effects on local, national, and international economies. as a consequence, regional outbreaks of emerging and re-emerging pathogens can have deleterious effects on global stability. thus, it is prudent that a concerted effort is employed to assimilate data that bridge both clinical and molecular information in investigations review of these pathogens. these efforts will not only provide considerable context in regard to the molecular events that potentiate clinical manifestations of pathogenesis but also better inform the design and implementation of novel therapeutics. to this end, global analyses of host molecular responses can provide considerable insight into the complex molecular events that underlie cellular responses. indeed, transcriptome analyses have provided important information regarding host transcriptional responses during emerging and re-emerging pathogen infection. these investigations often provide critical insight into the kinetics of host immune responses during the course of infection as well as mechanistic information regarding the cellular intermediates involved in these processes. however, the role of ptms in the regulation of these events cannot be captured by traditional transcriptome technologies. in particular, the role of kinase-mediated regulation of cell signaling pathways has remained poorly understood. given the central role of kinases in the regulation of cellular processes (e.g., homeostasis, metabolism, proliferation, and stress responses), it is of inherent importance that future investigations also address the role of the kinome in the cellular response to pathogen insult. furthermore, kinomics also provides a mechanism for the identification of novel therapeutic targets based on the direct assessment of the activation state of cell signaling pathways. for example, proinflammatory responses during early stages of infection, and in particular the dysregulation of specific cytokines or cell signaling events that contribute to these, may represent potential therapeutic targets in the early stages of highconsequence viral pathogen infection. however, the selection of immunomodulatory therapeutics that target these dysregulated host responses is complicated by the regulatory events (i.e., kinase-mediated cell signaling events) that occur upstream of changes in gene expression. in addition, mrna is subject to a variety of regulatory processes (including gene silencing, mrna stability, translational efficiencies, protein turnover, enzyme/substrate subcellular sequestration, and/or protein activation/repression ptms). thus, from the standpoint of therapeutic discovery, the sole reliance on technologies for the global investigations of host responses that do not account for these regulatory processes or the role of ptms in the modulation of cellular responses could impede the identification of efficacious therapeutics. to this end, kinome analysis may also facilitate the identification of immunomodulatory therapeutics that have gained licensure through analysis of a quantifiable biological event (kinase-mediated phosphorylation) or for identifying novel host therapeutic targets for which therapeutics could be designed/developed. furthermore, kinase inhibitors may serve as primary or adjunctive therapies for emerging infectious diseases. in addition, preclinical data and the increasing number of kinase inhibitors that have gained regulatory approval for cancer and other maladies suggest this approach is feasible and efficacious. from the perspective of this review, kinome investigations have identified several therapeutic targets and licensed kinase inhibitors that have impaired viral replication in vitro and reduced the severity of disease in vivo (table ) . for example, it has been demonstrated that the erk/mapk and pi k/akt/mtor signaling pathways have a role in viral propagation during mers-cov infection. indeed, licensed kinase inhibitors that targeted these pathways (i.e., everolimus, selumetinib, and trametinib) resulted in decreased viral replication in vitro when added prior to, or following, infection. furthermore, the pharmacologic inhibition of pi k and pkc following ebov infection provided partial protection in a lethal model of evd in mice. it should be noted that although the modulation of an individual kinase may have suppressive effects on infection (i.e., viral replication), this might not provide the level of inhibition required to completely negate viral escape. in addition, given the ability of many cell signaling pathways to signal through both canonical and noncanonical mechanisms, inhibition at a single intermediary point within a pathway may not provide the overall level of inhibition required to negate a deleterious response (i.e., viral replication, changes in cellular phenotypes, etc.). thus, although previous investigations have demonstrated that individual kinases or cell signaling pathways may represent novel targets for anti-infective therapies, it is prudent that future investigations also examine combinations of inhibitors for efficacy and anti-infective activities. furthermore, the targeting of cell signaling pathways at or near the origin point for the cell signaling cascade should also be examined as these likely represent stronger inhibitory targets given the generally reduced branching of cell signaling networks at or near the cell receptor. in addition to host-directed therapeutic targeting, kinomics also confers the ability to identify novel inhibitors of pathogens through detailed characterization of the viral life cycle. hostmediated ptms, and in particular kinase-mediated phosphorylation, have been implicated in the viral life cycle and pathogenesis for several members of the order mononegavirales, including ebov. − thus, therapeutic targeting of kinases may represent a novel therapeutic strategy that can be employed to modulate host-centric or pathogen-centric molecular events during infection. for example, in silico prediction of viral protein phosphorylation sites provides a mechanism for the construction and, ultimately, the annotation of viral protein ptms that are critical to the viral life cycle. furthermore, the use of kinome peptide arrays has extended beyond the human kinome and now extends to a variety of animal species. − it has been suggested that the interspecies phenotypic variability may reflect differences in phosphorylation sites found within the proteome. thus, the development of species-specific kinome peptide arrays provides additional utility for kinome analysis as peptide arrays representing traditional laboratory animal species (mouse, guinea pig, nonhuman primate) can be employed to detail the species-specific host response. the results from such analyses, and the overlap between these and those described previously from the analysis of human infections, may inform review the selection of appropriate animal models that meet regulatory approval through the fda animal efficacy rule. taken together, it is of inherent importance that future investigations of emerging and re-emerging pathogens address the complex nature of biological responses. thus, molecular investigations of pathogenesis 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syndrome coronavirus infection identification of novel cellular targets for therapeutic intervention against ebola virus infection by sirna screening inhibition of lassa virus and ebola virus infection in host cells treated with the kinase inhibitors genistein and tyrphostin productive replication of ebola virus is regulated by the c-abl tyrosine kinase pyridinyl imidazole inhibitors of p map kinase impair viral entry and reduce cytokine induction by zaire ebolavirus in human dendritic cells variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and src family tyrosine kinases the authors declare no competing financial interest. review key: cord- -g os m authors: martins-da-silva, andrea; telleria, erich loza; batista, michel; marchini, fabricio klerynton; traub-csekö, yara maria; tempone, antonio jorge title: identification of secreted proteins involved in nonspecific dsrna-mediated lutzomyia longipalpis ll cell antiviral response date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: g os m hematophagous insects transmit infectious diseases. sand flies are vectors of leishmaniasis, but can also transmit viruses. we have been studying immune responses of lutzomyia longipalpis, the main vector of visceral leishmaniasis in the americas. we identified a non-specific antiviral response in l. longipalpis ll embryonic cells when treated with non-specific double-stranded rnas (dsrnas). this response is reminiscent of interferon response in mammals. we are investigating putative effectors for this antiviral response. secreted molecules have been implicated in immune responses, including interferon-related responses. we conducted a mass spectrometry analysis of conditioned medium from ll cells and h after dsrna or mock treatment. we identified proteins. at h, proteins had an abundance equal or greater than -fold change, while the levels of proteins were reduced when compared to control cells. at the h time point, these numbers were and , respectively. the two most abundant secreted peptides at h in the dsrna-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (fkbp), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. the transcription profile of most candidates did not follow the pattern of secreted protein abundance. insect-borne diseases afflict billions of people throughout the world. these illnesses are caused by different pathogens such as helminths, protists, bacteria, and viruses, which are the etiologic agents of diseases such as filariasis, leishmaniasis, babesiosis and dengue. among the most important insect vectors are mosquitos and sand flies [ , ] . phlebotomine sandflies from the genera phlebotomus and lutzomyia are the main vectors of leishmania in the old and new world, respectively. in the old world, these dipterans are also proven virus vectors of phlebovirus, vesiculovirus and orbivirus [ ] [ ] [ ] . despite the lack of evidence proving the vectorial capacity of new world sandflies in the transmission of viruses to humans, the occurrence of diverse virus species naturally infecting lutzomyia spp. populations has been demonstrated [ ] [ ] [ ] [ ] . among insect transmitted diseases, the arboviruses are among the most devastating infectious illnesses in our planet. chikungunya virus (chikv), west nile virus (wnv) and dengue virus (denv) are three of a large number of human pathogenic arthropod-borne viruses [ ] . also, the world recently witnessed the reemergence of zika virus (zikv), which is associated with severe human neurological pathologies in adults and fetuses [ ] . the predominant strategy for controlling vector-borne diseases consists of targeting the vectors, mostly using insecticides. the deleterious side effects of insecticide usage are widely known. among these are consequences to ecosystems, animal and human health, and development of resistance, pointing to the urgency of developing new control strategies. the understanding of biological mechanisms involved in the relationship between pathogens and vectors can provide new approaches for the development of control strategies. the infection of aedes aegypti with wolbachia [ ] is an excellent example of a novel arbovirus control strategy. our group has been studying the interaction and immune responses of the sand fly lutzomyia longipalpis (diptera: psychodidae: phlebotominae), the most important vector of american visceral leishmaniasis [ ] , when infected with different pathogens [ ] [ ] [ ] . the establishment of continuous insect cell lines has been an important tool for the study of insect biology. cells from drosophila melanogaster (s ), aedes albopictus (c / ), and anopheles gambie (sua b), among other insect cell lines, have been employed to scrutinize diverse features of insect immunity [ ] [ ] [ ] . we have detected complex immune responses in the lutzomyia longipalpis ll embryonic cell line [ ] when challenged by different pathogens [ ] . we have also identified a non-specific antiviral response in these cells when transfected with non-specific double-stranded rna (dsrna) [ ] , in a way similar to interferon response in mammals [ ] . after dsrna transfection, regardless of nucleotide sequence, there was no replication of a west nile reporter virus that expresses luciferase and lacks structural genes, as indicated by a lack of luciferase production [ ] . we are presently searching for molecules responsible for this response. secreted proteins have a key role in biological processes involving cell signaling. naïve cells can be modulated by viral or host components transferred from neighboring infected cells via the release of extracellular vesicles and secretion of soluble molecules, leading to activation of host immune response and changes in the secreted protein repertoire [ ] [ ] [ ] . in order to investigate the effect of polyinosinic:polycytidylic acid (poly i:c) transfection on the protein secretion profile of l. longipalpis ll cells, we carried out mass spectrometry (ms) analyses comparing the protein composition of conditioned medium from poly (i:c) and mock-treated ll cells. l. longipalpis embryonic ll cells obtained from dr. robert b. tesh (university of texas medical branch, galveston, tx, usa) were grown at • c in l medium (sigma, mendota heights, mi, usa) supplemented with % fetal bovine serum (fbs; hyclone, logan, ut, usa), % tryptose phosphate broth, and % antibiotics (penicillin u/ml and streptomycin mg/ml, sigma). for mock transfection, ng/ml lipofectin transfection reagent (invitrogen, carlsbad, ca, usa) were added to l medium (leibovitz) containing % tryptose phosphate broth (tpb). the transfection mix contained the mock transfection mix added with ng/ml of polyinosinic:polycytidilic acid (poly i:c), a synthetic analog of double-stranded rna (invitrogen). these mixtures were added to × ll cells for h and supernatant was aspirated and stored. new medium was added and supernatant was aspirated and stored after h. for rna preparation, transfected and mock-transfected cells were collected after , , , and h incubations, as described above. cell viability was verified by trypan blue staining. more than % of cells were viable in all experiments. protease inhibitor cocktail × (sigma) was added to the media. dead cells and large debris were pelleted by centrifugation at × g for min. supernatants were centrifuged again at , × g for min to exclude remaining cell debris and microvesicles. the proteins in the supernatant were then precipitated using trichloroacetic acid (tca). one volume of tca % (sigma) was added to four volumes of conditioned medium and incubated for min at − • c. the samples were centrifuged at , × g for min. the supernatant was removed and the protein pellet was washed with cold acetone, vortexed and centrifuged at , × g for min. the material was submitted to mass spectrometry. the pellets were suspended in m urea, m thiourea, and mm -( -hydroxyethyl)- piperazineethanesulfonic acid (hepes). the proteins were reduced with mm dithiothreitol (dtt) and mm ammonium bicarbonate (abc), and alkylated with . mm iodacetamide and mm abc. before trypsinization, the samples were purified with detergent removal spin columns (thermo scientific). then, the samples were digested in mm abc with trypsin (promega, cat. v , madison, wi, usa) in a : trypsin to protein mass ratio by incubation at • c for h. after trypsinization, trifluoroacetic acid (tfa) was added to a final concentration of . %. peptides were desalted with homemade c spin columns. the peptides were analyzed in triplicate by liquid chromatography tandem-mass spectrometry (lc-ms/ms) in a thermo scientific easy-nlc system coupled to a ltq orbitrap xl etd (mass spectrometry facility rpt h/carlos chagas institute-fiocruz, curitiba, pr, brazil). peptide separation was carried out in -cm ( -µm inner diameter) fused silica, in-house packed with reversed-phase reprosil-pur c -aq -µm resin (dr. maisch gmbh, ammerbuch-entringen). chromatography runs were performed in a flow rate of nl/min from to % mecn in . % formic acid in a -min gradient, and a voltage of . kv was applied for peptide ionization. the mass spectrometer operated in a data-dependent acquisition mode. survey full-scan ms spectra (at a - m/z range) were acquired in the orbitrap analyzer with resolution of , at m/z (after accumulation to a target value of , in the c-trap). the ten most intense ions were sequentially isolated and fragmented in the linear ion trap using collision-induced dissociation at a target value of , . the "lock mass" option was enabled at . m/z in all full scans to improve mass accuracy of precursor ions [ ] . protein identification was performed with maxquant algorithm [ , ] version . . . . default parameters of the software were used for all analysis steps, unless stated otherwise. proteins were searched against an l. longipalpis protein sequence database (containing , protein sequences from the vectorbase protein database, downloaded on december ) and common contaminants, besides their respective reverse sequences to estimate the false discovery rate (fdr). carbamidomethylation of cysteine was set as a fixed modification, while methionine oxidation and n-terminal acetylation (protein) were allowed as variable modifications. in addition, an fdr threshold of . was applied at both peptide and protein levels. protein quantification was performed using a label-free approach, where the peptide peaks were detected as three-dimensional features-retention time versus signal intensity (extracted ion chromatogram, xic) versus mass/charge-and were aligned and compared across the runs, as previously described [ ] . the amino acid sequences of identified secreted proteins were subjected to bioinformatic analyses. the protein signal peptide (sp) from proteins was estimated using the software tool prediction of signal peptide "predsi" at http://www.predisi.de./home.html [ ] , under standard configuration. protein-protein interactions were performed using the search tool for the retrieval of interacting genes/proteins (string) database v . at http://string.embl.de/ [ ] . three biological replicates of × cells were transfected. the supernatant was removed , , , and h post-transfection and the cells, after being washed three times with pbs, were resuspended in ml of trizol (ambion, waltham, ma, usa). the rna was extracted following the trizol manufacturer's instructions. total rna was precipitated with isopropanol, resuspended in water and stored at − • c. rna was treated with dnase i (promega) and cdna was synthesized from µg of total rna using superscript iii first-strand synthesis (invitrogen). real-time polymerase chain reaction (qpcr) was performed using sybr green pcr master mix (applied biosystems, foster city, ca, usa) and the primers listed in table . expression levels were determined through (-delta delta c(t)) method (ddct) [ ] , normalized using rp gene expression [ ] , yielding the relative expression value. statistical analyses were done using graphpad prism software, version . (graphpad software, inc., san diego, ca, usa). for comparison of the transfected and mock samples at different times after transfection, an unpaired two-tailed student's t test was performed. * p ≤ . , ** p ≤ . , *** p ≤ . . table . list of primers used for qpcr. hemocytin basic transcription factor btf -f agtgcataagcaggcaacacca btf -r tacgtcgccaccgaaatgagtt juvenile hormone-inducible protein jhip-f ccttgctgagctccttgagaaact jhip-r tacacatggccattcccatcttcc eukaryotic translation initiation factor etif -f tcgataggcatctcacgtttccac etif -r tcttccccactgtatccaggatgt transmembrane protease serine -like repressor splicing factor rrm-f aatttgggaagctcaata rrm-r gaggatgtcgcaagccttct forskolin-binding protein proliferating cell nuclear antigen pcna-f catgaatctcgaccaggagcactt pcna-r tcacggcaaatgcgtgcaaa tubulin-specific chaperone a tbca-f cgtacgaaaaggaagcagatcagca tbca-r cttccttccggatcacgtgttcat ribosomal protein rp -f gaccgatatgccaagctaaagca rp -r ggggagcatgtggcgtgtctt comparative analysis of expression values was performed to verify the correlation between mrna levels of ll cells (samples collected , , and h post transfection) evaluated by qpcr, and secreted protein levels (samples collected or h post transfection) evaluated by mass spectrometry. linear regression, pearson's correlation coefficient (r), and goodness of fit (r ) were calculated using graphpad prism software (version . ). immune responses are triggered through the recognition of pathogen-associated molecular patterns (pamps) by host pattern recognition receptors (prrs). binding of pamps leads to the activation of signaling pathways: rnai, janus kinase/signal transducers and activators of transcription (jak/stat), immune deficiency (imd) and toll [ ] [ ] [ ] [ ] . the major insect innate immune response triggered by virus infection is normally the small interfering rna pathway. rnai is a molecular mechanism in which the presence of intracellular dsrna leads to the production of small rnas that suppress the expression of genes with matching sequences [ ] . nevertheless, alternative mechanisms exist. in previous work, our group identified an antiviral response when the ll embryonic l. longipalpis cell lineage was transfected with dsrna, which included the synthetic poly i:c. after dsrna transfection, cells did not support wnv virus like particle (vlp) replication, as evidenced by lack of luciferase reporter gene expression. this block in replication may be due to a cellular antiviral response triggered by dsrna. [ ] . a similar non-specific antiviral response was observed in the marine shrimps litopanaeus vannamei and penaeus monodon [ , ] . more recently, the occurrence of dsrna-mediated non-specific antiviral response was also described in two members of the apidae family: the honey bee apis melifera, and the bumblebee bombus terrestris [ ] [ ] [ ] . this non-specific response differs from what is seen in drosophila, where transfection with virus-specific dsrna triggers an rna interference (rnai)-mediated response against the virus, whereas non-related dsrnas fail to activate the antiviral response [ ] . in vertebrates, intracellular dsrna is recognized by the prr retinoic acid-inducible gene (rig-i) and the melanoma differentiation-associated protein (mda ). these are also known as rig-i-like receptors (rlrs) [ , ] . these receptors recruit the mitochondrial antiviral-signaling (mavs) protein that mobilizes kinases and ubiquitin ligases, leading to the activation of transcription factors, such as interferon receptor factor (irf- ) and nuclear factor κb (nf-κb) [ , ] . the transcription factors induce type i interferon (ifn) and pro-inflammatory cytokine production. dsrna, including poly i:c, is also detected by toll-like receptor , initiating the type-i interferon (ifn-α, β) signaling pathway via a toll/interleukin- receptor (tir)-domain-containing adapter-inducing interferon-β (trif) signal, which activates interferon receptor factor (irf- ) and nuclear factor κb (nf-κb), leading to ifn-β expression [ , ] . to investigate variations in the exoproteome profile of dsrna-transfected l. longipalpis ll cells, we carried out mass spectrometry (ms) analyses comparing the protein composition of conditioned medium from poly i:c and mock-treated cell cultures at two time points ( and h); in total proteins were identified (table s ) . only . % ( ) of all identified secreted proteins presented a signal peptide (sp). among proteins with ≥ -fold positive change variation, this percentage decreased to . %, with only six proteins with sp: at h and at h (table s ). the lack of signal peptide in the majority of secreted proteins suggests that unconventional protein secretion pathways are the main secretory mechanisms in ll cells, as already seen in other systems [ ] . this could be due at least partly to exosomal transport. we identified some of the known exosome markers among our secreted proteins, based on the exo carta exosome marker list (www.exocarta.org/exosome_markers). among these were glyceraldehyde- -phosphate dehydrogenase (gapdh), enolase (eno), fructose-bisphosphate aldolase (aldoa), moesin (msn), phosphoglycerate kinase (pgk), cluster of differentiation (cds) , and , the heat shock proteins (hsps) and , and annexins (table s ). these were not differentially expressed with a greater than -fold change and were present in both mock and poly i:c transfection samples. we performed an interaction analysis of the ll dsrna-transfected exoproteome. the network analysis of proteins presenting different abundance between mock and transfected groups (table s ) predicted that proteins (table s ) were connected by edges ( figure a) forming an intricate network. the prevised protein interaction (ppi) enrichment p-value was . , meaning that secreted proteins have more relations among themselves than what would be expected for a random set of proteins of similar size drawn from the genome. exoproteome functional enrichment analysis identified enhanced interactions related to specific biological functions. these interactions have a significant false discovery rate (fdr) of less than . . among these gene ontology (go) categories and pathways, we can quote: biological process go: (peptide metabolic process) fdr: we also carried out string interaction analysis ( figure b ) of proteins with ≥ -fold change variation detected on mass spectrometry analysis. from the protein sequences (table s ) shown in figure b ,c, were identified in the string database (table s ) and from those, (table s ) ( figure b) were predicted as connected at least to one protein. this interaction analysis displayed a significant ppi enrichment p-value of . × − . in this interactome we can distinguish two major protein groups: the first one with members is related to oxidative stress modulation and includes phospholipid scramblase (plscr ), thioredoxin (txn), glutaredoxin (glrx ), superoxide dismutase (sod ), and forskolin-binding protein (fkbp ), among others. the second group is comprised of proteins linked with peptide synthesis and cytoskeleton organization. among these are the riboproteins rps and rplp , the eukaryotic translation initiation factor (eif d), moesin (msn), cortactin protein (cttn), and others. two biological functions were enhanced in the ≥ -fold change secreted protein cluster: cellular component: go: (extracellular exosome) fdr: . × − and molecular function: go. (rna binding) fdr: . × − . these same biological function enrichments are found when we study the whole exoproteome. the complete fdr-filtered (p < . ) lists are shown in table s . the measurement of relative protein abundance revealed that the secretion of most proteins did not vary significantly between test and control supernatants (figure a ). only . % ( ) of all identified proteins at h presented variation of ≥ -fold change, and . % ( ) presented variation of ≤ -fold change ( figure b ). these percentages change to . % ( ) and . % ( ) , respectively, at h after dsrna transfection ( figure c ). we focused on the proteins with ≥ -fold change variation, which represent . % of the total identified proteins ( figure c ). the proteins with intensified secretion in the first h had their relative amounts decreased at h. most proteins more abundant at h had a basal detection level at h. interestingly only one protein, the enzyme carboxylesterase, presented secretion levels with ≥ -fold change at both time points. in insects this enzyme is related to detoxifying processes, including insecticide resistance [ ] . several proteins with higher secretion at h or h are related to immune responses. among the proteins with higher secretion at h we can highlight eight proteins related to immune response. phospholipid scramblase (plscr ) had a positive variation with a greater than -fold change. scramblases are conserved in all eukaryotic organisms. they are multiple palmitoylated, endofacial membrane proteins originally identified based on their capacity to promote accelerated transbilayer phospholipid movement in response to ca + [ ] . plscr was identified as an interferon and growth factor-inducible protein necessary for the ifn-dependent induction of gene expression and antiviral activity [ ] . in human and mouse plasmacytoid dendritic cells (pdcs), which are professional interferon-producing cells specialized in recognizing viral rna and dna through the endosomal toll-like receptors (tlrs) tlr and tlr , respectively, plscr was described as a tlr -binding protein that plays a significant role in type- interferon responses in pdcs by regulating tlr expression and trafficking [ ] . the direct interaction of plscr with human t-cell leukemia virus type- (htlv- ) leads to a reduction of virus titers [ ] . these reports demonstrate the significant, but not fully understood, role of phospholipid scramblase in antiviral response. another interesting protein with a greater than -fold increased secretion at h was the hexameric chaperone complex prefoldin (pfd). subunits of prefoldin have been found to be associated with cellular response to viral infection. the pfdn expression was positively modulated in both vaccinia virus-infected hek cells and in the rabv-infected n a cells [ , ] . pfdn was found directly bound to the hepatitis c virus (hcv) f protein [ ] . the string interactome maps were generated using default settings (medium confidence of . , with criteria for linkage: neighborhood, genefusion, co-occurrence, co-expression, experimental evidence, existing databases, and textmining). proteins were represented as nodes and their functional links were defined by solid lines. the thickness of the lines signifies the level of confidence of the reported association. the non-connected nodes were suppressed. no additional interplay proteins were added to the networks. the protein symbols and node colors are listed in table s . still within the proteins with higher secretion at h, we found forskolin-binding protein (fkbp ), an immunophilin family member, with a secretion level increase of greater than -fold. this is a highly conserved group of proteins which binds to immunosuppressive drugs such as fk , rapamycin, and cyclosporine [ , ] . fkbps are involved in several biochemical processes and participate in the immune response against viral infection. mitochondrial fkbp was identified as a tumor necrosis factor (tnf) receptor-associated factor (traf ) and tnf receptor-associated factor (traf ) binding protein. binding of fkbp to traf proteins facilitates the type-i interferon response induced by dsrna transfection or newcastle disease virus (ndv) infection in murine fibroblasts. depletion of fkbp reduced the expression of type inf in these cells [ ] . one interesting protein with a greater than -fold enhanced secretion at h is coatomer protein i (copi). copi-coated vesicles are associated with transport from the golgi to the endoplasmic reticulum [ ] , endocytosis [ ] , maturation of autophagic vacuoles [ ] , expression of cell surface receptors, and modulation of the plasma membrane lipid composition [ ] . studies demonstrated the involvement of copi subunits in diverse aspects of virus infection [ ] [ ] [ ] . depletion of the copi β subunit in human /ace cells led to a decrease in the severe acute respiratory syndromecoronavirus (sars-cov) replication [ ] . the string interactome maps were generated using default settings (medium confidence of . , with criteria for linkage: neighborhood, genefusion, co-occurrence, co-expression, experimental evidence, existing databases, and textmining). proteins were represented as nodes and their functional links were defined by solid lines. the thickness of the lines signifies the level of confidence of the reported association. the non-connected nodes were suppressed. no additional interplay proteins were added to the networks. the protein symbols and node colors are listed in table s . still within the proteins with higher secretion at h, we found forskolin-binding protein (fkbp ), an immunophilin family member, with a secretion level increase of greater than -fold. this is a highly conserved group of proteins which binds to immunosuppressive drugs such as fk , rapamycin, and cyclosporine [ , ] . fkbps are involved in several biochemical processes and participate in the immune response against viral infection. mitochondrial fkbp was identified as a tumor necrosis factor (tnf) receptor-associated factor (traf ) and tnf receptor-associated factor (traf ) binding protein. binding of fkbp to traf proteins facilitates the type-i interferon response induced by dsrna transfection or newcastle disease virus (ndv) infection in murine fibroblasts. depletion of fkbp reduced the expression of type inf in these cells [ ] . one interesting protein with a greater than -fold enhanced secretion at h is coatomer protein i (copi). copi-coated vesicles are associated with transport from the golgi to the endoplasmic reticulum [ ] , endocytosis [ ] , maturation of autophagic vacuoles [ ] , expression of cell surface receptors, and modulation of the plasma membrane lipid composition [ ] . studies demonstrated the involvement of copi subunits in diverse aspects of virus infection [ ] [ ] [ ] . depletion of the copi β subunit in human /ace cells led to a decrease in the severe acute respiratory syndrome-coronavirus (sars-cov) replication [ ] . we identified two juvenile hormone-inducible proteins at h with . -and . -fold increases. despite the absence of information on the possible involvement of juvenile hormone-inducible proteins in the insect antiviral response, the juvenile hormone itself and retinoic acid are terpenoids, with similar structure and biological effects in mammalian and insect cells [ ] . retinoic acid-inducible genes are related to type-i ifn response in mammals [ , ] . viruses , , of we identified two juvenile hormone-inducible proteins at h with . -and . -fold increases. despite the absence of information on the possible involvement of juvenile hormone-inducible proteins in the insect antiviral response, the juvenile hormone itself and retinoic acid are terpenoids, with similar structure and biological effects in mammalian and insect cells [ ] . retinoic acidinducible genes are related to type-i ifn response in mammals [ , ] . another virus infection-related protein found was kinesin, an atpase protein belonging to the class of motor proteins. kinesins move along microtubule (mt) filaments, and are power-driven by the hydrolysis of adenosine triphosphate (atp) [ ] . recently this protein has been associated with the intracellular transport of different viruses. hiv- trafficking to the nucleus is kinesin-and dynein motor-dependent [ ] . kinesin was also identified as a chandipura virus matrix binding protein [ ] . kinesin from lepidopteran trichoplusia ni binds directly to nucleocapsid proteins of the autographa californica multiple nucleopolyhedrovirus (acmnpv) [ ] , and the α herpersviral envelope protein pus uses kinesin to promote the anterograde axonal transport of herpes simplex virus (hsv- ) [ ] . these results demonstrate that kinesin protein interacts directly with the virus particle. the insect protein canopy is a homolog of the human protein associated with toll-like receptor (tlr) (prat ). prat is required for cell surface expression of the toll family of receptors and for the intracellular nucleic acid-sensing tlr / . this protein is essential for tlr-dependent immune response [ ] . the . -fold increased secretion of this prat homolog signals the participation of toll-like receptors in the ll antiviral non-specific immune response. the barrier to autointegration factor (baf) dna binding protein had an increase of . -fold at h. this protein is described as a potent antiviral effector against poxyviruses, retroviruses and the herpes virus. the baf antiviral property is regulated by kinases and phosphatases, with dephosphorylation activating its antiviral properties [ ] . notwithstanding the higher number of positively modulated secreted proteins at h when compared with h, the number of proteins described in immune response to viruses in interferon-like responses was smaller than what was seen at h. among them we quote the protein hemocytin, with a secretion increase of . -fold. hemocytin is an insect humoral lectin homologous to mammalian von willebrand factor, which is involved in platelet adhesion to subendothelium [ ] . hemocytin expression is induced by pathogens. lectins are adhesive molecules which bind carbohydrates and among other functions are involved in hemolymph bacterial clearance [ ] . also of interest is the occurrence of glutaredoxin (fold change of . ), thioredoxin (fold change of . ) and superoxide dismutase (fold change of . ), antioxidant enzymes involved in the control of oxidative stress at h. reactive oxygen species (ros) production, generated due to microbial invasion, has long been known to exert an antimicrobial effect. the activation of the antiviral and inflammatory signaling pathways has also been linked with the production of ros [ , ] . due to the high chemical reactivity of ros, cells possess scavenger antioxidant mechanisms that maintain redox homeostasis [ ] . real time pcr (qpcr) analysis of genes coding for significantly positive-or negatively-modulated secreted proteins revealed the existence of some genes with transcriptional profile correlated with the amount of protein detected in the supernatants ( figure a ). there were seven molecules that presented a correlation between mrna and secreted protein levels (represented in figure a ). three of them, hemocytin (hmc), lecithin cholesterol acyltransferase (lcat), and scramblase (scr), showed coordinated mrna and secreted protein levels. when mrna levels changed at or h post transfection (either by up-or down-regulation), we detected a corresponding change in secreted protein levels or h after the mrna level changes. hemocytin (hmc), an insect humoral lectin which plays an important role in a non-specific self-defense mechanism [ ] , had increased mrna and secreted protein levels at h and h. this indicates that the transcription, translation and secretion of this molecule are correlated and the prolonged increase of its expression suggests this molecule may be important for the ll cells response to poly i:c transfection. two other molecules, lecithin cholesterol acyltransferase (lcat) (a plasma enzyme which esterifies cholesterol) [ ] , and scramblase (scr) (a regulator of transbilayer lipid asymmetry in eukaryotic cell membranes) [ ] , were reduced both in mrna and secreted protein levels at or h post-transfection. these two molecules are involved in lipid metabolism or membrane formation and the reduction of their expression may suggest that ll cells reduce vesicle formation h after transfection challenge. three other molecules showed increased mrna levels at or h post transfection and increased secreted protein levels at h, with posterior decrease at h. one was the eukaryotic translation initiation factor (etif ), which in humans is directly recruited by the hepatitis c virus (hcv) internal ribosome entry site (ires) to promote the translation of viral proteins [ ] . in ll cells it showed increased mrna levels at h and h, and increased secreted protein levels h after mrna increase. curiously, h post transfection the secreted protein levels decreased drastically. juvenile hormone-inducible protein (jhip) showed increased mrna levels after h and increased secreted protein levels h after mrna increase. curiously, at h post transfection the secreted protein levels decreased drastically regardless of changes in mrna levels prior to this timepoint. basal transcription factor (btf ) has been reported to play a significant role in the transcriptional regulation related to eukaryote growth and development [ ] . in ll cells it showed increased mrna levels at h and increased secreted protein level h after mrna increase. at h post transfection the secreted protein levels decreased drastically regardless of changes in mrna levels prior to this time point. transketolase mrna levels increased at h post transfection and the secreted protein levels also increased at this same time point, suggesting a late effect. curiously, regardless of the maintenance of mrna levels from h to h, the secreted protein levels were considerably low at h post transfection. changes in transketolase activity are involved in a number of tumors and degenerative diseases [ ] . based on the data presented in figure a , we observed that in the majority of the molecules (six out of seven) there was a correlated change of secreted protein levels h to h after changes in mrna levels. the remaining genes investigated showed no correlation between the mrna levels and the detection of proteins in the supernatants. ( figure b ). the linear regression of mrna levels plotted against secreted protein levels revealed a weak correlation (figure ). the pearson correlation coefficient (r) and goodness of fit (r ) calculated for mrna levels ( h, h, h and h) versus secreted protein ( h and h) are shown in figure and table . the non-correlation between transcription levels and amounts of secreted protein indicates that secretion is regulated by some not-yet disclosed mechanism, involving transcription, translation, storage, sorting, and transport, as already reported for other systems [ ] . this non-correlation was also observed when the exoproteome of wrl- human hepatic cells infected with the chikungunya virus was analyzed [ ] . we observed that the majority of analyzed genes were transcribed in the first h ( figure a,b) . the results obtained with the signal peptide prediction, the secreted protein interaction network, the identified biological functions enriched pathways, and the relatively small number of proteins with significant abundance variation reveal the existence of finely-tuned protein secretion modulation in response to dsrna treatment. there is also a strong indication for the participation of the exosomal secretory pathway as the major ll cell secretion mechanism in this response. the increased secretion of proteins such as phospholipid scramblase, fkbp, juvenile hormone-inducible proteins, and protein canopy in the first h indicate that this non-specific antiviral response is in a way similar to an interferon-like response mediated by the toll pathway. this response occurs preferentially in the first hours after transfection. the increase of antioxidant enzymes at h indicates that the cells are working to control the oxidative stress level in the culture. we are presently in the process of validating these findings by silencing specific genes with subsequent analysis of nonspecific antiviral response. (table ) and samples collected at different time points posttransfection. quantification was normalized relative to the house keeping gene rp , and relative gene expression expressed as fold change was calculated relative to the mock group. bars represent mean with standard error (sem) of two biological replicates. tables represent protein fold change corresponding to each gene name. * p ≤ . , ** p ≤ . , *** p ≤ . . (table ) and samples collected at different time points post-transfection. quantification was normalized relative to the house keeping gene rp , and relative gene expression expressed as fold change was calculated relative to the mock group. bars represent mean with standard error (sem) of two biological replicates. tables represent protein fold change corresponding to each gene name. * p ≤ . , ** p ≤ . , *** p ≤ . . biology of phlebotomine sand flies as vectors of disease agents a new arbovirus isolated in india from patients with febrile illness isolation of chandipura virus from sandflies in aurangabad the genus phlebovirus and its vectors carajas and maraba viruses, two new v esiculoviruses isolated from phlebotomine sand flies in brazil viremia and immune response with sequential phlebovirus infections ecuador paraiso escondido virus, a new flavivirus isolated from new world sand flies in ecuador, is the first representative of a novel clade in the genus flavivirus factors 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transmission pathways in t lymphocytes are inhibited by complexes formed between an immunophilin and either fk or rapamycin calcineurin is a common target of cyclophilin-cyclosporin a and fkbp-fk complexes mitochondria-nucleus shuttling fk -binding protein interacts with traf proteins and facilitates the rig-i-like receptor-mediated expression of type i ifn structure of coatomer cage proteins and the relationship among copi, copii, and clathrin vesicle coats involvement of specific copi subunits in protein sorting from the late endosome to the vacuole in yeast early endosomes and endosomal coatomer are required for rnai screen of salmonella invasion shows role of copi in membrane targeting of cholesterol and cdc copi activity coupled with fatty acid biosynthesis is required for viral replication a role for the host coatomer and kdel receptor in early vaccinia biogenesis. proc. natl. acad. sci human host factors required for influenza virus replication a kinome-wide small interfering rna screen identifies proviral and antiviral host factors in severe acute respiratory syndrome coronavirus replication, including double-stranded rna-activated protein kinase and early secretory pathway proteins activation of mammalian retinoid x receptors by the insect growth regulator methoprene regulation of antiviral innate immune signaling by stress-induced rna granules myosin v motor proteins: marching stepwise towards a mechanism hiv- capsids bind and exploit the kinesin- adaptor fez for inward movement to the nucleus host interactions of chandipura virus matrix protein trichoplusia ni kinesin- associates with autographa californica multiple nucleopolyhedrovirus nucleocapsid proteins and is required for production of budded virus the basic domain of herpes simplex virus pus recruits kinesin- to facilitate egress from neurons a protein associated with toll-like receptor (tlr) (prat a) is required for tlr-dependent immune responses the barrier to autointegration factor: interlocking antiviral defense with genome maintenance cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von willebrand factor immune proteins and their gene expression in the silkworm, bombyx mori hsv infection induces production of ros, which potentiate signaling from pattern recognition receptors: role for s-glutathionylation of traf and reactive oxygen species activate nf-κb (p ) and p and induce apoptosis in rvfv infected liver cells the nrf cell defence pathway: keap -dependent and -independent mechanisms of regulation establishment and application of a high throughput screening system targeting the interaction between hcv internal ribosome entry site and human eukaryotic translation initiation factor exploring the roles of basal transcription factor in eukaryotic growth and development structure and functioning mechanism of transketolase control of cystic fibrosis transmembrane conductance regulator membrane trafficking: not just from the endoplasmic reticulum to the golgi differential analysis of the secretome of wrl cells infected with the chikungunya virus the authors declare no conflict of interest. key: cord- -hhcfrjrn authors: hall, charlotte a.; chilcott, robert p. title: eyeing up the future of the pupillary light reflex in neurodiagnostics date: - - journal: diagnostics (basel) doi: . /diagnostics sha: doc_id: cord_uid: hhcfrjrn the pupillary light reflex (plr) describes the constriction and subsequent dilation of the pupil in response to light as a result of the antagonistic actions of the iris sphincter and dilator muscles. since these muscles are innervated by the parasympathetic and sympathetic nervous systems, respectively, different parameters of the plr can be used as indicators for either sympathetic or parasympathetic modulation. thus, the plr provides an important metric of autonomic nervous system function that has been exploited for a wide range of clinical applications. measurement of the plr using dynamic pupillometry is now an established quantitative, non-invasive tool in assessment of traumatic head injuries. this review examines the more recent application of dynamic pupillometry as a diagnostic tool for a wide range of clinical conditions, varying from neurodegenerative disease to exposure to toxic chemicals, as well as its potential in the non-invasive diagnosis of infectious disease. the origin of the phrase "the eyes are the window to the soul" is attributed to the roman consul cicero, but during the past three decades the ability of the eye to act as a window into nervous system function has been exploited for a wide range of clinical applications, including mental health and neurodegenerative disorders, as well as exposure to toxic or illicit substances and trauma. the pupillary light reflex (plr) describes the constriction and subsequent dilation of the pupil in response to light, which not only serves as a major determination of retinal image quality [ , ] , but also provides an important metric of autonomic nervous system function [ ] . as such, measurement of the pupil's response to light serves as a non-invasive tool for basic neuroscience research and the study of parasympathetic and sympathetic balance. the pupil has a large dynamic range, typically from . - mm diameter at full mydriasis to . - mm diameter at full miosis, and is controlled by the antagonistic actions of the iris sphincter and dilator muscles [ ] . the sphincter and dilator are innervated by the parasympathetic and sympathetic nervous systems, respectively; thus, different parameters of the plr can be used as indicators for either sympathetic or parasympathetic modulation. factors which affect average pupil diameter include age, sex, iris colour, retinal and optic nerve health and optical media clarity [ ] ; however, the most powerful determinant of pupil size is ambient light level. figure . schematic of the pupillogram (blue line) and associated plr parameters. the light stimulus at time zero results in a rapid reduction in pupil diameter. latency (tl) is calculated as the elapsed time between light onset and the start of constriction. the pupil then rapidly constricts (maximal constriction velocity; mcv) from the baseline (d ) pupil diameter to the minimum (dmin) pupil diameter; the constriction time (tc) and maximum constriction amplitude (mca) are calculated as the time interval and size difference between these two values, respectively. at offset of light stimulus or during sustained light stimulation the pupil undergoes a period of rapid redilation or pupillary "escape" to a partially constricted state. subsequently the pupil slowly returns to the baseline diameter. response latency describes the delay in pupil constriction following the onset of a light stimulus, with the latency shortening as light intensity increases, to a minimum of - ms [ , ] . the latency period is due to the delay in iris smooth muscle contraction and to a lesser extent the temporal dynamics of retinal output and innervation pathways [ , ] . the latency period is followed by a period of rapid constriction of the pupil until it reaches the maximum constriction velocity (mcv), after which constriction slows until the minimum pupil diameter is reached. the onset of pupil contraction can be determined using velocity and acceleration analysis [ ] . the maximum constriction velocity varies with light stimulus intensity, duration, spectral composition, retinal size and location [ ] . the maximum constriction amplitude (mca) represents the difference between the baseline and minimum pupil diameter. however, the baseline pupil diameter can be affected by a number of factors and can influence the mca (a smaller mca is observed with a smaller baseline pupil diameter). therefore, the mca should be normalised to baseline pupil diameter to account for this effect [ ] . after this peak constriction, the pupil quickly redilates or "escapes" to a partially constricted state during a prolonged light stimulus lasting from - up to s, before slowly redilating to the initial size [ ] . in addition to the dynamic phases of the plr during light stimulation, there is also a sustained component [ , ] . both outer and inner photoreceptors contribute to the early sustained post- figure . schematic of the pupillogram (blue line) and associated plr parameters. the light stimulus at time zero results in a rapid reduction in pupil diameter. latency (t l ) is calculated as the elapsed time between light onset and the start of constriction. the pupil then rapidly constricts (maximal constriction velocity; mcv) from the baseline (d ) pupil diameter to the minimum (d min ) pupil diameter; the constriction time (t c ) and maximum constriction amplitude (mca) are calculated as the time interval and size difference between these two values, respectively. at offset of light stimulus or during sustained light stimulation the pupil undergoes a period of rapid redilation or pupillary "escape" to a partially constricted state. subsequently the pupil slowly returns to the baseline diameter. response latency describes the delay in pupil constriction following the onset of a light stimulus, with the latency shortening as light intensity increases, to a minimum of - ms [ , ] . the latency period is due to the delay in iris smooth muscle contraction and to a lesser extent the temporal dynamics of retinal output and innervation pathways [ , ] . the latency period is followed by a period of rapid constriction of the pupil until it reaches the maximum constriction velocity (mcv), after which constriction slows until the minimum pupil diameter is reached. the onset of pupil contraction can be determined using velocity and acceleration analysis [ ] . the maximum constriction velocity varies with light stimulus intensity, duration, spectral composition, retinal size and location [ ] . the maximum constriction amplitude (mca) represents the difference between the baseline and minimum pupil diameter. however, the baseline pupil diameter can be affected by a number of factors and can influence the mca (a smaller mca is observed with a smaller baseline pupil diameter). therefore, the mca should be normalised to baseline pupil diameter to account for this effect [ ] . after this peak constriction, the pupil quickly redilates or "escapes" to a partially constricted state during a prolonged light stimulus lasting from - up to s, before slowly redilating to the initial size [ ] . in addition to the dynamic phases of the plr during light stimulation, there is also a sustained component [ , ] . both outer and inner photoreceptors contribute to the early sustained post-illumination pupil response (pipr; < . s post-stimulus) [ ] . subsequently, the pipr, which may be sustained for up to minutes after light offset depending on the properties of the light stimulus, is solely controlled by the intrinsically photosensitive retinal ganglion cells that depolarise during light stimulation (dependent on both light intensity and wavelength) and then repolarise slowly after light offset [ , [ ] [ ] [ ] . the response of the pupil to light can be manually assessed using the swinging flashlight test or using a pupillometer device. pupillometers enable objective quantification of the plr and generally consist of an infrared-sensitive imaging sensor coupled with a digital interface for the automated recording, processing and reporting of pupil data. the acquired images are processed to yield a pupillogram with pupil size plotted as a function of time, from which plr parameters can be calculated ( figure ). there are several commercially available pupillometers and the relative performance of commercial systems is the subject of several publications [ ] [ ] [ ] . however, commercial systems are typically designed for specific applications and use proprietary software, which may limit their use for basic research. in response, a number of research groups have published methods for developing prototype automated infrared pupillometers, which use open-source software [ , ] . there is also increasing use of remote eye-tracking devices, such as tobii, that are capable of measuring pupil size and can be used in combination with open-source software for the analysis of plr [ ] . there are three major divisions of parasympathetic neurons that integrate the light stimulus to produce a pupil contraction: (i) an afferent division; (ii) an interneuron division; and (iii) an efferent division. the response latency, maximum constriction and pupil escape, and the corresponding constriction parameters (mcv, mca and rca; relative constriction amplitude) are dependent on the actions of the sphincter muscle and on the function of retinal photoreceptors, as well as the time consumed in the afferent and efferent pathway. they are thus under direct control of the parasympathetic nervous system. both mcv and rca parameters are dependent on the baseline pupil diameter in healthy individuals and it is important to normalise measurements with respect to baseline values [ , ] . there is also a strong linear relationship between mcv and mca [ ] ; thus, mcv and rca are considered to be the most robust parameters for detecting parasympathetic dysfunction [ ] . the afferent limb of the plr begins with photoreceptive inputs from rod, cone and intrinsically photosensitive retinal ganglion cells (iprgcs) located in the retina (summarised in figure ). under dark conditions, rod and cone photoreceptors exist in a constant depolarised state and continuously release glutamate [ , ] . however, when stimulated by light, the rod and cone photoreceptors undergo graded alterations in membrane potential and hyperpolarize, resulting in a reduction in glutamate release. rod and cone cells synapse with their respective bipolar cells and reductions in glutamate release results in either inhibition or disinhibition of the bipolar cells depending on whether they express excitatory ionotropic ("on") or inhibitory metabotropic ("off") glutamate receptors [ ] . these "on" or "off" bipolar cell subtypes synapse with the corresponding retinal ganglion cell (rgc) in the non-cholinergic regions of the inner plexiform layer (ipl). horizontal neurotransmission also occurs within the ipl and outer plexiform layer (opl) via amacrine and horizontal cells, respectively, and is critical in shaping the spatial and temporal aspects of photopic and scotopic vision [ ] . the third photoreceptor, iprgc, accounts for approximately % of the total rgcs [ ] [ ] [ ] . these cells have a role in non-image-forming visual processes, such as circadian photoentrainment, as well as in the plr [ ] [ ] [ ] . the iprgcs regulate pupil size through the integration of extrinsic signals from rods and cones but also through intrinsic (melanopsin) phototransduction [ ] . melanopsin is a g protein-coupled photopigment, which is maximally sensitive to nm wavelength light and, unlike rods and cones, depolarizes in response to light following activation of a phototransduction cascade involving gq/ and phospholipase c [ - ]. unlike rods and cones, which have their photopigment concentrated in specialised light-absorbing cellular domains (outer segment), melanopsin is distributed throughout the plasma membrane of iprgcs [ ] . the iprgcs also directly contribute to the pipr as a sustained constriction of the plr (> s) in response to high intensity, short wavelength light [ , , , [ ] [ ] [ ] . protein-coupled photopigment, which is maximally sensitive to nm wavelength light and, unlike rods and cones, depolarizes in response to light following activation of a phototransduction cascade involving gq/ and phospholipase c [ - ]. unlike rods and cones, which have their photopigment concentrated in specialised light-absorbing cellular domains (outer segment), melanopsin is distributed throughout the plasma membrane of iprgcs [ ] . the iprgcs also directly contribute to the pipr as a sustained constriction of the plr (> s) in response to high intensity, short wavelength light [ , , , [ ] [ ] [ ] . the axon terminals of the bipolar cells stratify at different depths of the inner plexiform layer, which is subdivided into the off outer sublamina (where off bipolar cells terminate) and the on inner sublamina (where on bipolar cells terminate). there are also on and off bands of melanopsin dendrites from the iprgcs, but both lie outside of the on and off cholinergic bands within the ipl. the bipolar cells are photoreceptor specific and the bipolar dendrites synapse exclusively with either rod or cone cells. the interneuron and efferent arms of pupil constriction are summarised in figure . the rgc axons form the first interneuron arm of the plr arc and carry the neuronal signal from the photoreceptors [ ] . the rod and cone cells are located in the outer nuclear layer (onl), which is adjacent to the retinal pigment epithelium (rpe). the horizontal cell, bipolar cell and amacrine cell somas are located in the inner nuclear layer (inl), whilst the ganglion cell somata are located in the ganglion cell layer (gcl). the axon terminals of the bipolar cells stratify at different depths of the inner plexiform layer, which is subdivided into the off outer sublamina (where off bipolar cells terminate) and the on inner sublamina (where on bipolar cells terminate). there are also on and off bands of melanopsin dendrites from the iprgcs, but both lie outside of the on and off cholinergic bands within the ipl. the bipolar cells are photoreceptor specific and the bipolar dendrites synapse exclusively with either rod or cone cells. the interneuron and efferent arms of pupil constriction are summarised in figure . the rgc axons form the first interneuron arm of the plr arc and carry the neuronal signal from the photoreceptors [ ] . . the parasympathetic nervous system is the main system responsible for pupil constriction in response to light. the integrated afferent input is transmitted along the axons of the retinal ganglion cells (rgc), which contribute to the optic nerve. at the optic chiasm, nerves from the nasal retina cross to the contralateral side, whilst nerves from the temporal retina continue ipsilaterally. the pupillary rgc axons exit the optic tract and synapse at the pretectal olivary nucleus. pretectal neurons are projected either ipsilaterally or contralaterally, across the posterior commissure, to the edinger-westphal nucleus. from there, the pre-ganglionic parasympathetic fibres travel with the oculomotor, or iii cranial nerve, and synapse at the ciliary ganglion. the post-ganglionic parasympathetic neurons (short ciliary nerves) travel to and innervate the contraction of the iris sphincter muscle via the release of acetylcholine at the neuromuscular junction, resulting in pupil constriction. at the optic chiasma, approximately half the rgcs from the nasal plane in each eye decussate to the opposite optic tract [ ] . at the terminus of the optic tract, the axons of rgcs responsible for the plr separate from the visual axons and carry the afferent pupillomotor signal through the brachium of the superior colliculus to synapse at the pretectal olivary nucleus in the dorsal midbrain [ ] . the pretectal neurons integrate the input signals (retinal, supranuclear and infranuclear), that modulate the plr and form the second interneuron of the reflex arc. these pretectal nuclei project to either the ipsilateral or contralateral edinger-westphal (ew) nucleus within the oculomotor nuclear complex, which contain the pre-ganglionic parasympathetic neurons that control the iris sphincter [ ] . the bilateral neuron projection results in a double decussation of pupillary fibres, first at the optic chiasm and then within the pretectal area, and ensures each ew nucleus receives information about the level of incoming light from each eye. therefore, unilateral light stimulation causes an equal direct and consensual pupillary constriction. however, contraction anisocoria, whereby the direct pupillary constriction is slightly stronger than the consensual reaction, may present if asymmetry occurs during crossing of fibres at the chiasm or pretectal olivary nucleus; this is normally clinically insignificant [ ] . from the ew nuclei, the efferent pre-ganglionic axons pass into the right and left fascicles of the oculomotor nerve (third nerve) to join the motor axons destined for the eye muscles ( figure ). the oculomotor nerve bifurcates into a superior and inferior division near the anterior cavernous sinus. the parasympathetic fibres travel with the inferior division through the superior orbital fissure toward the orbital apex and synapse at the ciliary ganglion (cg). the short ciliary (post-ganglionic) nerves pierce the globe around the optic nerve and pass between the choroid and sclera toward the iris. these nerves innervate the contraction of the iris sphincter muscle via the neurotransmitter acetylcholine (ach), resulting in constriction of the pupil. dilation of the pupil following a light stimulus occurs through two integrated processes driven by the sympathetic neurons and corresponds to the recovery phase of the plr and is summarised in figure . firstly, the parasympathetic innervation of the pupil sphincter is suppressed by supranuclear inhibition via central sympathetic neurons, resulting in relaxation of the muscle and pupil dilation. these sympathetic neurons primarily originate in the reticular activating formation in the brainstem and inhibit the pre-ganglionic parasympathetic neurons at the ew nucleus via α -adrenergic receptor activation. secondly, the iris dilator muscle contracts via excitation of the α -adrenergic sympathetic pathway. this peripheral sympathetic nerve activation greatly enhances the dynamics of pupil dilation in terms of speed and maximal pupil diameter attained. the sympathetic influence on the iris dilator muscle consists of a paired, three-neuron arc on both the right and left side of the central and peripheral nervous system, without decussations, extending from the hypothalamus to the iris dilator muscle (figure ) [ - ]. in this three-neuron arc, the synaptic transmission is mediated by ach at the first two junctions, while the post-ganglionic fibres innervate the dilator muscle via noradrenaline. nerves within the ophthalmic division of the trigeminal nerve provide sensory innervation to the iris and may play an additional role in modulating pupil diameter [ ] . mechanical and chemical irritation of the eye can cause a strong miotic response that is non-cholinergic and fails to reverse with autonomic-acting drugs. in addition to the neuronal mechanisms involved in pupil size control, circulating catecholamines and peptide hormones may act on the iris dilator or sphincter muscles, either directly through the bloodstream or potentially indirectly through the tears [ , ]. conditions that influence the integration of parasympathetic stimulation and inhibition, sympathetic stimulation and humoural release of neurotransmitters may each affect the dynamics of the plr and may be clinically diagnostic. pupil [ ] [ ] [ ] [ ] diseases, autism spectrum disorders [ , ] , as well as glaucoma [ , [ ] [ ] [ ] and autonomic neuropathies associated with diabetes [ ] [ ] [ ] [ ] [ ] . additionally, pupillometry has been applied to other clinical fields, such as monitoring of central states in anaesthesiology and analgesia, as well as monitoring and prognosis following head injuries, cardiac arrest and drug overdose [ ] . there is an established body of evidence to indicate that cholinergic hypofunction is a significant component of neurodegenerative diseases, such as alzheimer's and parkinson's diseases, which are due to ach and dopamine deficiencies, respectively. moreover, the majority of studies that analysed ach-dependent plr parameters have revealed significant differences between normal age-matched patients and those with alzheimer's [ - , [ ] [ ] [ ] or parkinson's disease [ , , ] . studies typically used light stimuli wavelength of nm with a light intensity of . cd/m . specifically, patients with alzheimer's [ ] or parkinson's disease, with or without any detectable cognitive deficits or psychiatric disorders [ , , ] , had significantly lower mcv and mca values. ferrario et al. [ ] did not observe a significant difference in mca between alzheimer's patients and controls; however, this may be attributed to the longer light stimulus used ( s) compared to the other studies ( - ms) [ ] . the mcv and mca parameters correspond to the first portion of the characteristic v-shaped pupillometric response ( figure ) and are considered the most sensitive markers of cholinergic activity [ ] . therefore, the mca and secondarily the mcv are the best plr predictors to discriminate between healthy individuals and patients with alzheimer's or parkinson's disease [ , ] . a significant increase in latency has been observed for subjects with parkinson's disease [ ] [ ] [ ] ; however, this was not observed consistently in those with alzheimer's disease [ , , ] . additionally, bittner et al. examined the pupil's response under repetitive light stimulation, which is systematically unstable in normal patients [ ] . however, these changes were not observed in patients with alzheimer's disease. a number of possible pathophysiological mechanisms have been proposed for the observed changes in pupillary response seen in alzheimer's and parkinson's patients [ ] , but fotiou et al. [ ] and others have concluded that the most significant factor behind these findings was likely to be the involvement of a central cholinergic deficit. pupillometry, including repeated light stimulation pupillometry, may serve as a useful diagnostic tool even at early, subclinical stages of autonomic nervous system dysfunction [ , ] . the plr is a well-established measurement in the management and prognosis of patients with acute brain injuries, in conjunction with other clinical parameters such as age, mode of injury and glasgow coma scale [ ] [ ] [ ] . typical light stimuli parameters are a nm wavelength with a duration of s and low luminance ( . cd/m ) used to stimulate rod cells or high luminance ( cd/m ) used to stimulate iprgcs. in particular, the location of the pupillomotor nuclei within the dorsal midbrain and efferent oculomotor nerve are important in the determination of brainstem compression and the onset of transtentorial herniation [ ] . morris et al. reported that loss of the plr or development of anisocoria or pupil asymmetry > mm in patients who sustained traumatic brain injuries was correlated with increased morbidity and mortality rates [ ] . however, manual examination using a penlight is subject to large inter-examiner discrepancies that can be as high as %, particularly when pupils are constricted [ ] , and these may be further confounded by a variety of factors, including alcohol, narcotics or hypothermia, which are common to many trauma patients [ ] . furthermore, couret et al. observed an error rate of approximately % even for intermediate-sized pupils ( - mm), with a % failure rate in the detection of anisocoria [ ] . additionally, larson and muhiudeen found complete failure in the detection of the plr by manual examination when the reflex amplitude was less than . mm [ ] . automated pupillometry using a pupillometer is a more sensitive technique that has smaller inter-examiner discrepancies compared to manual examination [ , ] . the ability of pupillometry to detect subtle changes in pupillary reaction even when pupils are constricted has potential clinical significance and may provide a useful tool in the early detection, monitoring and management of brain injuries [ ] . in support of this, several groups have demonstrated that use of a pupillometer was superior to manual assessment in predicting the -day outcome following cardiac arrest [ , ] . the cholinergic system is key to normal pre-and postnatal neurodevelopment, and numerous studies, ranging from neuroimaging data [ ] , to post mortem histopathological analysis of brain tissue [ , ] , animal models [ , ] and molecular genetic studies [ ] , have suggested that alterations in the cholinergic system may be a contributing factor to the aetiology of autism spectrum disorder (asd). moreover, an atypical plr is reported in both children and adults with asd [ , [ ] [ ] [ ] . common measurement parameters used include a light stimulus wavelength of nm and a duration of ms with a light intensity of . cd/m . typically, these differences are characterised by longer latency, reduced constriction amplitude [ , ] and reduced constriction velocity [ ] compared to children without asd. however, nyström et al. reported the opposite for high asd-risk infants, defined as those who had a sibling with asd. the difference in age among the studies' subjects, who ranged from -month-old infants [ ] up to children > years of age [ , , ] , may provide an explanation for the contrasting data. children without asd exhibit an age-dependent decrease in plr latency before reaching a plateau at > years of age [ , ] ; this correlates with similar trends in white-matter maturation rates as determined by flash visual evoked potential studies [ ] . however, in children with asd there was no age-dependent decrease in plr latency [ ] . moreover, brain-imaging studies have shown that the neurodevelopment trajectory of brain maturation is atypical in children with asd. initially, young children (< years of age) exhibit accelerated maturation and a larger brain volume compared to children without asd [ ] [ ] [ ] [ ] [ ] , but this is followed by a period of arrested maturation after years of age [ , ] , and then a possible decrease in brain volume in older children and adults [ ] . therefore, the hypersensitive plr observed in infants with a high risk of asd [ ] could be attributed to the accelerated white-matter maturation associated with asd, which then reverses after years of age, resulting in a hyposensitive plr response corresponding to the reduction in plr parameters [ , , ] . there is evidence to suggest that, apart from the cholinergic system, other neurotransmitter systems are altered in asd, such as glutamatergic and gabaergic transmission [ ] . therefore, the plr may also be affected as a result of altered bipolar cell signalling within the retina. overall, assessment of the plr by pupillometry may provide a rapid and non-invasive diagnostic tool for infants and children with a high risk of asd or other cholinergic-dependent neurological development disorders [ ] . pupillometry offers a reliable and convenient tool for illicit drug and alcohol screening [ ] [ ] [ ] . a number of studies have demonstrated the ability of pupillometry to differentiate between potentially drug impaired and normal subjects with - % accuracy [ , ] . the most significant parameters were rpa and mcv [ ] . additionally, pupillometry may offer benefits over urinalysis, particularly in the case of roadside testing, as it provides a measurement of function and impairment rather than a measurement of drug metabolites, which may be present in the urine for an extended period even though any drug-induced physiological effects or impairment have ceased. pupillometry may have a role in both the detection of alcohol intoxication and treatment management during alcohol withdrawal. data from chromatic pupillometry studies demonstrated a significant increase in both baseline pupil diameter and peak constriction amplitude following a nm wavelength light stimulus at exhaled breath alcohol concentrations of ≥ . mg/l [ ] . however, following a high dose of alcohol ( g/kg body weight) the opposite was observed, with significant decreases in pupil diameter, constriction amplitude and velocity compared to control groups, suggesting inhibition of parasympathetic nerve activity [ , ] . these apparently contradictory results are likely to reflect the acute, dose-dependent inhibition of the parasympathetic nervous system, which results in the predominance of sympathetic nerve activity [ ] . pupillometry may also have a role in the development of clinical management tools to prevent severe autonomic dysfunction during alcohol withdrawal [ ] . specifically, prolonged latency and decreased constriction velocity parameters were described for participants undergoing alcohol withdrawal. the reduction in parasympathetic innervation of the pupil is likely to be due to increased activation of the locus coeruleus, as previously described during alcohol withdrawal [ , ] . the pupillary response to , methylenedioxymethamphetamine (mdma) and tetrahydrocannabinol (cannabis) is characterised by an indirect central parasympathetic inhibition, resulting in significantly increased latency and decreased constriction amplitude and velocity [ , , ] . additionally, increased sympathomimetic activity due to increased noradrenaline and serotonin signalling was reported following mdma intoxication, resulting in mydriasis and a reduction in the plr recovery time [ ] . however, for cannabis intoxication there are conflicting reports regarding the effect of the drug on baseline pupil diameter. hartman et al. observed a significantly increased pupil size compared to control participants, under both scotopic and photopic light conditions as well as following direct light stimulation, suggesting increased sympathetic nervous system activity [ ] . this is in contrast to data reported by fant et al. and others, which demonstrated a significant cannabis-induced effect on the plr but either a small ( . mm) decrease [ , ] or no change [ ] in baseline pupil diameter. these differences may be attributed to study design, as the studies that observed little or no effect on pupil diameter used a defined, high dose ( mg ∆ thc) and a specified time duration between drug administration and pupil measurements, whereas hartman et al. used drug recognition expert examination data; thus, the exact dose and timings are undefined [ , , ] . changes in pupil size and response to light have been reported following exposure to toxic chemicals such as organophosphates, as well as bacterial toxins including botulinum toxin. ophthalmic manifestations are early and persistent signs of botulism. botulinum toxins (btx) block the release of ach at neuromuscular junctions, post-ganglionic parasympathetic nerve endings, and post-ganglionic sympathetic nerve endings that release ach, resulting in paralysis of the sympathetic and parasympathetic innervation of the iris [ , ] . this may result in transient pupil dilation and attenuation of the plr by uptake of btx into the parasympathetic ciliary ganglion or the parasympathetic neuromuscular junctions at the iris sphincter muscle [ ] . there are a number of reports in the literature describing mydriasis with an attenuated plr as a consequence of ingesting contaminated food [ ] , or following injection of btx [ , ] . organophosphates, a family of chemicals that includes nerve agents and pesticides, inhibit cholinesterase activity, resulting in increased levels of ach at the nerve synapses; they thus act as an indirect cholinergic agonist. published studies by dabisch et al. and others suggest that the majority of cholinesterase inhibition observed within the eye is a result of the nerve agent vapour acting directly on the ocular tissues, rather than distributing to the eye as a result of systemic absorption [ ] [ ] [ ] . the localised increase in ach leads to contraction of the pupillary sphincter muscle, resulting in dose-dependent miosis [ , [ ] [ ] [ ] . miosis is a highly sensitive index of exposure and can occur at exposure levels below those that cause systemic effects [ , ] . in relevant animal models, the amounts of sarin and cyclosarin required to produce miosis were up to -and -fold lower, respectively, than the amounts required for lethality [ ] . the plr is also reduced following organophosphate exposure, as a result of developing tolerance to cholinergic agonists and desensitization of muscarinic ach receptors within retinal tissue following prolonged exposure [ , , ] . the threshold dose required to attenuate the plr is similar to that required to produce miosis, but the duration of the response is very different [ ] . dabisch et al. [ ] observed rapid miosis and attenuation of the plr in a rodent model following a single low-dose soman vapour exposure, but while pupil size returned to normal after h, the plr took up to days to fully recover. similarly, exposure to dichlorvos vapour resulted in a dose-dependent transient miotic response in the guinea pig; however, a persistent enhanced pupillary response to light was observed [ ] . the recovery of pupil size is attributed to desensitisation of the muscarinic receptors rather than reactivation of cholinesterases within the eye, which may take up to days to recover. consequently, the plr is attenuated until muscarinic receptor function is regained [ ] . in support of this, oximes, which reactivate acetylcholinesterase, had no effect on sarin-induced miosis in animal models. moreover, tropicamide-a muscarinic receptor antagonist that competes with ach for binding sites, preventing receptor desensitisation-rapidly increased pupil size and restored plr [ ] . however, organophosphates inactivate cholinesterases at both muscarinic and nicotinic receptor sites and paradoxical pupil dilation or mydriasis may occur in certain circumstances due to dominant nicotinic effects at the pre-ganglionic fibres of the sympathetic nervous system, resulting in increased innervation of the dilator muscle [ , ] . an area of interest that currently remains unexplored is the response of the plr to infection and the potential diagnostic value of pupillometry. the brain monitors and modulates immune status through both humoural and neural pathways [ , ] . neuroendocrine responses control inflammation at the systemic level through the hypothalamic-pituitary-adrenal axis [ ] . the first branch of this pathway, the vagus nerve, is activated either directly by cytokines (released from innate immune cells) or indirectly through the chemoreceptive cells located in the vagal paraganglia [ ] . the release of pro-inflammatory cytokines needs to be carefully controlled, as excessive or uncontrolled release (also known as a "cytokine storm") may contribute to the pathogenesis of infections, including the novel coronaviruses sars and mers [ ] [ ] [ ] , influenza [ ] , and ebola [ ] , as well as potential bacterial biothreat agents such as burkholderia pseudomallei [ ] and yersinia pestis [ ] . signals from the vagus afferent fibres eventually project to the locus coeruleus region of the brain. the locus coeruleus exerts a dual influence on the plr, ultimately leading to pupil dilation [ , ] . firstly, it contributes to the sympathetic outflow that innervates the pupillary dilator muscle. secondly, it attenuates the parasympathetic outflow via inhibition of the ew nucleus. furthermore, experimental models of infection-including sepsis [ ] [ ] [ ] , pneumococcal pneumonia [ ] , endotoxaemia [ , ] , leptospirosis [ , ] and influenza a [ , ] -have also highlighted the significance of the cholinergic signalling pathway during infection. changes to cholinergic signalling are likely to influence the plr both directly, through ach receptors located on iris sphincter muscles, and indirectly, through altered parasympathetic nervous system function. therefore, measurement of the plr using dynamic pupillometry may offer the potential to detect systemic changes in parasympathetic and sympathetic system function in response to infection and inflammation. pupillometry shows promise as a non-invasive diagnostic technology for a wide range of conditions. however, there are a number of limitations that require consideration and further research is needed to enable translation into clinical settings. firstly, plr measurements can be influenced by the light stimuli used [ , ] , sex [ ] , age [ , ] and iris colour [ ] . changes in pupil size are also observed in response to other stimuli, including spatial structure patterns [ , ] , object nearness or accommodation reflex [ ] , and a variety of emotional and cognitive stressors [ , ] . therefore, it is critical that standardised protocols be developed to enable the use of pupillometry as a diagnostic tool and limiting factors should be considered as covariates or exclusion criteria in plr studies to enable inter-study comparisons [ ] . further research is also required to establish whether the observed changes in plr associated with different disorders are sufficient in terms of specificity and sensitivity to be used diagnostically. however, the ease of use, non-invasive nature and low cost mean pupillometry is well-placed for inclusion in early diagnostic screening assessments and could complement other sources of information to identify individuals at risk who warrant further clinic investigations. furthermore, there are approaches that have been exploited in particular fields-such as the use of chromatic pupillometry to measure the response of different sub-types of photoreceptor, specifically iprgcs, in diabetes [ ] and glaucoma [ , , ] -which could be applied to other conditions 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of iris color on the pupillary light reflex visual acuity measured with pupil responses to checkerboard stimuli spatial pattern as a stimulus to the pupillary system task-evoked pupillary responses, processing load, and the structure of processing resources iris mechanics. ii. influence of pupil size on details of iris structure the authors would like to thank philip lees for his assistance in finalizing this manuscript. the authors declare no conflict of interest. key: cord- - l lk x authors: hertzog, paul j; mansell, ashley; van driel, ian r; hartland, elizabeth l title: sculpting the immune response to infection date: - - journal: nat immunol doi: . /ni - sha: doc_id: cord_uid: l lk x this report describes advances in the understanding of how microbes elicit and evade immune responses and the sensing of pathogens by host cells that leads to the activation and production of intra- and extracellular signaling molecules. o n - february , approximately scientists met in lorne, located hours from melbourne on the great ocean road, victoria, australia, for the inaugural lorne infection and immunity conference. this multidisciplinary, international meeting was designed to stimulate collaborative research in infection and immunity by bringing together researchers specialized in the study of pathogen biology and host immune responses. the meeting built on the reputation of more than years of lorne annual conferences on proteins, cancer and genomes. sessions covered diverse topics, including innate and adaptive immune mechanisms, microbial-host genomics and adaptation, microbial invasion, evasion and molecular pathogenesis, as well as structural biology of host and pathogen molecules and their effect on immunity. here we summarize some of the newest ideas about infection and immunity research presented at the meeting that are shaping beliefs about how pathogens infect their hosts and how the host resists infection, as well as the translation of such ideas into the clinic. the human body has many innate receptors that probably evolved to sense microbial prod-ucts such as nucleic acids and microbial cellsurface macromolecules from both pathogenic and commensal organisms. these receptors are now also known to sense endogenous molecules that may be present in an inappropriate time, place or form. for example, several classes of receptors sense pathogen nucleic acids but also respond to endogenous dna that can arise from cell damage . e. latz (bonn, germany) opened the proceedings by introducing the nucleic acid-sensing receptors of the innate immune system and highlighted the importance of receptor-mediated trafficking of dna into intracellular compartments for certain inflammatory responses. the toll-like receptors (tlrs) are known from structural studies to facilitate the assembly of large aggregates of adaptors such as myd , which form a scaffold for assembly of the subsequent components in tlr signaling. indeed, the assembly of receptors and signaling molecules into large multimeric complexes is a common thread in the biology of signal transduction. the discovery and characterization of one such aggregate, called the 'myddosome' , has provided new insights into the mechanism and regulation of myd -dependent signal transduction by tlrs , . the myddosome is a highly oligomeric signaling scaffold assembled sequentially from death domains of the receptor signaling adaptor myd and protein kinases irak and irak -irak . it has a complex and variable stoichiometry and drives the formation of large signaling macro-complexes. the assembly process probably involves positive cooperation and takes place in lipid-rich membrane microdomains. these findings have important implications for the therapeutic targeting of tlr pathways in diseases as diverse as rheumatoid arthritis and neurodegeneration (n. gay, cambridge, uk). this suggests that effective therapies will need to target critical protein-protein interactions in the myddosome, such as the toll-interleukin (il- ) receptor domains at the heart of these assemblies, whose initial interactions are weak and are probably more readily disrupted. signaling specificity is probably achieved by the localization of kinase and ubiquitin ligase substrates to the protein scaffold. indeed, e ubiquitin ligases have key roles in regulating signaling in many biological processes, including the initiation, progression and maintenance of immune responses . using mice with targeted genes, m. pellegrini (melbourne, australia) has characterized the critical role of a newly identified e ligase in maintaining the immune system in a quiescent state. the absence of this e ligase causes late embryonic death due to overwhelming immune activation. it seems to function in dendritic cells (dcs) to terminate spurious responses by the transcription factor nf-kb, and deficiency in this ligase results in enhanced and protracted transcriptional responses driven by the nf-kb subunit p . other sensors of infection, belonging to the nod-like receptor family of proteins, are reported to recognize pathogens as well as sterile inflammatory stimuli such as crystals via activation of the il- b-generating inflammasome . a. kupz (melbourne, australia) has identified and characterized a previously unknown in vivo pathway by which salmonella enterica serovar typhimurium (s. typhimurium) elicits cytokine production by distinct lymphocyte subsets involving inflammasomemediated recognition of the bacteria by dcs. although it has been known for some time that distinct cytokines are critical for the control of clinical and experimental infection with s. typhimurium, the exact regulation of their secretion during such infection has remained unclear. this inflammasome-mediated mechanism invokes intricate crosstalk between dcs and the responding lymphocyte subsets. the activation of this pathway has a substantial effect on the ability of infected mice to control the replication of s. typhimurium in vivo. in discussing another gram-negative pathogen, helicobacter pylori, m. kaparakis (melbourne, australia) reported the use of bacterial outer membrane vesicles as a means of delivering bacterial products into the cytoplasm. the contents of outer membrane vesicles enter a compartment in which recognition by the cytosolic pattern-recognition receptor nod occurs, leading to autophagy, inflammation and immune responses in the gut. c. mackay (melbourne, australia) showed how important the chemoattractant receptor gpr is for the normal regulation of inflammatory responses, particularly in the lower gut, in preventing inflammatory bowel disease. gpr is expressed by innate-type immuneresponse cells and binds short-chain fatty acids such as acetate, which are produced in the gut through bacterial fermentation of dietary fiber. mackay proposed that diet, the makeup of the intestinal microbiota and bacterial production of acetate may account for changes in the incidence of asthma and autoimmune diseases in developed countries. interestingly, acetate is emerging as an important factor produced by certain probiotic strains of bacteria . although the innate immune system has evolved to generate a rapid and effective inflammatory response to invading infectious organisms, it is no surprise that many microbial pathogens have evolved to avoid detection by this system and/or to deactivate the immune response. c. roy (new haven, connecticut, usa) presented work on legionella pneumophila, the causative agent of legionnaire's disease, demonstrating that this opportunistic and accidental intracellular pathogen stimulates almost every pathway of the innate immune response as it infects and subsequently replicates in macrophages . indeed, l. pneumophila has proven a useful tool with which to study innate immune responses, particularly the nlrc inflammasome, which recognizes l. pneumophila flagellin . l. pneumophila is an environmental organism whose natural hosts are unicellular eukaryotes, and this bacterium has thus not evolved to avoid detection by mammalian immune systems. in contrast, the closely related organism coxiella burnetii, a cause of q fever, is highly adapted to the host and evades detection by the immune system via mechanisms that are poorly appreciated. both pathogens translocate virulence effector proteins into the host cell through the use of the dot/icm type iv secretion system, yet the effector protein repertoires of the two organisms are very different. the adaptation of c. burnetii to a mammalian host is evident in its ability to inhibit mammalian proapoptotic signaling. the c. burnetii dot/icm effector ankg binds the host protein gc qr (p ) and inhibits apoptosis in dcs, whereas l. pneumophila lacks this effector and induces rapid apoptosis in dcs in a p -dependent manner . further work on the c. burnetii dot/icm effectors will undoubtedly identify additional mechanisms of host adaptation and microbial evasion of the immune response. new information on host-cell invasion by the intracellular pathogen s. typhimurium presented by r. teasdale (brisbane, australia) showed that there is still much to learn about the dynamic interaction between host and pathogen that enables s. typhimurium to form its intracellular niche. after invading the host, s. typhimurium stimulates a process similar to macropinocytosis to mediate its uptake into cells. by screening host-cell proteins involved in macropinocytosis through the use of small interfering rna-mediated knockdown or pharmacological inhibition, researchers have discovered that phosphatidylinositol-( )kinase (pikfyve) is essential for the replication of s. typhimurium inside cells . the product of this kinase, phosphatidylinositol-( , )-bisphosphate, is required for the fusion of 'macropinosomes' (large vesicles filled with extracellular fluid that are formed through macropinocytosis) with late endosomes-lysosomes and proper maturation of the s. typhimurium-containing vacuole. further screening has also identified a role for the sh -px-bar-domain family of sorting nexins in macropinosome formation and the initial stages of s. typhimurium infection. several presentations described the ongoing battle between clearance by the immune system and subversion of the immune response by viruses. new aspects of the host response were reported by m. beard (adelaide, australia), who has used transcriptome analysis of liver infected with hepatitis c virus (hcv) to show that the interferon-stimulated gene product viperin and members of the interferon-inducible transmembrane family of proteins have distinct anti-hcv properties and act via inhibition of viral replication and entry, respectively, which thereby raises new possibilities for targeting chronic hcv infection. s. lemon (chapel hill, north carolina, usa) contrasted the ability of hcv to avoid immune responses and clearance with that of hepatitis a virus as a means of identifying and characterizing viral evasion strategies. both viruses use a series of virus-produced proteases to target critical elements of the innate response machinery, yet surprisingly, it seems that hepatitis a virus is more evasive in a chimpanzee model of hepatitis infection, eliciting a minimal interferon response. these results suggest that although both viruses have evolved evasion strategies to target the same innate immune mechanisms, their survival and subsequent clearance rely on distinct processes . further exploring the host-hepatitis virus interaction, z. yuan (shanghai, china) described the various strategies used by hepatitis b virus (hbv) to evade the host immune response. liver biopsies of patients infected with hbv obtained before and after treatment have shown that hbv uses at least two mechanisms to avoid the innate immune response. first, impaired tlr-induced cytokine expression correlates with higher expression of hbv surface antigen and inhibition of the jnk kinase pathway. second, hbv polymerase inhibits the phosphorylation, dimerization and nuclear localization of the transcription factor irf to dampen immune responses. together these studies suggest hbv targets signaling by the innate immune response to evade detection by the immune system and establish chronic infection. reporting a twist on evasion of the immune system by hepatitis viruses, g. ebert (munich, germany) harnessed the ability of ′-triphosphorylated small interfering rnas to trigger innate immune interferon responses. this research used small interfering rnas designed to silence hbv replication in concert with more interferon production to suppress hbv replication in both cell-culture and transgenic hbvinfection models, which provides hope for volume number july nature immunology new insight into immune regulation by 'old' molecules such as interferons is constantly being acquired. progress in understanding the biology of the type iii interferon ifn-l (il- -il- ) has led to considerable advances in the understanding of immunity. m. o'keefe (melbourne, australia) discussed the cellular specificity of ifn-l production and expression of the ifn-l receptor by cells of the immune response. her studies and those of others have shown that expression of this receptor is not ubiquitous, like that of receptors for other members of the interferon family, but instead is restricted to epithelial cells and, as described here, also plasmacytoid dcs (pdcs). in further contrast to other interferons, it seems that ifn-l is produced by restricted subsets of dcs in response to specific ligands such as the synthetic rna duplex poly(i:c), which makes ifn-l a nonubiquitous protein of the immune response. these subtle differences may account for the diverse immune functions now attributed to this newly identified cytokine. a. thompson (melbourne, australia) elaborated further on the role of ifn-l in the pathogenesis of and therapeutic responses to hcv. genome-wide association studies have identified polymorphisms in the region of il b (which encodes ifn-l ) on chromosome that correlate well with treatment outcome in patients infected with hcv genotype who are treated with ifn-a (with covalently attached polyethylene glycol polymer chains) and ribavirin . patients carrying the 'goodresponse' variant have enhanced phase-one kinetics and two-to threefold higher rates of viral eradication. furthermore, differences between patients of different ethnic background in allele frequency explain much of the recognized ethnic disparity in cure rates . moreover, il b polymorphisms have also been associated with spontaneous clearance of hcv infection . the underlying biological mechanisms, however, remain unclear, which provides an important area for future research. emerging and re-emerging infections further evidence of the adaptation of microbes to their host or environment is apparent from genomic approaches to understanding buruli ulcer, an emerging but neglected tropical disease. this is a disease of subcutaneous tissue that affects humans and other animals that is caused by infection with mycobacterium ulcerans and for which there is no vaccine. t. stinear (melbourne, australia) has used genomics to address some key research issues, including how m. ulcerans is transmitted to humans. using multistrain comparative genomics, the group has shown that m. ulcerans evolved from the closely related mycobacterium marinum by lateral gene transfer and genome reduction. genes found in m. ulcerans but not in closely related mycobacteria are predicted to encode m. ulcerans-specific antigens, and recombinant proteins derived from these genes seem to elicit specific serum antibody responses in humans living in a region in which buruli ulcer is endemic . this suggests it may be possible to use serology to assess exposure to the bacterium. k. holt (melbourne, australia) discussed a study of the microevolution of shigella sonnei, a common agent of endemic dysentery. wholegenome sequencing and phylogenetic analysis of a collection of over isolates from global sources has categorized the population into several distinct clusters, with most of the recent isolates restricted to a single cluster. analysis of isolates from ho chi minh city has shown nearly all local cases can be attributed to the local expansion of a single clone. similar rapid microbial adaptation is evident in sub-saharan africa, where a distinct sequence type of s. typhimurium has emerged as a frequent cause of invasive disease. m. levine (baltimore, maryland, usa) reported that the high disease burden and case fatality caused by this strain has made vaccine development an urgent priority. this emergence was discovered through a comprehensive surveillance program and highlights the importance of monitoring infectious diseases in vulnerable populations. microbes also form an unusual interaction with hosts such as bats, which serve as the reservoirs for many emerging viruses, including paramyxoviruses (hendra and nipah viruses), coronaviruses (severe acute respiratory syndrome coronaviruses) and filoviruses (ebola and marburg viruses). wild bat populations or experimentally infected bats, however, do not show any clinical signs of disease. work by l. wang (geelong, australia) is focused on understanding virus-bat interactions from the perspective of genomics and 'transcriptomics' (expression profiling). considerable progress has also been made in establishing bat cell lines and in functional studies of key molecules in bat innate immunity, such as tlrs and the interferon system. ultimately, these fundamental investigations may help to explain why bats seem to be asymptomatic carriers of some viruses that are deadly to humans. an effective immune response requires the ordered, sequential expression and secretion of inflammatory cytokines by the appropriate cells. j. stow (brisbane, australia) has used live-cell imaging to show that cytokines traffic through recycling endosomes in macrophages, which allows the orchestrated release of multiple cytokines through the secretory and lysosomal pathways. these studies have demonstrated that recycling endosomes coordinate cytokine release while allowing macrophages to engulf and destroy invading pathogens. macrophage activation and the induction of a robust immune response are critical for the generation of protective immunity to mycobacterium tuberculosis. b. saunders (sydney, australia) proposed that macrophages use a previously unknown communication strategy that involves the release of microparticles after stress induced by bacillus calmette-guérin. these microparticles contain m. tuberculosis antigens that are processed by antigen-presenting cells, thereby inducing antigen-specific t cell responses. therefore, microparticles may represent a mode of immunological transfer that induces cell migration and the inflammatory response to infection with m. tuberculosis. the signal-transduction programs of core cells of the innate immune response, such as macrophages, are mediated by key transcription factors such as the irf proteins. for example, irf controls the differentiation of m -type macrophages in both mice and humans . p. pitha (baltimore, maryland, usa) discussed the phenotype of irf -deficient mice, which includes b cell abnormalities and a propensity to develop autoimmunity. this finding underscores the role of key transcription factor-mediated genetic programs in the interface between the innate and adaptive immune systems. these factors mediate the innate responses of both mrna and microrna in cells of the innate immune system, such as macrophages, whose coordinated induction was presented by s. forster (melbourne, australia). additional functions for another innate cell type, the pdc, were reported by d. ang (melbourne, australia). although pdcs have a recognized role as producers of type i interferon during viral infection, here pdcs were also shown to combat the intracellular bacterial pathogen l. pneumophila. depletion of pdcs in mice infected with l. pneumophila exacerbates volume number july nature immunology this field and the second lorne infection and immunity conference in february . liver dis we apologize to the many presenters whose work was not included here because of space constraints. the lorne infection and immunity conference was hosted by the victorian infection and immunity network (the university of melbourne infection and immunity domain, monash university, walter and eliza hall institute, victorian infectious disease reference laboratory, the burnet institute, the commonwealth scientific and industrial research organisation) and was sponsored by pfizer centres of therapeutic innovation, roche, csl, gilead life research and bio-rad. the authors declare no competing financial interests. into how cd c hi and cd c lo-int cells regulate the development of il- -producing t helper type cells and immunopathology differently.how il- acts on a large number of genes in diverse cells is unclear at present. c. mccoy (melbourne, australia) and colleagues have built on published work showing that expression of the microrna mir- is inhibited by il- . this microrna has been linked to the modulation of inflammatory responses, inflammation-induced cancer and the pathogenesis of autoimmunity. this latest work suggests that through mir- , il- regulates a set of genes previously unknown to be regulated by this cytokine, which helps to explain its biological potency. this work also strengthens the case for the development of therapeutics based on mir- and surely other micrornas that regulate immune responses. the field of host-pathogen interactions has seen discoveries that extend far beyond greater knowledge of pathogen or immune-response factors. multidisciplinary approaches to understanding the complex interaction between the pathogen and the immune response during infection have led to a heightened appreciation of the adaptation of pathogens to their host as well as greater insight into so-called 'sterile' inflammation, innate and adaptive immune control and autoimmunity. the first lorne infection and immunity conference was established to bring together aspects of infection, the pathogen and the immune response. collaborations and ideas that result from discussions at these meetings are set to change the way researchers view infection, leading to improved basic knowledge and its translation into better treatment and prevention of infectious and inflammatory diseases. we look forward to further rapid developments in infection , whereas supplementation of pdcs into the lungs ameliorates infection by diminishing the bacterial load. notably, resistance to pulmonary l. pneumophila infection is independent of type i interferon signaling, which shows that pdcs control bacterial infection in the lungs by a mechanism distinct from antiviral pathways such as type i interferons. in summary, these presentations confirmed the idea that the presence of pathogen-associated stimulating molecules and their detection by cells of the innate immune system drive the signaling and production of cytokines, in a context-dependent manner, that shape the nature of the response that ensues.modulation of the adaptive response innate sculpting of adaptive immunity occurs through cytokines such as il- , a potent immunosuppressive cytokine with wide-ranging effects on t cells and b cells as well as cells of the innate immune response, such as macrophages and dcs. the tropical disease visceral leishmaniasis is characterized by splenomegaly, remodeling of the red and white pulp compartments of the spleen and local il- -mediated immunosuppression. p. kaye (york, uk) presented data that have extended published work to show that the cellular control of splenic remodeling is highly compartment specific. although inflammatory monocytes negative for matrix metalloproteinase and positive for the neutrophil marker ly g (mmp -ly g + ), not mmp + ly g + neutrophils, are responsible for regulating vascular changes in red pulp, neither of these populations has a substantial role in regulating the destruction of follicular dcs or fibroblastic reticular cells in the white pulp. through the use of a model in which mice were depleted of cd c + cells during chronic infection and then were reconstituted by adoptive transfer, this work has provided new insight key: cord- -drvjr gq authors: amankwah-amoah, joseph title: note: mayday, mayday, mayday! responding to environmental shocks: insights on global airlines’ responses to covid- date: - - journal: transp res e logist transp rev doi: . /j.tre. . sha: doc_id: cord_uid: drvjr gq the covid- pandemic in / ushered in a new turbulent and chaotic global environment where governments not only placed temporary restrictions on people’s movements, but also mandated limits on business activities. however, lacking in the contemporary scholarly discourse is a deeper understanding of how businesses respond to such pandemics. in this research note (rn), a conceptual framework of firms’ responses is advanced. using the global airline industry, the analysis delineates a host of internally generated and externally imposed firms’ strategic and tactical responses to the pandemic including in-flight service changes, flight cancellations, seeking emergency aids and financial supports, and firm closures. the analysis demonstrates that in responding to the crisis, many airlines sought to minimise erosion of long-developed knowledge, market capabilities, route networks, access to airports, customer base and relationships/trust with customers prior to covid- to equip them for recovery. the wider implications for academics, managers and governments are outlined as the effects of covid- continue to unfold. the epic transmission of covid- across multiple countries with such a precipitous negative shock on national economies and accompanying social distancing measures to halt human-to-human transmissions makes it one of the most unparalleled events in modern times (see world health organization (who), a, b). nowadays, the popular press is replete with covid- pandemic stories which continue to unfold across the globe affecting over nations and territories (lipsitch et al., ; the economist, a who, a who, , b who, , c worldometers, ; see also ; craighead et al., ; govindan et al., ; cai and choi, ) . in many service-oriented economies and industries such as air travel and tourism, the covid- pandemic and accompanying social distancing measures have virtually hampered business activities. in the midst of this epical changing global business environment, we are also often confronted with a myriad of unknowns and difficult challenges which force regulators, governments and businesses to respond. a plethora of research efforts has been directed at investigating pandemics (see also lipsitch et al., ; choi, ; loske, ; yu et al., ) and much of the prior academic literature on pandemics has tended to focus on the mortality rates, government policies and transmission mechanisms (see nigmatulina and larson, ). thus, a notable and significant shortcoming in the existing research is the very limited insights on businesses' response to health crisis/pandemics. as demonstrated by past studies, "how companies respond to unforeseen disruptions such as pandemics or outbreaks remains limited" (amankwah-amoah, a, p. (wenzel et al., ) . against this backdrop, the objective of this research note (rn) is to examine how airlines' have responded to covid- and factors that facilitate, shape or constrain their responses. the airline industry is one of the most global industries (doganis, ) , heavily impacted by governments' restrictions on people movement (detailed analysis will follow shortly) and as such represents a fertile ground for exploring how firms respond to crisis. this study is also motivated by the growing importance and somehow increasing frequency of global health pandemics and the need for a better understanding of how governments, companies and international organisations not only attempt to anticipate these challenges but also respond to their occurrences. more than million covid- cases and , fatalities have been reported globally, demonstrating the importance of this issue (who, b; worldometers, ) . the analysis makes pivotal contributions to the literature. first, in light of the evolving and unprecedented nature of covid- and its impact on businesses and way of life (lipsitch et al., ; who, a who, , b who, , c , this study contributes to the ongoing discourse by providing preliminary analysis of a host of airlines' responses to the crisis, shedding light on factors that facilitate, shape or constrain their responses to the crisis. in addition, although some studies have explored the issue of pandemics (see ivanov, ) , these have often been done in isolation from businesses' action and responses to such events. in this direction, an integrated framework of analysis was developed and utilised to analyse firms' responses to crisis, encapsulating the nature and timing of a host of strategic and tactical responses to the covid- pandemic. our analysis rectifies the oversight and offers insights into the short-and long-term responses adopted by airlines. furthermore, although there are studies on firms' responses to crisis (mishra, ) and the timing of responses (see wenzel et al., ) , these two streams of scholarly works have emerged and developed insolation. the paper extends the literature by integrating these two largely divergent approaches to contribute to the ongoing conversation of the effects of the covid- pandemic on global businesses by focusing the global airline industry. the sections that follow present a review of literature on firms' responses to environmental jolts. after using the review to develop an organising framework, we then present the main findings using the integrated framework, followed by discussion of the implications. environmental jolts can be defined as "transient perturbations whose occurrences are difficult to foresee and whose impacts on organisations are disruptive and potentially inimical" (meyer, , p. ) . during crisis, some firms may seek to preserve their key employees, market knowledge and resources, whilst concurrently striving to minimise the adverse effects of sudden changes in the business conditions (amankwah-amoah, a; wenzel et al., ) . a good example in the global airline industry is that following the covid- -induced crisis, british airways (ba) decided to bring forward its decision to discontinue boeing fleets as part of its recovery strategy. the airliner once dubbed the "queen of the skies", the "most recognisable" among the public as well as the preferred choice of global airlines for long-haul routes (flight international, ; specia, ) . by joining other global airlines such as qantas that have phased out or in the process of phasing out their s fleets (specia, ) , ba was able to usher in a new recovery approach to counteract the covid- effects. according to the flight international ( , p. nd), the withdrawal of the 's from service crucially eliminated a fleet that "represents higher operating costs from both fuel-burn and maintenance perspectives". this shift also epitomizes a new era where airlines are increasingly embracing new generation fuel efficient aircrafts (flight international, ; specia, ) . to explore the issue of firms' responses to jolts, we situate our analysis within the literature on timing and locus of causality of organisational actions/inactions/failure. time is a pivotal dimension in firms' responses to jolts/events and its effects may be difficult to determine. past studies have demonstrated that timing is a key resource which can grant an organisation a first-or late-mover advantage in the face of crisis montgomery, , ; makadok, ) and can also be harnessed in devising suitable responses by firms to environment-altering events (grzymala-busse, ). linked to timing is availability of information, resources and cost, which could determine whether the organisation, actors or decision-makers act in a proactive or reactive manner (grzymala-busse, ). thus, there is a shorter-term and longer-term dimension to firms' responses shaped by the duration or nature of the event (aguinis and bakker, ) . the duration of events can determine the nature of its effect on firms' activities as well as the availability or diversity of resources and expertise that firms can mobilise and deploy to contain/respond to crisis. grzymala-busse ( , p. ) observed that, "where negative externalities exist, early movers are advantaged" due to pioneering costs of responding to the event. late arrivals in responding to negative external events have the opportunity to observe and learn from other firms and therefore are more likely to be effective in devising their responses (see lieberman and montgomery, ; makadok, ) . over time, some firms may opt to scale back their operations (retrenchment) to reduce costs, and develop networks and markets in response to an unfolding crisis (bruton et al., ) . this approach, in tandem with curtailing the business scope (de figueiredo et al., ) , is likely to span over both a short-term and long-term strategy (see bluedorn and ferris, ; bluedorn and martin ) . broadly speaking, short-term responses may focus on meeting immediate environmental demands and ensuring immediate survival of the business, whereas, long-term responses are designed to focus on future periods and entailed enduring set of actions, which can be a number of years (see bluedorn and ferris, ; bluedorn and martin, ) . at the effects of crisis unfold, some firms that display ineffectiveness or failure to respond may be forced to exit the industry (amankwah-amoah, b). for new firms, survival is often paramount which often precipitate short-term oriented strategy. underpinning literature pertaining to the locus of causality is useful in understanding the issue. here, there are two main schools of thought: voluntaristic and deterministic views (amankwah-amoah, b; amankwah-amoah et al., ; mellahi and wilkinson, ; whetten, ; zhang et al., ) . the deterministic perspective views organisational actions as being driven by external factors such as government directions, economic recession, declining demand and luck, over which managers have limited or no control (amankwah-amoah, b). often government-mandated actions can drive small and financially weak firms into bankruptcy. given that legitimacy is conferred by organisational stakeholders, they can also force a firm to act or adopt a set of measures to maintain its existence (perrow, ) . thus, government pressures can force firms to adopt a course of action irrespective of the impact on the firms' operations. businesses are viewed as victims of crisis or unpredictable circumstances in their business environment e.g. natural disasters such as flash flooding, tornadoes, landslides, earthquakes, sinkholes, volcanic eruptions, heat waves and droughts. these events can suddenly alter the business environment, culminating in closure, redesign of the business model or firms actually closing down. many firms are often unable to fully capitalise on their existing resources and expertise to identify and neutralise environmental threats, which often forces closure of the business (delmas and toffel, ) . in contrast to the other perspective, the voluntaristic perspective traces actions, inaction and firm failure to managers, workers and characteristics of the organisation such as resource and expertise (amankwah-amoah et al., ; mellahi and wilkinson, ) . extending previous research, we contend that firms' ability to respond to a global outbreak is predicated on its firm-specific assets which denotes resources and expertise of the firms including relationships nurtured over time. from this literature, we contend two types of firm responses: internally designed/generated and externally imposed. in keeping with insights from the timing literature, we contend two dimensions of timing: short-term and long-term. thus, businesses respond to crisis either in the short term or long term via utilisations of their own expertise and resources or requirements/set of actions imposed on an industry by external entities/parties, as shown in fig. . externally imposed responses are driven by external/ institutional factors such as governments, industry bodies and societies to ensure standardisation of responses and securing wider participation of all firms in the industry for the proposed course of action. it stems from actions of external actors of the organisation or simply an outcome of regulatory, political, social and economic changes. however, externally imposed courses of action tend to be detached from organisation-specific problems and shortcomings which can amplify organisational problems. with internally generated responses to crisis, the organisation has direct ownership and influence in devising and carrying out the course of action with the aim of ensuring its long-term survival. they are carefully designed organisational approaches and sets of actions informed by the organisation's knowledge, experiences and market knowledge. anchored in the internally generated course of action is deployment of firm-specific key resources and expertise to respond to or neutralise the external threats (mccutchen, ) . when crises/jolts emerge, they often release the constraints on organisational-decision makers and their latitude to act, thereby opening up a wider range of strategic options, ammunitions and actions for firms (bryson, ; wenzel et al., ) including different engagements with governments, customers, clients and suppliers. firms are likely to face imposed responses as well as sets of actions during crisis. it is, therefore, expected that firms may be motivated to embrace internally initiated as well as externally imposed responses to develop new relationships with political actors to secure access to financial and political resources in both the short term and long term to ensure survival of their businesses. the above dialogue suggests that during crisis, different firms in an industry would likely exhibit some of these different responses. crossing the pillars produces the × matrix of firm responses to external environmental shock. as demonstrated in fig. , internally generated short-term responses (quadrant i) describes the short-term operational and tactical responses to the unfolding organisational crisis. this is where early-warning signals or cues necessitate modifications in the processes, routines, strategy and structures of the organisation. externally imposed short-term responses (quadrant ii) is where early-warning signals or cues of crisis lead to an external actor-imposed course of action to curtail the negative effects. the internally generated long-term responses (quadrant iii) is where the crisis demands long-term strategic and operational responses within the firms to prepare for the post-crisis environment. the externally imposed long-term responses (quadrant iv) focuses on long-term political network development and leveraged overtime to enhance the competitiveness of the firms. online supplementary appendix (amankwah-amoah, , ; amankwah-amoah and debrah, debrah, , bbc, c; chua, a chua, , b chua, , c cirium, a cirium, , b dunn, a dunn, , b european commission, ; frost, ; ft, ; harper, ; hsu and flitter, ; iata, iata, , a janzen, ; johnson, ; kaminski-morrow, a nats, ; puhak, ; salaudeen, ; the canadian transportation agency, ; the economist, b, d; waldron, ; walsh, ; wolfsteller, a, b) provides a more general overview of the global airline industry and covid- as the research setting. following and applying fig. , we delineate the responses adopted by airlines around the globe, and the internal and external factors that facilitated or impinged on responses. online supplementary appendix provides additional details on the dimensions of the quadrants with illustrative examples. quadrant i displays a situation where firms deduced internal responses to crisis with a largely short-term focus. in the preliminary stage, most airlines sought to make modest changes, focusing on environmental scanning associated with the virus and governments' directives on travel to the affected area and who directives. in january , turkish airlines was amongst others who conveyed their decision to assess the situation (dunn, c) . some of the early efforts by airlines focused on providing additional safety measures and suspending some services to minimise the risk of transmission. given that the coronavirus transmits via human-to-human contact and fig. . a network model of effects of social distancing on airline business model. microscopic droplets through coughing and sneezing, it makes it difficult to deliver people-to-people services (hester, ) . several tactical responses were adopted during this phase including deep cleaning planes before take-off. for many airlines, cleaning staff were required to put on protective suits in helping to disinfect aircraft against the coronavirus (cornwell, ) . such initiatives were further backed by airlines such as cathay pacific who reassured their customers of measures being taken by highlighting the "intensifying disinfection of aircraft after landing, making cabin crews don gloves and masks, removing blankets, magazines and pillows, and adding safeguards to the in-flight food and drink service" (lee, , p. nd) . as the crisis unfolded, many airlines started moving towards introducing some elements of in-flight social distancing, compulsory temperature checks and demanding that passengers put on masks (lee, ) . generally, the social distancing measures adopted by airlines sought to curtail social interaction between employees, employees and customers as well as between customers, with the aim of halting the elevation of potential risk to people. without effective social distancing measures to stem or curtail coughing and sneezing in close proximity of others (chaudhary and maidment, ) , airlines can potentially expose employees (in-flight crew) to the health risks stemming from the coronavirus outbreak. this was particularly relevant to the industry as the threat of virus transmission to the airlines' in-flight crew might force them to opt for self-preservation over customer/passenger care, thereby compromising their duty of care. some airports also adopted similar measures to reduce interaction between people and restrict large gatherings at their premises to curtail the spread of the virus. nevertheless, the social distancing approach recommended by many governments was "nearly impossible to accomplish on an airplane" (hester, , p. nd) . in-flight attendants work in tight spaces (often within feet of their and customers, i.e. the distancing recommended by us centers for disease control and prevention), and make regular contact with passengers, pushing beverage carts up narrow aisles, reaching over customers to serve food and beverages and standing at arm's length from customers to perform mandatory safety demonstrations (hester, , p. nd) . in addition, flight attendants generally do not possess the expertise of medical doctors and healthcare professionals, and are therefore incapable of dealing with in-flight medical emergencies associated with someone with coronavirus (hester, ) . the effects of social distancing on the global airline business model are reflected in fig. . online supplementary appendix provides examples of multiple airlines that adopted this approach during the crisis period. quadrant ii demonstrates a situation where a set of actions are imposed on the industry. owing to the outbreak, a new directive was issued from who for the aviation industry and aviation personnel focused on the operational considerations to help halt the transmission of covid- . the proposed measures for in-flight and all personnel re-emphasised hand hygiene, social distancing, respiratory etiquette and seeking medical advice on suspected cases (who, c). in line with who's guide to hygiene and sanitation in aviation, some of the operational responses emphasised enhanced cleaning and disinfection which covers airports and service providers (who, c). in addition, it re-emphasised post-event cleaning procedures and disinfecting contaminated surfaces following notification of suspected cases (who, c) . in relation to prior analysis, the observation about in-flight social distancing was further buttressed when india's directorate general of civil aviation (dgca) suggested that airlines adopt in-flight social distancing by keeping at least one metre between passengers at airport check-ins and security counters as well as keeping the middle seats empty on flights empty to minimise contact between passengers (indiatoday, ) . for instance, in an attempt to avert second outbreaks in china, the government limited inter-china flights for both chinese and foreign airlines by allowing just one flight a week and each flight was not to exceed % capacity (bbc, d) . the effects of the crisis are further demonstrated when in europe, for instance, largely due to government-imposed measures, all but a few "essential routes" on domestic and neighbouring markets remain functional, largely to allow critical travel, cargo, medical supplies and repatriation of nationals (dunn, d) . in march , following the australian government's guidelines and suggestions, qantas and jetstar suspended some scheduled international flights (cirium, d) . for the qantas group, the grounding of around aircraft reduced its international capacity by around % and domestic by % (cirium, d) . quadrant iii focuses on strategic and operational activities developed and employed by firms to respond to and prepare for the postcrisis period and recovery of services. in the past, most firms were assumed to be passive recipients of events in their external environment. however, it was observed that in the face of a partial or full lockdown in most nations, airlines engaged in corporate political activities where they engage with policymakers to enact the "rule of the game". our analysis indicates that at the same time, many sought to lobby governments for aid packages to help them overcome the sharp decline in demand. following the cessation of flights and governments' restrictions on flights, many airlines starting burning through cash reserves, further diminishing their financial positions and making failure more likely (cirium, f) . accordingly, some airlines such as virgin australia and air new zealand sought government financial support to help them overcome their predicament (cirium, f) . as the industry group, the iata made calls for support and cautioned of dire consequences facing the industry with global revenues from ticket sales falling as much as £ bn (bbc, a (bbc, , b . to offset the negative effects of these dramatic changes culminating in loss of revenue, some airlines pursued cost-reductions via offloading workers, terminating affected routes and encouraging some employees to work from home. the state-owned, and one of the world's biggest long-haul airlines, emirates, adopted measures such as a temporary - % basic salary reduction for employees to help them keep highly skilled employees as well as minimise or avoid job losses (cornwell, ; klar, ) . this was also designed to enable it to mobilise employees and resources swiftly to resume services for customers when conditions improve and the outbreak is controlled (cornwell, ; klar, ) . quadrant iv demonstrates the wider and long-term effects of the imposition of action on firms and living through this post-covid environment. here, measures such as in-flight social distancing policies, government-mandated air travel restrictions and other externally imposed measures are key. long-term consequences are predicated on the effects of short-term imposed directives and measures on the industry. in this direction, the international air transport association in collaboration with the world health organization have developed guidelines to guide cabin crew and airport workers, e.g. captains are required to inform air traffic control of suspected communicable disease (iata, b). indeed, the iata represents around airlines, accounting for % of overall air traffic, and develops industry-wide policies on pivotal issues. fig. presents graphically the changes and shift from pre-covid to post-covid environment and firms' recovery strategy. in this research note, we set out to examine how airlines' have responded to covid- and factors that facilitate, shape or constrain their responses. a unified conceptual framework was developed to capture the internally generated and externally imposed strategic and tactical responses over both the short and long terms. the study highlighted the host of factors that impinged on and shaped airlines' decisions and responses to respond to the threat such as government-mandated actions in terms of travel restrictions, quarantines and social-distancing schemes. the government-mandated and other external constraints curtailed firms' strategic investment and route network decisions, thereby altering the competitive positions of many airlines and further weakening already weak airlines and financially healthy ones. one notable innovation was the introduction in-flight social distancing into the airline business model with long-term implications in terms of in-flight arrangements, high-density seating and in-flight services. taken together, in responding to the crisis, airlines sought to minimise the erosion of long-developed market capabilities, route networks, and prior relationship of trust with customers. from a practical standpoint, the analysis underlines the need for the aviation industry and governments to ensure that the new in-flight social distancing policies do not translate into expensive procedures that make quality inflight arrangements and high-density seating difficult to achieve. in interpreting the present study, one must bear in mind some notable limitations. first, it is worth pointing out that the crisis is ongoing and therefore the analysis only represents a snapshot of the current state of affairs and firms' actions. thus, future studies could systematically examine the post-crisis and recovery strategies adopted by airlines. second, the × matrix offers opportunity for future research to examine the effects of different approaches among different types of airlines such as traditional legacy airlines and low-cost carriers. future studies could also examine the effects of in-flight social distancing on the traditional hub-and-spoke and point-to-point networks, in-flight catering services, fleet utilisation and seat allocation. this is important given that in-flight social distancing has potential of curtailing high-density seating that typify short-haul services and some long-haul services. to stimulate a much deeper understanding of the effects of covid- on businesses, future studies could pursue these fruitful avenues. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work 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grateful to transportation research part e -editor professor choi and the five anonymous reviewers for their constructive comments, which have helped enormously to elevate the arguments and analysis. much appreciated. supplementary data to this article can be found online at https://doi.org/ . /j.tre. . . key: cord- -sq mq f authors: ciabattini, annalisa; garagnani, paolo; santoro, francesco; rappuoli, rino; franceschi, claudio; medaglini, donata title: shelter from the cytokine storm: pitfalls and prospects in the development of sars-cov- vaccines for an elderly population date: - - journal: semin immunopathol doi: . /s - - - sha: doc_id: cord_uid: sq mq f the sars-cov- pandemic urgently calls for the development of effective preventive tools. covid- hits greatly the elder and more fragile fraction of the population boosting the evergreen issue of the vaccination of older people. the development of a vaccine against sars-cov- tailored for the elderly population faces the challenge of the poor immune responsiveness of the older population due to immunosenescence, comorbidities, and pharmacological treatments. moreover, it is likely that the inflammaging phenotype associated with age could both influence vaccination efficacy and exacerbate the risk of covid- -related “cytokine storm syndrome” with an overlap between the factors which impact vaccination effectiveness and those that boost virulence and worsen the prognosis of sars-cov- infection. the complex and still unclear immunopathological mechanisms of sars-cov- infection, together with the progressive age-related decline of immune responses, and the lack of clear correlates of protection, make the design of vaccination strategies for older people extremely challenging. in the ongoing effort in vaccine development, different sars-cov- vaccine candidates have been developed, tested in pre-clinical and clinical studies and are undergoing clinical testing, but only a small fraction of these are currently being tested in the older fraction of the population. recent advances in systems biology integrating clinical, immunologic, and omics data can help to identify stable and robust markers of vaccine response and move towards a better understanding of sars-cov- vaccine responses in the elderly. . , while the m:f ratio of deaths is around . , suggesting that, despite being more frequently infected, females are more capable of dealing with the infection [ , ] . it is widely reported that most deaths occurred among patients with at least one underlying disease, such as hypertension [ ] and diabetes mellitus [ ] . a meta-analysis of seven clinical studies performed in china identified chronic obstructive pulmonary disease (copd), cardiovascular disease, and hypertension as risk factors for severe disease and intensity care unit (icu) admission [ ] . analysis of risk factors associated with more than ten thousand deaths by covid- in the uk confirmed that age was linearly correlated with risk of death and that obesity, diabetes, severe asthma, respiratory disease, neurological disease (including stroke), recent (< years) hematological malignancy, and recent (< year) cancer diagnosis were all associated with higher death risk. as for hypertension, the hazard risk was higher only for the population < years old, even if hypertension itself was strongly associated with other risk factors such as obesity and diabetes [ ] . importantly, these epidemiological studies identify the categories of subjects who are at higher risk of developing severe sars-cov- infection and that should be prioritized in vaccine administration. the massive effort for the development of a vaccine against sars-cov- could be frustrated by the poor responsiveness to vaccination that characterizes a large proportion of the elderly population. in this rush against the time, we risk to pay a dear toll for the lack of knowledge in the response to vaccination of the elderly, a well-known issue, neglected notwithstanding its evident urgency and the annual reproposal through the seasonal influenza epidemic above all. interestingly, there is a consistent overlap between the factors hampering vaccination effectiveness in the elderly and those that boost the virulence and worsen the prognosis of sars-cov- infection. a common characteristic of the elderly people is the onset of a sterile low-grade increase of the basal inflammatory state named "inflammaging," which is considered a universal etiological agent of most of the age-related diseases [ ] . it is likely that some specific components of the inflammaging phenotype could both influence vaccination efficacy and then increase the risk of the early massive production of inflammatory cytokines, termed the "cytokine storm syndrome." this is a condition reported in severe covid- cases during which the patient's immune system spins out of control and starts damaging healthy organs owing to the increased vascular permeability, vascular paralysis, and hypovolemic shock [ ] . angiotensin-converting enzyme (ace ) has been identified as the receptor for sars-cov- , and it has been suggested that differential levels of ace in the cardiac and pulmonary tissues of younger versus older adults may be at least partially responsible for the spectrum of disease virulence observed among patients with covid- [ ] . here, we analyze the different aspects that tackle sars-cov- vaccination in the elderly population, considering immunologic, genetic, and socio-economic factors that impact on the age-related changes of immune responses. a view of the current available vaccine platforms with a special focus on the clinical trials including older adults is reported. how the elderly condition can affect covid- disease progression and the response to vaccination immunosenescence for many reasons, it is difficult to clearly define what immunosenescence is: (i) immunosenescence is quite complex and involves cellular and molecular changes occurring lifelong (from newborns to centenarians) in both the innate and the adaptive immune systems; (ii) these changes can be at the same time detrimental and beneficial/adaptive [ ] ; (iii) it is difficult to identify a unique common marker of immunosenescence, due to the overwhelming number of biological and non-biological factors that can impinge lifelong on the immune system of each individual; (iv) the changes occurring with age in the immune system are deeply correlated with the profound environmental, epidemiological, lifestyle, societal, medical, and public health changes, including vaccination policies and practices, that occurred in the last century. accordingly, immunosenescence is highly contextdependent [ ] , different in different geographical and historical settings and in men and women, correlated to socioeconomic position, and sensitive to psychological stressors. indeed, both the adaptive and the innate immune systems have the capability of "remembering" all immunological stimuli a person has been exposed to lifelong. we have conceptualized this situation with the term immunobiography, which should help in understanding the enormous heterogeneity of the immune phenotype in old people. this is also the reason why there is a sort of imprinting in the immune responses favoring those towards antigens that have been experienced early in life [ ] . the complex biological processes of aging are the result of alterations in gene regulation and protein expression, signaling pathways, and biological networks. complex changes, including pervasive epigenetic and metabolic modifications, affect most of the subsets of naïve, memory, regulatory effector t cells, and b cells [ ] [ ] [ ] . despite the challenging complexity, a universally observed hallmark of immunosenescence is the decrease of naive t cells (particularly cd + t cells) in peripheral blood [ ] consequent to thymic involution responsible for the early decline in the output of naïve t cells to the periphery and for the related shrinking of the t cell repertoire [ ] [ ] [ ] . other important aging-related alterations are (i) the shift in the bone marrow maturation of hematopoietic cells towards myelocytic differentiation [ ] , concomitant with a reduced lymphopoiesis, mainly due to changes in progenitor cells in the bone marrow [ , ] ; (ii) the increased numbers of memory cells owing to large clonal expansion towards epitopes of persistent viral infections (cytomegalovirus [cmv] and epstein barr virus [ebv]) [ , ] ; (iii) the compromised ability of cd + t cells to differentiate into functional subsets, resulting in a multitude of dysregulated responses, such as a reduced cognate help to b cells with consequent reduced humoral immunity, and the increased ratio of the proinflammatory th cells with respect to the immunosuppressive t regulatory cells, thus favoring a basal proinflammatory status [ , ] ; (iv) accumulation of differentiated exhausted t cells, induced by a repeated pathogen encounter during chronological aging, and end-stage differentiated senescent t cells, characterized by a progressive reduction of telomere length leading to a state of proliferative arrest [ ] . with aging, health conditions associated with immune senescence, comorbidities (particularly noncommunicable diseases such as heart disease, cancers, and metabolic and autoimmune diseases), and pharmacological treatments affect the immune responses to both vaccines and infectious diseases. overall, as a result of immunosenescence, the elderly population is more susceptible to infections, particularly to influenza, streptococcus pneumoniae rsv, and group b streptococcus but also to opportunistic, re-emergent chronic infections such as herpes zoster as well as antibiotic-resistant nosocomial pathogens. the reduced adaptive immune response, together with altered innate cell function, such as chemotaxis, phagocytosis, signaling pathways, and intracellular killing, prevents the appropriate control of the initial inflammatory response elicited upon viral infection. for rna virus, such as coronavirus, different pattern recognition receptors (prr) are triggered on the innate cells during the early phases of infection. these include the endosomic toll-like receptor and and the cytosolic rig-i/mda- molecules, which recognize viral rna [ ] , and the cgas-sting pathway, which recognizes cytosolic dna [ ] activated by cellular damage and mitochondrial dna release caused by viral infection [ ] . the stimulation of these prr leads to the expression of type i ifn, a factor that limits viral replication through the stimulation of interferon-stimulated genes, and other inflammatory cytokines [ ] . for middle east respiratory syndrome (mers)-cov, the timing of type i ifn production appears to dictate the outcome of infection in mouse models, and its administration within day after infection was protective against lethal infection, while a delay in ifn production caused an inability to control viral replication, leading to cellular damage of airway epithelia and the lung parenchyma and an eventual lethal inflammatory cytokine storm [ ] . the latter response often predominates in older individuals and in aged mouse models of sars-cov- infection [ , ] . induction of innate immune responses is a crucial step in the pathophysiology of covid- disease (fig. ) . on one hand, it triggers the anti-viral host defense mechanisms necessary for elimination of infection, but on the other hand, it may contribute to hyperinflammation and tissue damage during the later stages of the disease in a minority of patients [ ] . this can be particularly relevant in the elderly population in which inflammaging, the state of chronic low-grade sterile inflammation [ ] , characterized by high serum concentrations of c-reactive protein (crp), il- , il- , and tumor necrosis factor (tnf)-α, can be present. tissue damage in covid- is mainly mediated by an excess of immune response to the virus, which results in a cytokine storm, with activation of the il- signaling pathway. the pathophysiology of sars-cov- infection has strong similarities to other severe viral lung infections caused by sars-cov- and mers-cov. one of the first published studies on clinical features of covid patients hospitalized in wuhan showed that proinflammatory cytokines and chemokines, such as tnf-α, granulocyte-colony stimulating factor (g-csf), interferon g a m m a -i n d u c e d p r o t e i n - ( i p - ) , m o n o c y t e chemoattractant protein- (mcp- ), and macrophage inflammatory proteins -α (mip- α), were significantly higher in patients admitted to the intensive care unit (icu) compared to those who were not in icu [ ] . immune pathology in the form of vascular and cutaneous lesions has also been widely reported [ , ] . the role of a dysregulated inflammatory response was proven in an animal model of sars-cov- infection using aged macaques. aged animals are more prone to develop severe disease and activate more readily the innate response, in particular the nf-kb pathway and proinflammatory cytokines such as il- and il- β, while not inducing significantly ifn-β response. the innate immunity activation is not due to the viral load, which is comparable among young and aged macaques [ ] . transcriptomic analysis performed in samples from subjects with severe covid- revealed the presence of low levels of type i and type iii interferon genes together with elevated levels of proinflammatory cytokines and chemokines, such as il- , il ra, ccl , ccl cxcl , cxcl , cxcl , and cxcl [ ] . which type of cells elicits this cytokine storm and the virological mechanisms behind this inflammatory reaction are still unclear [ ] . lung epithelial cells, alveolar macrophages, dendritic cells, and endothelial cells can effectively release the proinflammatory cytokines and chemokines, thus attracting monocytes, macrophages, and t cells to the site of infection [ ] . the overproduction of proinflammatory cytokines in the lungs can damage the tissue infrastructure, recruit macrophages that infiltrate air spaces, and generate the respiratory failure from acute respiratory distress syndrome (ards), which is recognized as the leading cause of mortality. meanwhile, the direct attack on other organs by disseminated sars-cov- , the immune pathogenesis caused by the systemic cytokine storm, and the microcirculation dysfunctions together may lead to multi-organ damage, even though whether sars-cov- can directly target organs other than the lung and how it can happen are aspects that need to be further investigated [ ] (fig. ) . together with the hyperinflammatory response, a significant lymphopenia, mainly related to cd + t and cd + t cells, which correlates with the severity of viral infection, was reported [ ] [ ] [ ] . the causes of this adaptive immunity suppression are still unclear. pulmonary recruitment of immune cells from the blood and the infiltration of lymphocytes into the airways may explain the reduction in blood. the well- systemic and local (lung) immune responses and their pathological role, following sars-cov- entry into the host are schematically represented. induction of innate immune responses is a crucial step in the pathophysiology of covid- disease, contributing to hyperinflammation and tissue damage during the later stages of the disease. infiltration of immune cells in the lungs causes overproduction of proinflammatory cytokines, which eventually damages the lung infrastructure, accumulation of macrophages in the air spaces and diffuse alveolar damage leading to acute respiratory distress syndrome (ards). furthermore, elevated levels of circulating proinflammatory cytokines can cause septic shock and multi-organ dysfunction. together with the hyperinflammatory response, overt disseminated intravascular coagulation has been reported and a significant lymphopenia, mainly related to cd + t and cd + t cells, has been observed, possibly due to pulmonary recruitment of lymphocytes from the blood. a possible immunopathological role can be mediated by non-neutralizing antibodies produced by b cells, which may enhance sars-cov- infection through antibody-dependent enhancement (ade), further exacerbating organ damage known age-related alteration of the immune function of t cell and b cells could lead to insufficient control of viral replication, thus increasing the macrophage infiltration and the lung injury (fig. ) . finally, a possible immunopathological role can be mediated by non-neutralizing antibodies produced by b cells that may enhance sars-cov- infection through antibodydependent enhancement (ade), further exacerbating organ damage. it has recently been shown that sars-cov- and the mers-cov take advantage of non-or subneutralizing antibodies and enter cells via surface cd a receptors, an fc receptor expressed on the surfaces of monocytes and alveolar macrophages. the antibody-cd interaction facilitates viral entry and infection, and activates intracellular signaling to upregulate proinflammatory cytokines [ ] . the complex and still unclear immunopathological mechanisms of sars-cov- infection, together with the progressive age-related decline of innate and adaptive immune responses, and the lack of a clear correlate of protection, make the design of vaccination strategies for older people extremely challenging (fig. ). an emerging class of instruments in the aging research is the development of markers capable of assessing the speed of the aging process. age is a major risk factor for a high number of diseases, and in general, it affects the fitness of each individual, including the capability of responding to vaccine administration and counteracting a severe infection [ ] . however, it is also evident that the elderly population is extremely heterogeneous, so while chronological age is useful to identify macroscopic risk classes, it is poorly informative within age classes to get individual information. biological age is thus useful to evaluate clinical parameters and health risks on the basis of the individual aging pace, which tend to be more heterogeneous in the elderly population. several established biological age markers have been generated based on both classical anthropometric, clinical, and biochemical parameters as well as on innovative molecular characterizations such as dna methylation and the composition of the n-glycan shell of circulating proteins [ ] . such biomarkers have shown in a number of studies that the aging pace is higher in the vast majority of the different elderly conditions, thus demonstrating that biological age assessment should be a critical information in a broad spectrum of clinical practices and in the development of strategies to tackle healthcare burden and emergencies. the detailed description of available biological age markers is out of the scope of the present manuscript, and an extensive overview is available in the review by jylhävä et al. [ ] . to date, biological age has not been assessed in the sars-cov- clinical setting, but it is noteworthy that biological age has been associated with all the most important risk factors related to a poor prognosis of sars-cov- infection. the field of elderly vaccination could benefit from biological age information, but also in this case, the available data are rare. in a study from gensous et al. [ ] , the whole genome methylation profile of pbmc was assessed in a group of volunteers of different ages who underwent influenza vaccination. the relationship between the vaccination response and the methylation profile was studied. while no difference in terms of biological age emerged in the study, an agedependent epigenetic remodeling emerged in elder non-responders. the study is limited owing to the very low number schematic interconnection between the main immune mechanisms elicited by the vaccination process, with the peculiarity of the elderly immune system-affected by both inflammaging and immunosenescence-and the still undefined correlates of protection from sars-cov- infection. the complex and still unclear immunopathological mechanisms of sars-cov- infection, together with the progressive agerelated decline of innate and adaptive immune responses, and the lack of a clear correlate of protection make the design of vaccination strategies for older people extremely challenging of analyzed subjects but confirmed that dna methylation is an informative instrument to be exploited in vaccination studies and strategies. immunobiography refers to the comprehensive immunological, clinical, socio-economic, and geographical history of each individual, and accounts for the large heterogeneity observed in the elderly regarding their health status, mirrored by their large individual variation in the responsiveness to vaccines. a major advantage of immunobiography is that it incorporates the most advanced conceptualization of immunosenescence which, according to the most recent literature [ ] , has to be considered as a context-and population-dependent phenomenon. accordingly, in order to be properly interpreted, agerelated changes of immune parameters occurring in an elderly person necessitate a variety of other additional data regarding sex/gender, demographic cohort, population/country, individual immunological history, anthropometric parameters, socioeconomic status and education, cmv serostatus, morbidity and co-morbidity, among others. it is of critical importance taking in consideration the elderly vulnerability to direct the rational design of vaccines designed for this target population. gender is a critical issue in both vaccination of the elderly and in the sars-cov- pandemic. the pandemic epidemiological data show clearly that the risk of severe disease and mortality is sharply higher in men than in women. men's hospitalization exceeds women by about %, indicating a significantly higher susceptibility of men towards severe sars-cov- infection. available data show that men outnumber women to times in terms of icu admissions [ ] [ ] [ ] . these numbers are concordant with the fatality rate that ranges between . and . men deaths for one women death. moreover, this unbalanced pattern is mirrored by the vaccine uptake, responses, and outcome in older-aged individuals. elderly women are indeed more responsive than men for several vaccine protocols recommended in older-aged individuals such as those against influenza, tetanus, pertussis, shingles, and pneumococcal infections [ ] . on the other hand, an influenza vaccination study reported that aged men antibodies had higher affinity than those produced by women. moreover, men seem to respond better to pneumococcal vaccination in two independent studies [ , ] . there is an impaired vaccination response in both old men and women with sex-specific weaknesses. the most striking data, however, is related to infection and all-cause mortality: indeed, in a number of reports, vaccine administration produces a sharper decrease of specific and all-cause mortality in vaccinated women compared to men, indicating that women have higher benefit from vaccination in the elderly [ ] [ ] [ ] . these data indicate the need to consider sex-specific vaccination protocols for the elderly population [ , ] and that the lack of such instruments could be critical in the sars-cov- pandemic since old men are both the most susceptible to severe sars-cov- infection and are those less likely protected by a possible sars-cov- vaccine [ ] [ ] [ ] . another factor that could affect vaccine response is the intestinal microbiota that plays a crucial rule in the regulation of the immune system and is highly affected by age [ ] [ ] [ ] [ ] . microbial community composition indeed is influenced by age, environmental and socio-economic factors, diet, gender, chronic infections, immunosuppressive chemotherapy, antibiotic treatment, or probiotic use [ , [ ] [ ] [ ] . the improvement in the nucleic acid sequencing obtained in the last years hits massively the microbiological research and promotes the analysis of heterogeneous microbiological ecosystems such as those that reside in humans. the characterization of such ecological niches opens to the new conceptualization of humans as metaorganisms (organisms composed of different organisms) to stress the tight interdependencies between the host and the microbiological species residing in different anatomical districts. gut microbiota changes with age and that is likely an important contributor and modulator of the inflammaging phenotype [ , ] . elderly people have less diverse gut microbiota and reduced beneficial microorganisms [ ] . the general imbalance of gut microbiota, called "dysbiosis," is associated with both frailty, a geriatric syndrome leading to increased vulnerability for adverse health outcomes, and systemic inflammation. since a hyperinflammation status has been observed in most severe cases of sars-cov- infection, it is possible that gut dysbiosis may influence the clinical manifestation in covid- infection [ , ] . interestingly, the gut microbiota has been shown to also affect pulmonary health through a bidirectional cross-talk between the gut microbiota and the lungs [ ] . along this "gutlung axis," microbial products can reach the lung through blood and modulate pulmonary immune responses [ ] , while inflammation processes occurring in the lung can impact on the gut microbiota [ ] . some studies have demonstrated that respiratory infections are associated with a change in the composition of the gut microbiota [ ] and the antibiotic treatment of mice for removing some gut bacteria has led to increased susceptibility to influenza virus infection in the lungs [ ] . since one of the severe clinical manifestations of covid- is pneumonia and progression to acute respiratory distress syndrome (ards), especially in elderly and immunecompromised patients [ ] , it can be speculated that sars-cov- infection can affect this gut-lung cross-talk which might influence the outcome of the clinical manifestation [ ] . moreover, even though respiratory symptoms represent the principal clinical presentation of covid- , clinical evidence suggests that the intestine may be another viral target organ. indeed, a high expression of ace has been observed in the brush border of intestinal enterocytes [ ] and, using a human small intestinal organoid system, it has been demonstrated that sars-cov- readily replicates into the enterocytes, resulting in the production of large amounts of infective virus particles [ ] . some reports show that sars-cov- rna can be detected in the stool of some patients of covid- [ , ] , and patients often present gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain [ ] . therefore, the characterization of the gut microbiota in patients with active sars-cov- intestinal infection could represent a striking aspect to investigate. these considerations on inflammaging, immunobiography, biological age, gender, and microbiota pertain to every vaccination strategy, but are particularly relevant for the development of vaccines against sars-cov- since it more seriously affects the elderly population and immunopathology is a crucial factor for the severity disease. need for the design of a sars-cov- vaccination strategies tailored for the elderly sars-cov- vaccines are urgently needed, and their design should take into consideration that the elderly population is the main target population for vaccination. while older adults are most likely to be severely affected by covid- , they also may be less responsive to vaccination. efficacy of vaccination in the elderly is indeed strongly reduced compared to that of younger adults [ , ] . sars-cov- vaccination strategies, tailored for the elderly, should take into consideration the delicate balance between immunosenescence/ inflammaging and the immunopathological aspects of the covid- disease (fig. ) . vaccine adjuvants and vectors should be specifically designed for stimulating the elderly immune system without exacerbating the inflammatory status [ ] . despite these considerations, the elderly are rarely included in vaccine clinical trials; in the last decades, the vast majority of randomized control trials did not include older adults and in particular frail older adults who are mostly at risk. we currently do not have full knowledge on the mechanisms of immunity to protect this population from sars-cov- [ ] . the development of a sars-cov- vaccine is extremely challenging, since we are faced with a novel virus, just emerged in humans, and correlates of protection have not yet been fully identified, even though the induction of neutralizing antibodies is presumed to be a crucial target for an effective vaccination (fig. ) . protection in older individuals against influenza virus appears to require higher neutralization titers than in younger individuals [ ] , and this issue might need to be addressed for sars-cov- . the knowledge obtained from the vaccine development efforts for mers and sars-cov- can be of high value for sars-cov- , although no vaccines are licensed for these coronavirus strains [ ] . memory cd + t cells, induced by infections with other coronavirus and capable of responding to sars-cov- , have been detected in - % of sars-cov- unexposed donors [ , ] . the characterization of these cross-reactive t cells in the elderly and their impact on the immunogenicity of vaccine candidates should be taken into consideration in the ongoing covid- vaccination studies. sars-cov- vaccine candidates based on different vaccine platforms have been developed, and about candidates have been tested in pre-clinical experiments, according to the who landscape documents of covid- candidate vaccines (https://www.who.int/publications/m/ item/draft-landscape-of-covid- -candidate-vaccines) (fig. ) . information on the specific sars-cov- molecules selected as vaccine antigens is limited, even though most candidates aim to elicit neutralizing antibodies against the spike (s) protein and its receptor-binding domain (rbd), as already performed with the sars and mers vaccines. a wide range of both innovative and traditional technology platforms has been deployed, including nucleic acid (dna and rna), recombinant viral vectors (replicating and non-replicating), recombinant protein combined with adjuvants, and live attenuated or inactivated virus [ ] . some of these platforms were already tested in human studies for sars-cov- virus, such as inactivated virus, dna and soluble s proteins [ ] [ ] [ ] , or for mers-cov [ ] . the most advanced candidates for sars-cov- entered in human clinical testing with unprecedented rapidity employ nucleic acid (both mrna and dna), recombinant vaccine vectors (human or chimpanzee adenovirus vectors), subunit s protein combined or not with different adjuvants, and inactivated sars-cov- virus. other novel platforms based on the use of synthetic modified antigen presenting cells (apc) or cytotoxic t lymphocytes are also under study (fig. ) . the platforms using mrna, nonreplicating viral vectors, and inactivated sars-cov- virus have already reached the clinical trial phase iii. some of the different platforms used may be tailored for specific population subtypes, such as the elderly, children, pregnant women, or immunocompromised patients [ ] . in this regard, some of the ongoing clinical studies have specifically taken into consideration the older population, by including vaccination arms with people aged > years. a schematic diagram of the ongoing phase i and ii clinical trials that have included older adults is reported in fig. . enrolling older adult volunteers will help to better understand vaccination outcomes among the older population, who are most at risk of complications from covid- . the ongoing clinical studies based on mrna technology (mrna- from moderna n. nct , and bnt from biontech se, n. nct ) aim to evaluate the safety, tolerability, immunogenicity, and potential efficacy of different sars-cov- rna vaccine candidates in the adult population, with a specific attention to older people (n.-n. releases. nih clinical trial of investigational vaccine for covid- begins. . https://www.nih.gov/newsevents/news-releases/nih-clinical-trial-investigationalvaccine-covid- -begins). the lipid nanoparticleencapsulated mrna- vaccine, which encodes for the full-length s protein, is currently evaluated in a dose-ranging study in the adult population ( - years old), and in participants from to and > years of age (fig. ) . similarly, the large dose-finding study with the bnt biological component ( estimated participants) based on the administration of mrna coding for the full-length s protein, or for the two smaller receptor-binding domains, is going to test the immunogenicity in adults ( - years) and older adults ( - years) . an ongoing phase i/iia trial (n. nct ) is also aimed at evaluating the safety, tolerability, and immunological profile of the ino- vaccine that, exploiting the dna technology, contains a plasmid encoding the full-length s glycoprotein. the ino- vaccine is administered by intradermal injection followed by electroporation in healthy adults aged to years. another platform that is currently specifically tested in older people is based on the adenovirus type vector that encodes the s protein from the sars-cov- strain (trials n. - - ; pactr ; nct ; chictr and nct ; fig. ). different studies are ongoing, and one conducted in canada is a doseescalation designed study, from the younger adults ( to < ) to the older adults ( to < ). another huge phase / study (n. nct ) is aimed at determining the efficacy, safety, and immunogenicity of the candidate covid- vaccine based on the chimpanzee adenovirus vector (chadox ncov- ) in healthy uk volunteers, specifically divided in adults ( - years old), elderly (over the age of ), and children ( - years old). the chadox platform has already been shown to be effective in the established rhesus macaque model of sars-cov- infection [ ] . in this pre-clinical study, a single dose of chadox ncov- has protected six [ ] . moreover, the chadox has been used to develop investigational vaccines against several pathogens, including the closely related coronavirus responsible for the mers [ ] . adenovirus-based vectors are characterized by a broad range of tissue tropism that covers both respiratory and gastrointestinal epithelium, the two main sites that express the ace- receptor of sars-cov- , even though a possible immunodominance mediated by vector genes rather than the transgenes should always be considered [ ] . using the traditional recombinant protein technology to express the spike protein, a trial sponsored by clover biopharmaceuticals aus pty ltd. (n. nct ) is assessing the safety, reactogenicity, and immunogenicity of multiple doses of scb- administered with as adjuvant, or with cpg plus alum adjuvants. data will be separately analyzed on adult ( to years of age) and elderly ( - years of age) healthy subjects enrolled in the study. in another study, the s protein has been administered with the advax adjuvant (n. nct ), a potent and safe immunopotentiator composed of delta inulin [ ] . four trials are testing in the elderly population the inactivated sars-cov- virus (n. nct ; chictr ; chictr ), and one of these has been specifically performed only in people > years (n nct ; fig. ). numerous other vaccine developers have indicated plans to initiate human testing in . despite the several vaccine candidates (fig. ) , challenges including the need for optimizing antigen design and adjuvant formulation define the number of doses needed, induce the optimal immune response without exacerbating the inflammatory and antibody-dependent response involved in possible lung disease, and fully define correlates of protection and duration of immune responses have to be considered [ ] . finally, a general consideration for the sars-cov- vaccine development regards safety issues that could arise with covid- vaccines developed under the strong pressure of the pandemic situation. animal studies on vaccines for sars-cov- and mers-cov report possible adverse effects mediated by vaccine-induced antibodies that have poor or no neutralizing activity [ ] . safety and efficacy are two indissoluble properties of a vaccine to be administered to billions of people globally and need to be accurately evaluated for every sars-cov- candidate. the efforts in the development of covid- vaccines can benefit from the availability of most advanced tools and high-throughput technologies to decipher the effective immune responses in the older population and the correlates of protection. recent advances in systems biology integrating clinical, immunologic, and omics data can help to identify stable and robust markers of vaccine response and move towards a better understanding of sars-cov- vaccine responses in the elderly. machine/statistical learning applied to multi-omics data from clinical studies promises to revolutionize vaccine development by illuminating the mechanistic drivers of protective immunity. the high-performance data acquisition methods in molecular and cellular biology push the field of bioinformatics for the development and use application of the immunobiography approach could inform the stratification of elderly subjects and guide the implementation of vaccination strategies designed for specific elderly population clusters [ ] . mathematical modeling allows the combination of different networks involved in biological aging such as epigenetic networks, cell-cell networks, and population genetics and can allow to generate hypothesis on response to treatment or vaccination [ ] . recent progress in mathematical modeling can be utilized to generate biomarker models for prediction of disease and also response to vaccination taking into consideration biological age. currently, computational models have been applied to immunology data, for example, for the analysis of a high-dimensional dataset in vaccination studies [ , ] , but these models are limited to particular aspects [ , ] . there is the potential for these models to become more sophisticated and to predict how responses to pathogens and vaccines are affected by pre-disposing factors [ , ] . the systems vaccinology approach has been applied to characterize the immune response to different vaccines providing the proof-of-concept evidence of the capacity of systems approaches to delineate "molecular signatures" predictive of vaccine responses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this approach has also been applied to identify molecular signatures induced by immunization with the rvsv-zebov ebola vaccine, recently approved for human use. systems analysis has been conducted integrating clinical, immunologic, and omics data in clinical trials with different doses and in different continents (vianello et al. submitted, santoro et al. submitted). despite the great efforts made, unfortunately, many of the most useful clinical and multi-omics datasets are siloed in local databases to protect participant privacy and data confidentiality. creation of secure, faircompliant, federated learning databases in which predictive biological and mathematical models based on ai/ machine/statistical learning can be developed, refined, and tested on distributed datasets would have an enormous impact in supporting a rational vaccine development. sars-cov- vaccines are urgently needed, and their design should take into consideration that the elderly are the main target population for vaccination. the pandemic is stimulating the research on vaccine development, and this should be a tremendous opportunity to specifically include age and gender as critical factors for vaccination approaches and effectiveness. while older adults are most likely to be severely affected by covid- , they also may be less responsive to vaccination. in the ongoing tremendous efforts for covid- vaccine development, only a limited number of clinical trials have included the older fraction of the population in the study design, and the platforms used are not specifically designed considering the peculiarity of the elderly immune system. indeed, vaccination strategies tailored for the sars-cov- infection in the elderly should take into consideration the delicate balance of immunosenescence and inflammaging with the immunopathological aspects of the sars-cov- infection, such as the cytokine storm reported in severe covid- . therefore, the possible overlap between the factors hampering vaccination effectiveness in the elderly and those that boost the virulence and worsen the prognosis of sars-cov- infection should be carefully taken into consideration. thus, vaccine formulations, such as adjuvants and vectors, should be specifically designed for stimulating the elderly immune system without exacerbating the inflammatory status. the ongoing efforts in covid- vaccine development should fully exploit the availability of high-throughput technologies and recent advances in systems biology to decipher the effective immune responses in the older population and identify correlates of protection to guide towards sars-cov- vaccine strategies optimally designed to protect the older population. authors' contributions ac, pg, and dm, fs drafted the work; dm, rr, and cf revised it critically for important intellectual content; all the authors approved the version to be published. funding open access funding provided by università degli studi di siena within the crui-care agreement. this work was supported by commission of the european communities, horizon framework programme, grant number (transvac ), and russian ministry of science and education agreement no. - - - . conflict of interest the authors declare that they have no conflict of interest. consent for publication the authors are responsible for the correctness of the statements provided in the manuscript. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to 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affiliations key: cord- -kira mz authors: strike, philip c.; wardle, jane; steptoe, andrew title: mild acute inflammatory stimulation induces transient negative mood date: - - journal: j psychosom res doi: . /s - ( ) - sha: doc_id: cord_uid: kira mz objective: this study aims to assess the mood changes induced by mild acute inflammatory stimulation (typhoid vaccination). methods: using a double blind study design, healthy volunteers underwent baseline assessments of mood, financial strain and work stress and were randomised to injection of salmonella typhi vaccine or placebo injection. mood, symptoms and body temperature was assessed by a modified version of the profile of mood states at , , , , and h post injection. results: typhoid vaccination induces no increases in physical symptoms or temperature. mood improved over the day in the placebo but not in the vaccine condition. negative changes in mood following injection were correlated with chronic stress (financial strain) in the vaccination condition (r=−. , p<. ). conclusion: a mild acute inflammatory stimulus induces transient negative mood, and responses were modulated by chronic stress. implications for depressed mood in physical illness are discussed. people with infections or inflammation can exhibit profound changes in behaviour. initially, this was thought to be a psychological response to feeling unwell, but more recently the cytokines released as part of the inflammatory response have been implicated. ''cytokine associated sickness behaviour'' is the term used for this collection of symptoms such as malaise, lethargy, anorexia, hyposomnia, inactivity, impaired cognitive function and anhedonia [ , ] . this theory is supported by the fact that the syndrome can be reproduced by infusion of either recombinant cytokines, or molecules that induce cytokine synthesis (e.g., lipopolysaccharide) [ - ] . agents which antagonise cytokines abolish the syndrome [ ] . in animal models, the cytokine interleukin- (il- ) plays a central role, but il- and tumour necrosis factor alpha (tnfa) are also involved. inflammatory responses may also be relevant to depression. it has been known for several years that major depression is frequently associated with activation of the inflammatory response system, involving increased produc-tion of il- , il- receptor antagonist (il- ra), il- and tnfa [ ] , although the evidence relating subclinical depressive symptoms with inflammatory responses is equivocal [ , ] . proinflammatory cytokines affect the brain's metabolism of hxdroxy tryptophan ( -ht) by either directly or indirectly stimulating the enzyme indoleamine , -dioxygenase leading to a peripheral depletion of -ht providing a possible intermediate in the link between inflammation and depressive symptoms [ ] . these inflammatory responses may not be epiphenomena, but actively contribute to depressive symptoms and negative mood states. this has been documented most consistently in studies of mood responses following cytokine-based treatments for cancer. immunotherapy with il- or interferon a causes sadness, loss of interest and other depressive symptoms, and responses correlate with the magnitude of responses of endogenous cytokines [ , ] . depressive symptoms have also been reported following interferon a treatment of hepatitis c [ ] . these effects are rapid, and may be mediated by the effects of cytokines on the hypothalamic -pituitary-adrenocortical (hpa) axis [ ] . there is also evidence that antidepressants have antiinflammatory effects [ ] . taken together, these findings raise the intriguing possibility that depressive symptoms in as disparate illnesses as coronary heart disease, multiple sclerosis and rheumatoid arthritis may be due in part to cytokine activation. the effects of cytokines on mood can be investigated experimentally by administering stimuli that acutely induce inflammatory cytokine release. reichenberg et al. [ ] assessed the effects on mood and cognitive function of salmonella abortus equi endotoxin injection in volunteers. rapid increases of -to -fold in il- and tnfa occurred within - h of administration in endotoxin but not placebo-injected groups. increases in anxiety and depressed mood were reported over the same time-scale in the endotoxin group, and the changes were correlated with the magnitude of cytokine responses. however, a problem with this model is that endotoxin administration leads to fever and malaise. a larger increase in body temperature was recorded by reichenberg et al. [ ] in the endotoxin than placebo groups and although no symptoms of sickness were reported, appetite was substantially inhibited. many participants report flu-like symptoms in response to endotoxin. this makes it difficult to know whether behavioural changes were due to the inflammatory challenge or mild illness induced by the endotoxin itself. in the present study, we used a milder model of experimental inflammation, involving standard typhoid (salmonella typhi) vaccination. this method has been used by vallance et al. to induce inflammatory responses that have been studied in relation to atherogenesis [ ] . increases of four-to sixfold in il- occur, together with il- ra increases of averaging -fold after h [ ] . importantly, body temperature is not affected, and participants do not report any sickness or malaise. the use of vaccine rather than endotoxin may afford a better insight into the changes in mood in response to immune challenge in human healthy volunteers. the first aim of this study was therefore to examine the effects of typhoid vaccination on the subsequent mood of healthy volunteers in a placebo-controlled double blind trial. based on the evidence linking proinflammatory cytokine release with negative moods, we hypothesised that typhoid vaccination would induce a negative mood response. since moods are not constant over the day, changes in mood following vaccination cannot be definitively ascribed to the effects of an intervention in the absence of a comparison group. half the participants were therefore injected with placebo, and the mood changes in this group taken as the reference against which the active vaccination group could be compared. in the light of the kinetics of proinflammatory cytokine responses observed in earlier studies, we hypothesised that compared with placebo, vaccination would induce transient negative moods in the - h following treatment, in the absence of rises in systemic body temperature or symptoms of illness. psychological stress plays an important role in the way the immune system functions [ ] . plasma concentrations of inflammatory cytokines vary with acute stress and with chronic stressors such as care-giving and work strain, and increase with acute stress [ - ] . in a study of experimentally induced upper respiratory infection, background stress was found to correlate positively with il- responses to infection [ ] . it is conceivable that chronic stress might influence cytokine responses to vaccination, and thereby moderate mood responses. the supplementary aim of this study was therefore to assess associations between chronic stress and mood responses to typhoid vaccination. there are many aspects of chronic stress that could potentially be investigated, but in the interests of brevity and appropriateness for the population we limited measurement to two commonly assessed variables-financial strain and job demands. we hypothesised that background chronic stress would modulate cytokine responses, and thereby associations with mood responses in the vaccination but not placebo conditions. twenty-six volunteers ( female, male) between and years old were recruited from university staff. volunteers were all nonsmokers, had no recent illnesses, were on no regular medication, and were not pregnant. none had any history of any mental illness or had received typhoid vaccination within the last months. no volunteers had any allergies or had had any previous problems with vaccinations. all felt well on the morning of the study and had had no stressful events that morning. volunteers refrained from excessive exercise and from any caffeine or alcohol from the day before the study. the study was presented to participants as an examination of immune responses to vaccination (as assessed by saliva samples). ethical approval was given by the university college london medical research ethics committee. two measures of chronic stress were included in this study. the first was the financial strain questionnaire originally developed by pearlin et al. [ ] and utilized in the whitehall ii and other studies [ , ] . eight items (e.g., ''do you have enough money for the kind of clothing you and your family should have?'') were presented, with response options ranging from = no difficulty to = very great difficulty. ratings were summed, so scores could range from to , with higher ratings indicating greater financial strain. the second measure was an index of work demands derived from the demand/control model of work stress, as used in the whitehall ii study [ , ] . four items (e.g., ''do you have to work very intensively?'') were administered, each of which was rated on a four-point scale ranging from = often to = never/almost never. scores were converted to a scale from - , where indicates minimum and maximum demands. mood and symptoms were assessed with a modified version of the profile of mood states (poms) [ ] as described elsewhere [ ] . this consisted of items, each of which was rated on a five-point scale from = not at all to = extremely. six high loading items were taken from the vigour, tension -anxiety, depression -dejection, and confusion scales of the poms, and five items from the fatigue scale. in addition, there were four symptoms (feverish, aching joints, nauseated, and headache), and three filler items. participants were asked to rate how they felt at that moment. body temperature was measured with a sublingual digital thermometer, and blood pressure and heart rate using an electronic sphygmomanometer. all studies began at the same time ( a.m.) to ensure uniformity. the study was performed in a double blind, placebo controlled manner. after initial measurements of temperature, blood pressure, heart rate and mood, randomisation to either vaccine or placebo ( . ml of normal saline) was performed by a coordinator and . mg salmonella typhi vaccine (typhim vi, aventis pasteur msd) or placebo in identical -ml syringes was administered intramuscularly by a qualified nurse or doctor into the nondominant deltoid muscle of the volunteers. both vaccine and placebo were stored in the fridge before administration. there were no complications of vaccination or placebo injection. thereafter administration of questionnaires and clinical observations was performed by researchers blinded to the experimental group of participants. volunteers were assessed at , , , , and h after vaccination. at each of these times, they filled in the modified poms scale and had temperature, blood pressure and pulse measurements. scores for the five poms scales were computed for the baseline sample by summing ratings on individual items. the scores on the fatigue dimension were scaled up to conform with those of the other moods. ratings of the four symptoms were summed to produce a total score. vaccine and placebo groups were compared on baseline mood, symptoms, chronic stress and physiological measures using t tests. changes in symptoms, body temperature, blood pressure and heart rate over the study were analysed with repeated measures analysis of variance with group (vaccine, placebo) as the between-subject factor, and time (base, , , , , and h) as the within-subject factor. to avoid the multiple comparisons when assessing mood responses, we calculated total mood scores as recommended in the poms manual, by summing the negative items (tension, depression, confusion, and fatigue), and subtracting them from the positive vigour score. changes in mood from baseline were then computed and analysed by repeated measures analysis of variance. finally, correlations between chronic stress measures and changes in mood were computed separately for the vaccine and placebo groups. all analyses were carried out using spss v. . results are presented in terms of means fstandard deviation. the background characteristics and baseline measures are summarized in table . there were no significant differences between vaccine and placebo groups in age, financial strain, work demands, or in physiological measures. the mood scores showed a high positive vigour score coupled with low ratings on negative scales, indicating that participants were generally in positive moods. mood scores did not differ significantly between groups, with the exception of fatigue (t = . , p <. ), where ratings were higher in the placebo than vaccine group. however, the scores in both groups were extremely low (possible range - ), so the difference was trivial. none of the participants reported appreciable symptoms preinjection. the mean symptom ratings and body temperature assessed at the seven time points are shown in fig. . there was no significant change over time or difference between groups in either variable. the absence of any rise in body temperature with vaccination indicates that the procedure did not induce even mild illness. the mean symptom scores never exceeded one at any time. since the symptom scores theoretically range from a low of zero to a maximum of , it is evident that neither treatment elicited physical symptoms throughout the study. there were some variations in cardiovascular measures. heart rate showed a significant cubic trend over the study period [ f ( , ) = . , p < . ]. the mean heart rate at baseline ( . f . bpm) decreased to a low of . f . bpm after h. this was followed by a modest rise to . f . bpm after h, with a final fall to similarly, there were no differences between groups in systolic or diastolic pressure over the day. the analysis of changes in total mood score between baseline and later samples show a significant quadratic product moment correlations between mood change and the two background stress variables (financial strain and job demands) were computed separately for the vaccination and placebo groups. no significant effects were found in placebo condition. in the vaccine group, there was a significant negative correlation between change in mood over the first hour following injection and financial strain (r = À . , p < . ). this effect is illustrated in fig. . participants who reported high financial strain showed negative changes in mood over the first hour following vaccination, while positive mood changes were apparent in most participants who reported low financial strain. this suggests that background chronic stress levels may have affected the mood responses to typhoid vaccination. the principal hypothesis tested in this study was that the mild inflammatory response induced by typhoid vaccination would induce transient negative mood. it was necessary to test this hypothesis in a double-blind placebo-controlled fashion, since researchers' and participants' expectations might have influenced the pattern of results. in addition, mood can change over the day irrespective of stimuli such as vaccination, so the effects of this stimulus were tested against the fluctuations observed in the placebo group. mood was assessed using a standard measure, and we analysed the total poms rather than individual scales to provide a global impression of mood change. the results confirmed our expectations about the effects of the inflammatory stimulus. mood improved over the day in the placebo group, reaching a plateau - h postvaccination ( : a.m. to : p.m.), before deteriorating slightly later in the day. the increase in positive mood from the start of the study may have been due partly to time of day, and partly to the relief at having completed the injection. by contrast, mood did not improve over the day in the vaccination condition. the difference between groups was greatest over the first h following vaccination, with some convergence later in the day. by the end of the study ( : p.m.), mood had deteriorated below baseline in the vaccination group, while remaining elevated in the placebo condition. thus, relative to the placebo condition, the vaccination group had a more negative mood following injection. the mood differences were significant but modest. this compares with result of reichenberg et al. [ ] , in which both depression and anxiety increased in absolute terms in response to endotoxin. the difference may be due to the greater intensity of the inflammatory stimulus in this earlier study. in the present study, there was no change in body temperature, no febrile responses, and symptom reports were low and stable. in addition, the study by reichenberg et al. [ ] was more invasive in nature, involving venous cannulation and continuous temperature measurement through a rectal probe. it is notable that anxiety increased in the placebo as well as endotoxin condition in this earlier study. our results suggest that vaccination may be a preferable way of studying the relationship between immune challenges, mood and behavioural responses. a previous study of rubella vaccination has shown increases in depressed mood in a proportion of the young women tested who were initially seronegative [ ] . cytokines were not measured in this study, so it cannot be definitely concluded that mood changes were due to their activation. we are currently carrying out a study to address this issue. however, the time course of responses is consistent with the cytokine changes that would be expected. in previous studies using typhoid vaccination, the maximum rise in il- and il- ra occurred at -to -h postvaccination [ , ] . cytokine levels then fall gradually, but do not reach baseline values even -h postvaccination. these responses parallel the changes in mood observed in the present investigation. the second important finding was that the magnitude of mood change following vaccination was associated with one of the two measures of chronic stress. participants reporting greater financial strain showed larger negative changes in mood in the hour after vaccination. no such association was observed in the placebo group, so it is not a nonspecific response caused by negative affectivity reporting biases. there was also no association with the second measure of chronic stress, job demands. the explanation may be that job demands were relatively low, while financial strain was high on average in this sample compared with other population samples in which the measures have been used. the association with financial strain needs to be confirmed in a larger study involving cytokine measures, so as to discover whether inflammatory or some other responses to vaccine mediate effects. if the association is confirmed, it could be due to one of two processes; either background stress magnifies inflammatory responses to standard challenges such as vaccination thereby increasing the mood change, or it affects the extent of mood change following a fixed inflammatory response. individual differences in mood response to rubella vaccination have also been reported, with greater depressed mood in young women of lower socioeconomic status [ ] . a number of studies of responses to fixed doses of coronavirus or influenza have shown that the likelihood of infection is affected by psychosocial factors such as life stress, negative affect, and social isolation [ , ] . the observation by cohen et al. [ ] that the il- increases following infection with influenza were correlated with psychological stress is consistent with the possibility that the present finding was mediated through individual differences in il- response. studies relating negative moods with inflammation may be relevant to depression in coronary heart disease. inflammation is now thought to be a fundamental factor in the pathophysiology of vascular disease [ ] . acute coronary syndromes are also associated with marked inflammatory responses, with larger increases in il- h followed by raised il- [ ] . the acute increase in il- ra and il- over the first days following hospitalization with unstable angina is associated with adverse clinical outcomes [ ] . depression in the days following admission with acute coronary syndromes has also been shown to predict poor outcome in many studies [ ] , and several mechanisms to explain the association are being investigated [ ] . an additional possibility is that depressed mood is due to inflammation in these patients, and that heightened levels of inflammation underlie the association between depression and adverse cardiac outcomes. we studied a relatively homogeneous group of university workers, the majority of whom were postgraduate academics. they were relatively young, physically fit and active. the study was small, with insufficient numbers to test sex differences in responses. future studies of larger samples will involve cytokine as well as mood measures, to determine whether the magnitude of inflammatory responses is directly associated with mood changes following vaccination. behavioral effects of cytokines cytokine-induced sickness behaviour: mechanisms and implications cytokine-induced sickness behavior: where do we stand? lowered serum dipeptidyl peptidase iv activity is associated with depressive symptoms and cytokine production in cancer patients receiving interleukin- -based immunotherapy acute effects of recombinant human interleukin- on 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and antidepressive treatment cytokine-associated emotional and cognitive disturbances in humans acute systemic inflammation impairs endothelium-dependent dilatation in humans prevention of inflammation-induced endothelial dysfunction. a novel vasculo-protective action of aspirin the effects of psychological stress on humans: increased production of pro-inflammatory cytokines and a th -like response in stress-induced anxiety life stress, mood disturbance, and elevated interleukin- in healthy older women inflammatory cytokines, socioeconomic status, and acute stress responsivity immune dysfunction associated with chronic professional stress in nurses psychological stress, cytokine production, and severity of upper respiratory illness the stress process hardship and depression health inequalities among british civil servants: the whitehall ii study low job control and risk of coronary heart disease in whitehall ii (prospective cohort) study manual for the profile of mood states the effects of exercise training on mental well-being in the normal population: a controlled trial influence of socioeconomic status on behavioral, emotional and cognitive effects of rubella vaccination: a prospective, double blind study prevention of inflammation-induced endothelial dysfunction: a novel vasculo-protective action of aspirin social ties and susceptibility to the common cold psychological stress and susceptibility to the common cold atherosclerosis-an inflammatory disease inflammation and atherosclerosis. circulation increasing levels of interleukin (il)- ra and il- during the first days of hospitalization in unstable angina are associated with increased risk of in-hospital coronary events depression in patients recovering from a myocardial infarction depression as a risk factor for cardiac mortality and morbidity: a review of potential mechanisms this study was supported by the british heart foundation and the medical research council. we are grateful to bev murray, lindsay emmerson and lena brydon for assistance in vaccination. key: cord- -dkm y authors: tam, theresa w. s. title: preparing for uncertainty during public health emergencies: what canadian health leaders can do now to optimize future emergency response date: - - journal: healthc manage forum doi: . / sha: doc_id: cord_uid: dkm y it is clear that the risk for epidemics with high health and socio-economic impacts remains but there will be many unknowns at the start of future responses to these events. this article highlights principles and practices to assist health leaders in preparing for uncertainty, including integrating scalability to ensure response activities can be more easily adapted to suit evolving needs; assessing risk and capabilities to inform planning for appropriate response measures; and considering overall flexibility and adaptability of plans, systems, and resources. ultimately, being prepared for “disease x” is about applying the approaches that we have learned from previous events, using evidence-based practices to develop and strengthen foundational capacities, so that we are able to respond to the unanticipated in proportionate and appropriate ways. lessons from emerging health events, past and present, remind us that threats to human health are always present and will continue to be influenced by factors such as climate change, the human-animal interface, and international travel. however, emerging diseases come with many unknowns and even known diseases can behave in unexpected ways. during the initial preparation of this article, a novel coronavirus causing disease in humans was emerging at the humananimal interface in china. the covid- is a harsh reminder that uncertainty is part of the emergence equation and we will always be challenged to rapidly confirm the knowns and to respond as best we can, despite the unknowns. it helps to prepare with this in mind. severe acute respiratory syndrome (sars) was our first "disease x" of the st century. the world health organization recently coined this term to represent uncertainty as a critical planning element in preparedness for a serious international epidemic and specifically to encourage preparedness activities that account for uncertainty. there are many sources of uncertainty that are essentially the "who, what, when, where, why, and how" characteristics of a public health emergency. the "who" might be unexpected at-risk groups, such as persons with obesity who developed severe illness during the h n influenza pandemic. the "what" could be unexpected outcomes such as microcephaly in infants born to mothers with zika virus infection during pregnancy. the "where" could be an unexpected location for disease emergence, such as the h n pandemic influenza that began in north america, rather than asia, as had been anticipated and planned for. finally, in the event of an unknown pathogen such as sars in and now with covid- , the outbreak response must run parallel with a rapid gathering of international evidence (clinical, laboratory, epidemiological, etc), meaning that the level of uncertainty is dynamic and the response will need to be adjusted as and when we know more. the purpose of this article is to identify some basic principles for dealing with uncertainty in the context of a public health emergency, to provide some examples of how these principles in combination with past experience have advanced preparedness within the health sector in canada, and to stimulate thinking regarding what health leaders can do to further improve preparedness across the health sector in the immediate as the covid- situation unfolds and going forward in the longer term. in terms of preparedness planning, assumptions help establish a "starting point"-a direction to quickly proceed (eg, using specific established/routine practices) until a need to adjust the course is identified. they give responders an indication of what real-time data to collect, or what to watch for, to either validate the planning assumptions or signal that a change in approach is needed. for example, an assumption used in influenza pandemic planning posits that the novel influenza virus will be transmitted from person to person in the same way seasonal influenza is transmitted. healthcare providers will thus know what infection prevention and control (ipc) precautions to utilize, which builds confidence in responders when dealing with unknowns. this starting point also helps to inform how ipc measures can be scaled up or down if reality proves to be different from the planning assumption. therefore, assumptions are a foundational component for the development of preparedness and response plans and essential for incorporating flexibility. in the emergency preparedness and response context, scalability is used to convey the need for response activities to be dynamic. to manage demands and risks by scaling up (eg, adding more resources, enhancing active surveillance activities) or scaling down when there is evidence to indicate that specific response actions are no longer needed to achieve response objectives. a key lesson learned from past responses is that uncertainty and/or risk aversion can lead to overcompensation during a response (eg, inappropriate use of limited resources, responder burnout, or angst when trying to de-escalate). overcompensation can be avoided by ensuring there is sufficient content in guidance, plans, and emergency exercises to demonstrate how and when the response will be scaled up or down based on risk assessments and specific data analyses that build confidence and reduce risk aversion. another key principle involves taking a risk management approach to preparedness and response by conducting risk and capability assessments, to inform planning and response measures, and to identify gaps or enhancements that need to be addressed. specifically, this involves making an assessment of current resources available to mitigate and respond to the risk. assumptions can be used as a starting point to create scenarios against which risks and capabilities can be identified and assessed. for example, a planning scenario might include a person who presents to the emergency department complaining of nausea, weakness, and fever. to prepare for uncertainty, planners must consider variables in the scenario. for example, if this person had recently returned from a country with an ongoing ebola outbreak, would the existing triage system be sufficient to identify, assess, and rapidly contain a possible ebola case? this type of assessment helps identify specific risks, such as not including ebola in the intake differential diagnosis for ill travellers returning from an affected area. likewise, scenarios can facilitate a discussion regarding what capabilities are currently in place to mitigate this risk. however, it is also important to identify what might change the risk profile in the scenario. for example, the toronto sars case that triggered the first hospital-based outbreak was initially missed because they had no travel history. it was only learned later that the patient had been exposed to the true index case, who did have a significant travel history but was not seen in a healthcare setting. , this flagged a gap in our risk mitigation-that the capability to rapidly identify, and therefore contain, a sars case that had no travel history was lacking. to close this gap, triage questions also needed to include questions about close contact with an ill traveller. unfortunately, not all risks can be accounted for so it is important to consider what unknowns might significantly impact a risk and what planning can be done to account for them. emergency planners with expertise in risk and capability assessment need to work with healthcare leaders to determine how changes to risk levels will be addressed in real time, when to change course, and whether the capabilities are in place to deal with the requirements of the "new course." public health preparedness efforts have been largely based on previous infectious disease outbreaks, models, and scenarios. it is important to consider the flexibility and adaptability of current plans, systems, and resources when preparing for any health emergency; this is a key principle in "all-hazard" emergency preparedness. the preparedness efforts and response resources that have been developed and used for infectious disease outbreaks are now being utilized for other public health threats. borne out of the sars and h n experience, new federal/provincial/territorial governance structures were established to oversee the overall public health response. these governance structures have in turn been leveraged to respond to non-infectious disease national response, including most recently for the national epidemic of opioid-related deaths in canada. adaptable response systems are agile enough to incorporate learnings in real time and make adjustments to response activities through feedback loops. such systems can quickly establish new inter-sectoral connections to meet immediate specific response needs while increasing general response capabilities. specifically, the urgency of the opioid crisis led to the mobilization of new and pooled resources that ultimately established a timely surveillance and reporting network with coroners and medical examiners. built on an infectious disease outbreak response model, this network can potentially be leveraged for rapid mortality surveillance for emerging health events beyond opioids. there are also international efforts underway to invest in platform technologies for vaccines and therapeutics that can be adapted to target new pathogens once they are identified. utilizing sustainable, flexible governance structures and resources ensures the response system is well exercised and able to adapt to uncertainties, while supporting readiness for other health threats and emergencies. the idea of identifying "lessons learned" after the conclusion of an emergency response is also a key principle of preparedness for future events. the challenge for health leaders is to ensure that the lessons identified actually translate to better understanding and ultimately an improved ability to respond. importantly, this process has to ensure that learnings are not forgotten during peacetime or lost over time with staff turnover. the importance of risk communication, building and maintaining public trust and confidence, and cross health sector engagement are just a few of the key lessons that have been identified from past emergency responses. the covid- response is now showing us in real time the growing role of the internet and social media in risk communication. early, frequent communication of uncertainties is vital to building and maintaining public trust and confidence. we have learned that perception is reality and that being transparent in risk communication is essential. this means it is vital for health leaders to be forthcoming from the outset, clearly stating what we know and what we do not know, while reassuring the public that we will provide new information as and when we know it. despite the relatively limited spread of sars in canada, it served to highlight the importance of supporting and maintaining cross health sector preparedness for a seamless and comprehensive health response. this starts with increasing the number of astute frontline practitioners who are sensitized and equipped to practice "think, tell, test." this means the first line of defence is primed to think about the possibility of an emerging pathogen, promptly tell local public health authorities, and efficiently work with clinical colleagues, hospital administrators, public health, and laboratory partners to ensure early detection and rapid containment through appropriate and timely testing. continuing to build and maintain a skilled and engaged workforce is essential to a robust and flexible response system. health leaders can ensure that the lessons learned from past experiences are passed on through regular training and exercises to those entering or taking on new roles within the workforce. engaging across the health sector on a regular basis, in order to re-confirm roles and responsibilities, conduct joint risk and capability assessments, foster research, and provide updates on the status of preparedness activities, is also key to ensuring health sector preparedness and maintaining an ongoing state of readiness. there are operational (eg, medical evacuation and domestic transportation) and logistical (eg, supply chain and stockpiling issues) aspects of a response that require crosssectoral engagement to address, ideally in advance of an emergency. we have seen the benefit of having contracts in place, for example, for influenza pandemic vaccine supply during the h n pandemic and more recently for international medical evacuation capacity during the ebola outbreak in west africa. we have also witnessed the need for advanced preparedness to engage in real-time research to ensure the response is as evidence based as possible. this has translated into advanced planning by organizations such as the canadian institutes of health research, for example, to foster rapid ethics reviews during an emergency response and supporting the canadian immunization research network to conduct vaccine clinical trials in real time. extending the health sector preparedness to include other sectors/disciplines (eg, social services, critical infrastructure, regulatory authorities, public safety, justice) is critical to a seamless response. engaging communities and considering their contexts, culture, and perspectives in the preparedness for public health events is essential to the building of trust and public cooperation with health authorities during a response. post-sars, emergency management practices have been adopted more widely by the health system in canada. there is also a greater recognition of the significant social and economic impacts of public health emergencies and the importance of mitigating these impacts through mechanisms that enhance capabilities. health leaders would be wise to ensure that emergency management, multi-sectoral coordination, and mutual aid capabilities are well integrated and exercised within their institutional response planning. health facilities must also develop and practice their business continuity plan as a complement to their pandemic plan, given that the health of the workforce may be significantly impacted while workload demand is high. the public health agency of canada, established following sars as the national coordinating body for health emergencies, has made significant investments in order to increase emergency preparedness and response capacity in canada, build on the lessons learned from past experiences, and facilitate cross-sector preparedness and resiliency. this work has been multi-focal, ranging from the production and updating of plans, protocols, and technical guidance to conducting training, stockpiling vaccines, and therapeutics, to running exercises to test current knowledge and capabilities. many of these efforts are intended to increase the level of preparedness across the breadth of the healthcare sector, not just public health, and not just for emergencies originating in canada. examples include enhancing public health laboratory and border screening capacity; establishing mutual aid and information sharing agreements ; and clarifying roles, responsibilities, and procedures (eg, how to request and receive aid and emergency provisions) during emergencies. canada has met the international health regulations core capacity requirements and was ranked th in the world in the global health security index, assessing global health security and capabilities. although there is a strong existing system in place, ongoing work is still needed to achieve a state of flexible and scalable readiness for the next public health emergency. as we begin a new decade, we must maintain constant vigilance as epidemics are predicted to become more frequent, more complex, and harder to prevent and contain. health leaders need to prepare for uncertainty during an emergency response by developing, enhancing, and exercising resources-whether it be plans, people, or other resources-that can be flexible, scalable, and that are built on lessons learned and evidence-based practices. health leaders are well poised to see gaps and reflect on persistent challenges and recurring themes, while looking beyond their scope of influence to find creative solutions. working from the ground up, health leaders should train staff in emergency management principles, share corporate memory, incorporate lessons learned, and build confidence through regular exercises. exercises and training should consider the response to complex health emergencies, including pandemics, which are rapidly evolving and may last many months. health leaders should also consider engaging with health professional regulatory bodies to explore whether and how regulatory requirements might be adapted, streamlined, or otherwise expedited during an emergency. finally, staff must be encouraged to contribute to contingency planning by identifying concerns and repositioning them as uncertainties to be addressed. although it can be difficult to convince decision-makers to invest upstream in non-specific emergency preparedness resources, it is important to present the downstream benefits including risk reduction, medium-to long-term cost savings, and operational resilience. one means of fostering support is to build the understanding that investment of time, energy, and resources can pay off during normal operations, not just during large-scale health emergencies. emergency planning can help ensure business continuity whenever there is an unexpected surge in resource demands against the backdrop of everlimited, finite supplies. finally, recognize that the opportunity to address critical gaps is never more urgent than during an emergency. every event is an opportunity, given heightened political attention and investment in health capacity during a crisis. we need to build on these gains to both improve routine practice and better prepare us for future response. throughout this call to action to strengthen health security, i would urge health leaders to integrate a health equity lens and seek meaningful engagement from the communities they serve in order to build trust and enable a collaborative and effective response during times of uncertainty. the covid- is the latest "disease x" but it will not be the last. health leaders, now more than ever, need to gather new knowledge, adapt response activities, and meaningfully engage with all partners across government, research, and the public at large to respond, as flexibly and effectively as possible, to this new health threat in canada and around the world. available at: https:// www.who.int/activities/prioritizing-diseases-for-research-anddevelopment-in-emergency-contexts risk factors for severe outcomes following influenza a (h n ) infection: a global pooled analysis world health organization. zika epidemiology update lessons learned review: public health agency of canada and health canada response to the h n pandemic sars: lessons learned from toronto severe acute respiratory syndrome (sars): a year in review canadian pandemic influenza preparedness: planning guidance for the health sector fifteen years post-sars: key milestones in canada's public health emergency response covid public health emergency of international concern (pheic) global research and innovation forum: towards a research roadmap multi-lateral information sharing agreement (mlisa) joint external evaluation of ihr core capacities of canada mission report global health security index: building collective action and accountability the author would like to thank jill sciberras, jeannette macey, and teresa leung for their assistance in preparing this manuscript. theresa w. s. tam, bmbs (uk), frcpc https://orcid.org/ - - - key: cord- - q wkwrf authors: chattopadhyay, saborni; chen, jui-yi; chen, hui-wen; hu, che-ming jack title: nanoparticle vaccines adopting virus-like features for enhanced immune potentiation date: - - journal: nanotheranostics doi: . /ntno. sha: doc_id: cord_uid: q wkwrf synthetic nanoparticles play an increasingly significant role in vaccine design and development as many nanoparticle vaccines show improved safety and efficacy over conventional formulations. these nanoformulations are structurally similar to viruses, which are nanoscale pathogenic organisms that have served as a key selective pressure driving the evolution of our immune system. as a result, mechanisms behind the benefits of nanoparticle vaccines can often find analogue to the interaction dynamics between the immune system and viruses. this review covers the advances in vaccine nanotechnology with a perspective on the advantages of virus mimicry towards immune potentiation. it provides an overview to the different types of nanomaterials utilized for nanoparticle vaccine development, including functionalization strategies that bestow nanoparticles with virus-like features. as understanding of human immunity and vaccine mechanisms continue to evolve, recognizing the fundamental semblance between synthetic nanoparticles and viruses may offer an explanation for the superiority of nanoparticle vaccines over conventional vaccines and may spur new design rationales for future vaccine research. these nanoformulations are poised to provide solutions towards pressing and emerging human diseases. vaccination is a process of introducing antigenic material to activate an individual's immune system to develop adaptive immunity to a pathogen. it has proven to be the most successful and cost-effective prophylactic measure against infectious diseases. vaccines have been responsible for eradicating or effectively managing many major diseases, including smallpox, measles, mumps, rubella, diphtheria, tetanus, pertussis, polio, and yellow fever [ ] . despite the many examples of successful vaccines, many disease threats, such as human immunodeficiency virus (hiv), tuberculosis, dengue, and malaria, lack an effective prophylactic measure. thus, development of new vaccine formulations and technology remains an ongoing quest [ ] . conventionally, vaccine formulations are comprised of biological materials in the form of attenuated viruses, killed pathogens, or subunit protein antigens. each platform has its distinct advantages and shortcomings, frequently presenting a trade-off between safety and efficacy. for example, live attenuated vaccines are excellent at inducing long lasting protective immunity and strong immune response, but their "live" nature poses safety concerns, especially to individuals who may be immunocompromised. on the other hand, subunit vaccines are safer to administer, but they are less immunogenic and less effective at eliciting cellular immunity for disease protection (fig. ) . emerging technology and formulations to combine the advantages of live attenuated and subunit vaccines thus continue to be developed with the aim of maximizing vaccine safety and potency. in the last decade, advances in materials engineering have opened up new avenues for ivyspring international publisher innovative vaccine designs. in particular, synthetic nanoparticles have been widely adopted for vaccine development [ , ] . these particles, typically to nm in diameter, have shown effective immune potentiation in vivo, capable of inducing strong humoral and cellular immune responses against antigen targets. compared to live attenuated viral vaccines, synthetic nanoparticles promise better safety profiles because of their non-replicating nature. they are also readily amenable to different infectious pathogens [ ] . from a holistic view, many advantages of nanoparticle vaccines may be attributed to their intrinsic semblance to natural viruses (fig. ) . many viral features, such as nanoscale morphology, repetitive multivalent antigen display and controlled antigen/adjuvant delivery are conducive to immune processing on both physiological and cellular levels. as our immune system has evolved to effectively respond to infectious viral nanoparticles, it should not come as a surprise that nanoformulations adopting virus-like features can be more potent than conventional subunit formulations. to this date, numerous nanoparticle-based platforms have been examined for vaccine applications, and many have demonstrated encouraging efficacy against many pressing infectious threats, including malaria, influenza, ebola, and hiv [ ] [ ] [ ] [ ] . also worth noting is the rapidly expanding field of nanoparticle-based anticancer vaccines, which exploits nanotechnology for enhanced induction of antigen-specific cellular immune responses against oncologic malignancies [ ] [ ] [ ] [ ] . many design considerations in nanoparticle vaccines can be traced to the fundamental interaction dynamics between viruses and the immune system [ ] . upon close scrutiny, virus-like mimicry in terms of size, geometry, antigen display, and adjuvanticity can be commonly observed among vaccine nanoformulations. in light of the emerging landscape of nanoparticle vaccine research, this review highlights some of the underlying principles behind the advantages of nanoparticle vaccines. in the first section, commonly used biomaterials such as lipids, polymers and inorganic compounds are reviewed in the context of vaccine applications. in the sections that ensue, various nanoparticle design aspects are explored from a virus-mimetic perspective including lymphatic delivery, antigen display, and interaction with immune cells. lastly, we highlight conjugation strategies that couple subunit protein antigens with nanocarriers. this review also summarizes the various strategies for conjugating antigens/adjuvants with nanocarriers. the review aims to provide a holistic view of the recent advances for the next generation of immunomodulatory vaccines. towards mimicking viral features, preparing nanoparticulates in the range of to nm in size is the first step prior to subsequent vaccine development. it is therefore imperative to have an understanding of the various materials and their properties for nanoparticle preparations. the diverse range of nanomaterials for biomedical research encompasses a wide range of organic and inorganic matters, such as phospholipids, polymers, metal, silica, and carbon (fig. ) . these nanomaterials can be modified to provide a functionalized and stable interface for different biomedical applications. in the context of vaccination, many nanocarriers have been modified to trigger specific immune responses analogous to natural defense mechanisms against viral invasion. the following section summarizes the different synthetic nanoparticles used in vaccine applications with an overview on their functionalizability and immunological adjuvanticity. liposomes are self-assembled, spherical vesicles consisting of a phospholipid bilayer and an aqueous inner core [ , ] . they are prepared from phospholipids with fatty-acid chains of defined length and saturation. the choice of phospholipids and the addition of cholesterol influence liposomal stability and performance [ ] [ ] [ ] . the vesicles can have a unilamellar structure with one layer of phospholipids or a multi-lamellar structure with several concentric phospholipid shells. this platform is highly versatile for cargoes delivery and allows for incorporation of hydrophilic molecules in the inner core and hydrophobic molecules within the phospholipid bilayers. antigens and adjuvants may thus be incorporated into liposomes based on their lipophilicity [ , ] . the lipid bilayered structure of liposomes is structurally analogous to enveloped viruses, which are formed from budding of infected cells and are wrapped in pieces of cell's plasma membrane. the inherent semblance to viral particles may help explain the innate adjuvanticity of liposomes upon incorporation with protein and peptide antigens [ ] . numerous antigen targets, ranging from toxoid, viral antigens, and bacterial antigens, have been observed to elicit enhanced humoral responses following liposomal incorporations [ ] . mechanistic studies have shed light on the liposomes' adjuvanticity. for instance, liposomes have been shown to be capable of modulating cd + t cell mediated immune responses [ ] , reflecting facilitation of antigen processing via the classical mhc i and mhc ii pathways. liposomes have also been reported to promote the development of t cell-independent b cell immune responses [ ] and have potential to promote long-term immunity through the development of t-cell memory [ ] . in addition to their innate adjuvanticity, liposomes have been incorporated with a plethora of adjuvants ranging from small molecules [ , ] , glycolipids [ ] [ ] [ ] [ ] , oligodeoxynucleotides [ ] [ ] [ ] [ ] , to cyclic dinucleotides [ , ] . the structural fluidity of liposomes offers flexibility in platform modification towards virus mimicry. for example, the development of virosomes, which are fusion vesicles prepared from viral particles and synthetic liposomes, is an adaption that highlights the fundamental similarity between liposomes and viruses. derived from the membrane vesicles of viruses, virosomes consist of liposome-like lipid vesicles and viral envelope glycoproteins. the fusion vesicles retain some viral characteristics (i.e. epitope presentation) and have been applied for both drug delivery and vaccination purposes [ , ] . the applicability of this platform as a vaccine candidate has been demonstrated extensively using influenza virus-derived virosomes [ ] [ ] [ ] [ ] . in studies that apply virosomes for anti-influenza vaccination, derivatized influenza virosomes were shown to be non-infective as their genetic materials were removed. upon in vivo administration, these virosomes were rapidly uptaken by antigen presenting cells and in turn activated numerous other immune cells [ ] [ ] [ ] [ ] . immunization with virosomes was reported to reactivate influenza-specific memory cd + t cells that subsequently supported the proliferation of antigen-specific effector cells [ ] , leading to enhanced anti-influenza immune responses [ ] . besides influenza-based virosomes, induction of cytotoxic t lymphocyte responses has been demonstrated with a sendai virus-based carrier system loaded with ovalbumin (ova). it was demonstrated that sendai virosomes fused with ova elicited stronger ctl responses against the model antigen [ ] . liposomes may also be modified to enhance the stability of carriers in a manner analogous to how viruses employ viral matrix proteins to stabilize their lipid envelope [ ] . in a study by moon et. al., interbilayer-corsslinked multilamellar vesicles have been prepared by covalently crosslinking multiple layers of liposomes via thiol chemistry [ ] . these multi-lamellar liposomes showed added stability owing to short covalent bonds that crosslinked adjacent lipid layers within the vesicle walls. this modification served to address some of the shortcomings of liposomes, facilitating enhanced antigen encapsulation and increased particle stability. also belonging to the class of lipid-based nanoparticles are an emerging class of lipoplexes, which consist of cationic lipid derivatives for the complexation with nucleic acids [ , , ] . immunostimulatory rnas or mrnas encoding specific antigen targets have been formulated into lipoplexes to trigger immune responses. the function of these lipoplexes can be likened to the immune response induction by rna viruses [ ] . as rnas are delivered intracellularly by lipoplexes, they activate innate immune receptors, leading to an upregulation of type i interferons, which may further trigger a multitude of downstream immunological pathways [ ] . concurrently, these rnas are translated into antigens of interest, thereby promoting an antigen-specific immune response. lipoplexes carrying the mrna of target antigens have recently been shown in a phase i clinical trial to induce strong cellular responses against tumor antigens in humans [ ] . a wide variety of polymers have been applied to the development of nanoparticle vaccines. synthetic polymeric nanoparticles are typically solid particles between nm to nm. they have been an attractive platform for vaccine delivery as antigens and adjuvants can be either surface attached to or interior loaded inside these nanoparticles [ ] [ ] [ ] [ ] [ ] . in particular, controlled release of biomolecules is one of the strongest advantage of polymeric nanoparticles, the release kinetics of which can be regulated by tuning of the copolymer composition and molecular weight [ ] . typically, polymeric nanoparticles are formed via self-assembly of amphiphilic copolymers under an emulsion or nanoprecipitation process [ ] [ ] [ ] . notable polymeric nanoparticles in vaccine development are as follows. poly(lactic-co-glycolic acid)(plga) is one of the most commonly used polymers for biomedical applications [ ] . plga-based nanoparticles are known to be biodegradable, non-toxic and non-immunogenic. upon administration, the polymer is degraded into lactic acids and glycolic acids in vivo to be safely metabolised in the body. in vaccine applications, plga nanoparticles provide a robust platform for antigen functionalization and have been used to carry antigen derived from various pathogens. through surface conjugation or interior encapsulation, antigens including those derived from plasmodium vivax [ ] , hepatitis b virus (hbv) [ ] , bacillus anthracis [ ] , tetanus toxin [ ] , model antigens such as ovalbumin [ ] have been associated with plga-based delivery systems for enhanced immune potentiation. adjuvants have also been encapsulated or chemically attached to the plga polymer backbone for controlled delivery to enhance immune responses [ ] [ ] [ ] [ ] . natural polymers based on polysaccharide, such as pullulan [ ] [ ] [ ] , alginate [ ] , and chitosan [ ] [ ] [ ] [ ] [ ] [ ] , have been explored for nanoparticle vaccine preparations. in particular, chitosan-based nanoparticles have been widely studied due to their biocompatibility, biodegradability, non-toxic nature and their ability to be easily modified into desired shapes and sizes [ ] [ ] [ ] . more intriguing is the recent discovery of chitosan's immune potentiating mechanism via the dna sensing cgas-sting pathway [ ] . the sting pathway is triggered in the host cells by many viral pathogens and plays a major role in both the innate and adaptive immunity [ ] . upon interaction with dendritic cells, chitosan induces type i interferons in a cgas and sting-dependent fashion, mediating cellular maturation and the promotion of th responses. chitosan-based nanoformulations have been widely adopted for vaccine development, examples of which include vaccines against clostridium botulinum type-a neurotoxin [ ] , neospora caninum [ ] , hbv [ ] , and newcastle disease [ ] . the polysaccharide polymer has also been applied to enhance the potency of dna vaccines against viral, bacterial, and parasite infections [ , , ] . other polymeric nanoparticle platforms include poly(γ-glutamic acids)(γ-pga) nanoparticles [ , ] , polystyrene nanoparticles [ , ] , and poly-alkyl acrylate based nanoparticles [ , ] . γ-pga are comprised of amphiphilic poly(amino acid)s, which self-assemble into nano-micelles with a hydrophilic outer shell and a hydrophobic inner core. they are generally used to encapsulate hydrophobic antigens. polystyrene nanoparticles are solid particles consisting of polymerized styrene monomers that can be conjugated to a variety of antigens. poly-alkyl acrylate based nanoformulations have been prepared with poly (methylmethacrylate) (pmma), poly (ethylacrylic acid) (peaa), poly (propylacrylic acid) (ppaa) and poly (butylacrylic acid) (pbaa). studies have shown that polyacrylate-based nanoparticles show an inherent adjuvanticity with several model antigens [ , , ] . the type of polymer used in the nano-formulation strongly affects the structure, properties and applications of the particles. as gold is chemically inert, gold nanoparticles (aunps) have been studied extensively for biomedical applications. aunps can be synthesized reproducibly with a high level of precision, offering an ultrastable metallic core for further modifications. capable of being modified with a plethora of chemical functional groups such as thiols, phosphines, amines, and by extension protein antigens, aunps have been used broadly in many vaccination studies. a multitude of biomolecules, ranging from toxin, viral antigens, bacterial antigens, parasite proteins, and tumor antigens, have been coupled with gold nanoparticles to enhance immune responses. aunps vaccines have been explored in clinical trials for hepatitis b and malaria vaccinations [ , ] , and they also allow anchoring of nucleic acids for dna vaccine applications [ ] . aunps have been modified with different adjuvants as well, such as chitosan [ ] and cpg oligodeoxynucleotides [ , ] , and many alternative surface functionalization strategies have been explored to further enhance immune potentiation. aunps surface modified with cetyltrimethylammonium bromide (ctab), poly(diallydimethylammonium chloride) (pddac), and polyethyleneimine (pei), have been used as a dna vaccine adjuvant for human immunodeficiency virus (hiv) [ ] . these modifications were found to further boost the adjuvanticity of the aunp carrier. gold's unique malleability makes aunps an attractive platform for vaccinology studies [ ] . aunps can be fabricated into myriad of shapes (i.e. sphere, rod, cube, etc.) [ ] with tunable yet sharply distributed size range between - nm [ ] . this morphological tunability adds an additional dimension of virus mimicry with the introduction of non-spherical particles. gold nanorods, for instance, have been used as a vaccine vector for the delivery respiratory syncytial virus (rsv) f protein [ ] . these non-spherical particles bear resemblance to rod-like viruses that can also be frequently observed among different virus genera [ ] . the size modularity of aunps also allows for examining the impact of particle size on vaccine delivery, which will be discussed in details in later sections of this review. also worth noting is the more recent discovery that aunp size and shape can modulate the inflammatory responses at the cellular level [ ] . in a study that compares gold nanospheres, gold nanorods, and gold nanocubes, niikura et al. showed that whereas the nanospheres and the nanocubes induced tumor necrosis factor-α (tnf-α), il- , il- , and granulocyte macrophage colony-stimulating factor (gm-csf), the nanorods induced interleukin- β and interleukin- via an inflammasome-dependent mechanism [ ] . this shape-dependent immunological property may be due to the differing surface energies associated with different nanoscale features, which may promote varying levels of stress upon cellular uptake [ ] . silica nanoparticles offer a range of particle sizes and shapes via controlled synthesis using sol-gel chemistry. an abundance of silanol groups on silica nanoparticle surface allow for functional modifications for increasing specific cellular recognition, facilitating attachment of specific biomolecules, and modulating cellular uptake [ ] [ ] [ ] . nanoscale pores can be integrated into silica nanoparticles, yielding mesoporous silica nanoparticles (msns) with more versatile cargo-carrying capacity for vaccination purposes [ , ] . the pore size and surface functionalization of msns can be modified to control the encapsulation and release of antigens or adjuvants of choice [ ] [ ] [ ] [ ] . use of silica nanoparticles in vaccine applications include formulations against snake venom, e. coli [ ] , porcine circovirus [ ] , hiv [ ] , and other model antigens [ ] . carbon-based nanoparticles, such as carbon nanotubes and graphene, have also been studied as vaccine carriers [ , ] . owing to their high aspect ratio and large surface area, these carbon-based nanoparticles may carry a high proportion of antigens for immune activation. in a study on anticancer vaccination, villa et al. conjugated single-wall carbon nanotubes (swcts) with wilm's tumour protein, an antigen upregulated in many cancers. antigen conjugated swcts showed good uptake by dendritic cells and macrophages in vitro, and subcutaneous immunization with these swcts promoted induction of antigen-specific igg. in contrast, the free peptide formulation failed to induce an antibody response against the tumor antigen [ ] . in another study, graphene nanosheets were used to deliver antigens to facilitate antigen cross-presentation to cd + t cells [ ] . xu et al. also demonstrated the use of a dual polymer-modified graphene formulation as an effective adjuvant to enhance the immunogenicity of h. pylori derived antigen (alum-ure b) [ ] . semiconductor quantum dots have been applied for vaccine applications, offering a versatile platform for nanoparticulates antigen delivery with the added benefit of particle tracking. in a study by cambi et al., antigen-conjugated quantum dots of virus-like dimension were shown to combine antigen delivery and bioimaging functionalities, enabling immune cell tracking following antigen uptake. it presented the possibility of tracking antigen-presenting cells in vivo [ ] . along similar lines, sen et al. showed that quantum dots can induce t cell proliferation and ifn-γ production in vivo while being traceable within lymph nodes [ ] . other nanoparticle platforms with imaging functionality, including polymeric upconversion nanoparticles [ ] , and iron oxide nanoparticles [ ] , have also been studied as antigen carriers for vaccine applications. these platforms offer the capability for examining the function mechanisms behind the benefits of nanoparticle vaccines. one of the biggest advantages of nanoparticle vaccines is their ability to efficiently drain and accumulate to lymph nodes for enhanced immune processing. the lymphatic system is a subset of the circulatory system that consists of a complex network of vessels, tissues and organs. the lymphatic vessels conduct lymph between different parts of the body. as blood exits the blood vasculature to become interstitial fluid, the lymphatic system provides a return route for this interstitial fluid to the blood vessels in the form of lymph. in the process, it regulates fluid balance within tissues [ ] . underlying the lymphatic system are numerous lymph nodes that scatter throughout the body. these lymph nodes are the homing sites of migratory dendritic cells that present engulfed antigens. resident in these peripheral lymphoid organs are an abundance of specialized macrophages and other lymphocytes which play a major role in antigen capture and processing for adaptive immune responses. as lymph passes through lymph nodes, resident macrophages further capture passing antigens [ ] . in other words, the lymphatic system functions as a filter system of bodily fluids, trapping antigens in the lymph nodes for immune processing. viruses and virus-like nanoparticulates can accumulate in lymph nodes via both cell-mediated lymphatic delivery and convective lymphatic transport. the cell-mediated transport is mediated primarily by migratory dendritic cells, which take up antigens outside of the lymphatic system (i.e. skin and lung) and enter lymph nodes via either high endothelial venules or lymph vessels [ , ] . in comparison to small protein antigens, viruses and nanoparticle vaccines are more favorable to this hitchhiking mechanism. their particulate nature promotes receptor-mediated, complement-mediated, or other intracellular uptake mechanisms [ , ] . antigens associated with nanocarriers are routinely observed to be more efficiently uptaken by dendritic cells compared to soluble antigens, thereby enabling more effective lymph node delivery and cross presentation [ , [ ] [ ] [ ] [ ] . on the other hand, the nanoscale morphology of viruses and virus-like particulates allows them to move freely in lymphatic vessels to draining lymph nodes. upon lymph node entry, a special subset of macrophages is responsible for the capture of these nanoparticulates. in a study on lymphatic tracking of viruses by junt et al., macrophages in the subcapsular sinus and in the medulla of lymph nodes were shown to be responsible for the lymph node accumulation of subcutaneously administered inactivated vesicular stomatitis virus, adenovirus, and vaccinia virus [ ] . depletion of these macrophages resulted in an enhanced virus level that circulated back to the blood, highlighting both viruses' convective transport in the lymphatic system and the macrophages' role in virus filtration. this gatekeeper function by the lymph node-resident macrophages serves to limit blood-borne infection and promote immune processing. exploiting the same transport mechanisms aimed at detaining viruses, nanoparticle vaccines can effectively target immune cells in lymph nodes, delivering antigens or adjuvants following administration in peripheral tissues [ , , ] (fig. a) . the benefit and focus on lymph node targeting also explain why particulate vaccines are most commonly administered subcutaneously as opposed to the conventional intramuscular route for subunit vaccines; free lymphatic drainage and access to immune cells in lymph nodes following injection into the interstitium likely outweigh the "depot effect" afforded by the intramuscular route [ , ] . lymphatic targeting by nanoparticles have also been observed following administration via different delivery routes, including pulmonary [ ] , oral [ ] , intra-peritoneal [ ] routes. such favorable distribution profile allows tailoring of nanoparticle vaccines towards targeting specific immunological compartments against different infectious threats. studies on the influence of nanoparticle size on lymph node targeting began in the s as scientists aimed to maximize lymphatic delivery of drugs for treating metastatic cancer. it was generally observed that following subcutaneous injections liposomes smaller than nm were able to enter the lymphatic capillaries whereas larger liposomes remained at the injection sites [ ] [ ] [ ] . in a study by oussoren et al. using isotope labelled liposomes between to nm in diameter in lymphatic tracking, lymphatic uptake was found to be inversely proportional to the liposome size. nm, nm, nm and nm liposomes had approximately %, %, %, and % of the injected dose entering the lymphatic system, respectively. curiously, despite higher lymphatic entry by smaller liposomes in the study, liposome accumulation in the draining lymph node was similar across the differently sized formulations. it was found that the majority of the small liposomes, which consisted of egg-phosphatidylcholine and egg-phosphatidylglycerol, passed through the lymph node and were ultimately captured by the liver and the spleen [ ] . the result highlighted the dynamic relationship between particle size and lymph node accumulation; as smaller particles are more likely to enter the lymphatic system, they are also more likely to evade the filtering mechanism of lymph nodes. the authors showed that incorporating phosphotidylserine, a lipid more susceptible to macrophage recognition and capture, increased the lymph node accumulation by -fold. other lipid modification strategies, such as steric stabilization and ligand functionalization, have also been reported to influence the lymph node accumulation of liposomes following lymphatic uptake [ ] . later studies using solid nanoparticles on examining the effect of particle size on lymph node delivery echo earlier findings based on liposomes. using polystyrene beads, manolova et al. confirmed the size-dependent particle transport in the lymphatic system. upon subcutaneous delivery, large particles between to nm were found to be associated with dendritic cells from the site of injection, and small nanoparticles between to nm could drain freely in the lymphatic system, effectively targeting lymph node-resident dendritic cells and macrophages [ ] . in a study by reddy et al. on examining nanocarriers as a vaccine delivery platform, pluronic-stabilized polypropylene sulfide nanoparticles of well-defined sizes were investigated. using fluorescence microlymphangiography, the investigators showed a clear distinction between nm and nm particles regarding their lymphatic uptake. following injection into mouse tails, nm particles were efficiently drained to the lymphatic vessels, whereas the interstitial transport of nm particles was less efficient. in contrast to prior studies with liposomes, the nm particles also showed higher lymph node accumulation, resulting in a -fold enhancement in lymph node delivery as compared to the nm particles (fig. b, c) [ ] . such enhanced lymph node delivery, which was absent in earlier liposomal studies, could be attributed to both increased colloidal stability of the polymeric nanoparticles and the particles' ability to elicit complement activation. as the polypropylene sulfide particles were functionalized with surface hydroxyl groups to trigger the proteolytic cleavage of c complement protein, the danger signal associated with the complement activation could facilitate macrophage uptake upon lymph node entry [ ] , thereby reducing particle escape from the lymph node's filtering mechanism. the size-dependent lymph node targeting was also observed in other solid particle platforms. in a study by gao et al. that compared nm and nm gold nanoparticles as antigen carriers, nm gold nanoparticles yielded . -fold higher lymph node accumulation in terms of total gold delivery. upon conversion to particle number and total particle surface area, nm gold nanoparticles had an enhancement of -fold and . -fold as compared to nm particles, respectively [ ] . in general, nanoparticles between and nm, a length scale that coincides with viral particles, can exploit interstitial flow for lymphatic delivery. within this length scale, smaller particle size tends to favor lymph node accumulation. given the privilege of nanocarriers in lymphatic transport, nanoparticles have been shown to enhance the delivery of target antigens to lymph nodes and resident immune cells for processing and immune activation. in the aforementioned study on polypropylene sulfide particles, for instance, reddy et al. demonstrated increased resident dendritic cell activation in the lymph node by ovalbumin-conjugated nanoparticles. in their animal study, strong anti-ovalbumin humoral response was observed [ ] , highlighting the benefit of nanoparticle-mediated lymph node delivery on enhancing antigen processing. moon et al. also showed nanoparticle vaccines can promote preferential accumulation of antigens in the draining lymph nodes and enhance expansion of antigen-specific t cells. using interbilayer-crosslinked multilamellar vesicles (icmvs), a lipid-based nanoformulation consisting of multiple layers of lipid vesicles interconnected via thiol chemistry, the investigators demonstrated enhanced antigen delivery to total dcs, macrophages and plasmacytoid dcs in the lymph nodes [ ] . interestingly, liposomes of comparable sizes were much less effective in shuttling antigens to draining lymph nodes in the same study. this observation highlighted the importance of nanoparticle stability in vaccine design as the icmvs were more colloidally stable than liposomes. icmv-mediated antigen delivery resulted in significantly higher humoral and cellular responses as compared to the free antigens and the liposomal formulations. the effect of nanoparticle carrier on antigen transport was also shown in a study by chen et al., who demonstrated effective vaccination against coronaviruses using gold nanoparticle-adsorbed viral antigens. these antigen-coated nanoparticles were structurally analogous to coronaviruses in terms of size and antigen display. immunofluorescence quantification showed that viral spike proteins delivered with nm gold nanoparticles increased lymph node delivery by approximately -fold compared to free spike proteins. these virus-like particles showed high immunogenicity in both murine and avian models and enhanced anti-viral iga and igg titers and cellular immune responses in comparison to free protein antigens and a commercial wiv vaccine [ ] . in addition to delivering antigens for more effective immune processing, nanoparticles have been extensively applied to localize immunological adjuvants to lymph nodes for improved safety and potency. while conventional adjuvants such as alum have been widely employed clinically to promote humoral responses [ ] , more recent development in adjuvant research has identified many pathogen-associated molecular patterns (pamps) as promising adjuvant candidates towards promoting both humoral and cellular responses [ ] . these molecular danger signals are often similar to viral pathogens regarding their immune potentiating mechanisms, triggering innate immunity and in turn facilitating adaptive immune responses. many pamps (i.e. cpg-odn, poly(i:c), and cyclic dinucleotides) as well as other molecular agonists of toll-like receptors (tlrs) (i.e. imiquimod and resiquimod) are known to induce strong immune responses. however, their potency poses safety concerns over the likely induction of systemic inflammation. nanoparticle-based delivery thus offers a desirable strategy in guiding these immunological modulators to lymph nodes, increasing their effective concentration and reducing their systemic reactogenicity. in one example, nunh et al., constructed a ph-degradable nanogel platform ligated with imidazoquinoline (imdq), a tlr / agonist, and showed retention of the adjuvant at the injection site and the draining lymph node. the adjuvant in its free form elicited systemic inflammatory responses, but this side effect was largely obviated with the nanogel formulation. the targeting effect of the nanoformulations also resulted in recruitment of monocytes to the draining lymph node. a large number of immune cells, including b cells, dcs, and macrophages were shown to readily take up these adjuvant-loaded nanogels [ ] . in another study by ilyinskii et. al., synthetic vaccine particles encapsulating resiquimod (r , a tlr / and tlr ligand) augmented humoral and cellular immune responses to both soluble and nanoparticle-delivered proteins compared to that observed with free adjuvants. the adjuvant-loaded nanoparticles promoted local cytokine induction in the lymph nodes and reduced systemic cytokine production observed with free r . moreover, while injection of the nanoformulation led to sustained expressions of ifn-γ, il- , and il- β in lymph nodes after hours, free r induced only modest levels of il- and ifn-β [ ] . cpg-odn, an agonist of tlr- , is another adjuvant that's frequently coupled with nanocarriers for vaccination studies [ , , , , , ] . some of the primary advantages of nanoparticle-based cpg formulations include strong t cell responses, dosage sparing, and reduced systemic side effects. such formulations have been commonly applied in anticancer vaccination efforts owing to the need for high cell-based immune responses for effective tumor containment. transport of viral antigen and immune-potentiating adjuvants by viruses to immune cells is a highly coordinated event as viral particles shuttle both antigen targets and adjuvanting nucleic acids simultaneously. such antigen/adjuvant coordination, or its absence, has been shown to strongly influence immune cell activation [ ] . in addition, the immune system can respond to viruses through multiple pamps, including glycoprotein, dna, dsrna, and ssrna, activating a broad spectrum of signalling pathways for heightened antiviral immunity [ ] . the synthetic flexibility of nanoparticles has thus been exploited to emulate the co-delivery capacity of viral pathogens to boost immune responses. in some cases, antigen and adjuvant are localized on the same nanoparticles for synchronized delivery. ma et al. incorporated a hepatitis b surface antigen (hbsag) and cpg adjuvant onto plga nanoparticles via conjugation with dopamine, allowing the particles to display both the viral antigen and the immune activator. the study showed that the pathogen-mimicking particle enhanced the recruitment of immune cells to the injected site, activated bone marrow derived dendritic cells, and induced strong humoral and cellular immune responses [ ] . in another study conducted by kuai et al., a disc-like synthetic high-density lipoprotein (shdl) was applied to integrate both target antigens of cd + t cells and cpg-odn for anticancer vaccination. the nanodisc drastically improved the co-delivery of antigens and adjuvants to lymph nodes compared to soluble vaccine and induced a -fold enhancement in antigen-specific cd + cytotoxic t-lymphocyte response as compared to the free peptide and adjuvant control. besides incorporating antigens and adjuvants on the same particle for co-delivery, a number of studies demonstrated that delivering antigen and adjuvant in separate but similar nanocarriers can also elevate antigen-specific immune responses. in one example, an hiv antigen (hivgp ) was anchored on the surface of liposomes and co-delivered with a liposomal formulation of cd-gmp, a potent agonist of the sting pathway. the study showed a substantial accumulation of the sting agonist in draining lymph nodes [ ] , and it is expected that the liposome-bound peptide antigen was delivered in a similar fashion. as a result, enhanced activation of antigen presenting cells and increased levels of antibodies against the hiv antigen were observed. immune stimulation by formulations containing separate antigen-and adjuvant-loaded nanoparticles was also reported in a study exploring the benefit of multi-adjuvant loaded particles. by incorporating multiple distinctive activators of tlrs, including monophosphoryl lipid a (a tlr agonist) and r (a tlr agonist), kasturi et al. demonstrated adjuvant synergism that triggered elevated antigen-specific humoral responses [ ] . using ovalbumin and hemagglutinin of influenza viruses, the investigators demonstrated long-lasting humoral responses and evidence of memory b cell formation following immunization with the nanoparticle vaccine. these examples highlight the functional versatility of synthetic nanoparticles, which can facilitate different modes of virus mimicry for immune activation. in addition to the role of lymph nodes in trapping nanoscale particles for immune processing, the immune system has also adapted to the repetitive antigen display on viruses for effective potentiation. virus surfaces display antigenic epitopes in an ordered and highly repetitive fashion, and the presentation of repetitively arranged and appropriately spaced antigens on the surface of virus or virus-like particles has been linked to enhanced immune responses [ ] . many reports have shown that numerous components of the mammalian immune system have evolved to respond strongly to the repetitive antigen patterns frequently found on pathogens [ ] [ ] [ ] . in comparison, non-repetitive antigens are usually less effective in inducing immune responses [ ] [ ] [ ] [ ] . repetitive motifs on viral surfaces are also found to activate the complement system [ ] engaging the cd -cd complex, which further facilitates b cell activation and amplify other immune processing pathways [ ] . understanding the link between structural features of antigen display and immunological induction is vital in designing nanoparticulate vaccines. multivalent interactions promote b-cell receptor (bcr) clustering and signaling and facilitate receptor-mediated internalization of antigen. antigen features, such as epitope affinity, valency, or co-receptor recruitment can impact b and t cell signaling. in a study that used antigen-conjugated polymer to assay the impact of antigen valency on b cell activation, puffer et al. showed that clustering of bcrs by multivalent antigens is crucial for antigen-dependent signaling. the antigen-conjugated polymers clustered unbound bcrs and contributed to enhanced intracellular signalling [ ] . whereas the multivalent antigen-polymer conjugates elicited antibody production, free antigens failed to trigger humoral responses (fig. a) . highly repetitive surfaces are also known to bind strongly to natural igm antibodies through multivalent, high-avidity interactions [ ] . such antibody binding also facilitates cellular uptake of particles by macrophages and dendritic cells, which can in turn enhance immune processing through increased antigen presentation. it is also worth noting that many important components in the humoral arm of innate immunity, such as complement c q, pentraxins, ficolins and collectins, are multimeric structures that favor high-avidity interactions with repetitive pathogen surfaces [ ] (fig. b) . these observations highlight how the immune system has been primed to respond to repetitive motifs frequently found on viral particles. unlike free subunit antigens, nanoparticle vaccines present a high concentration of antigens on their surfaces. a nanoparticle can be surface functionalized with up to hundreds of antigens, effectively emulating the antigen display on viral surfaces [ , ] . the multivalent antigen display on nanoparticles enhances antibody responses by efficiently cross-linking bcrs, activating complement, and facilitating antibody binding. these factors play synergistic roles in promoting b cell differentiation and stimulating dc-mediated t cell priming. although synthetically prepared nanoparticles have yet to show the level of ordered antigen arrangement found on viruses and virus-like particles derived from cell culture systems [ , ] , ongoing studies continue to demonstrate emerging techniques to couple antigens of interest to nanocarriers. in the last section of the review, we review commonly used methods for coupling antigens with nanoparticles. these strategies may spur novel approaches for preparing nanoparticle vaccines. association of protein antigens with nanoparticles can be divided into particle encapsulation and surface association. while many studies show excellent nanoparticle vaccine potency with encapsulated antigens, surface associated antigen may benefit from the innate immune factors and immune processing mechanisms described in the previous section. techniques to associate protein or peptide antigens can be categorized into multiple categories. the different modes of antigen association are highlighted as follows. thiol and amine groups on protein or peptide antigens are frequently exploited for bioconjugation with nanoparticles. the thiol group on a cysteine amino acid is a powerful nucleophile with the capacity to form covalent linkage. several linker groups, such as maleimide and succinimidyl -( -pyridyldithio)propionate (spdp), facilitate the conjugation between thiol-containing antigens with nanoparticles [ ] . in a maleimide-thiol reaction, the nucleophilic thiolate anion attacks the π-bond of maleimide, forming the enolate intermediate and yielding the desired conjugate. this technique has been widely used to assemble nanoparticle vaccines [ ] [ ] [ ] . in a study on refining a liposomal formulation of hiv vaccine, thiol chemistry was exploited to control the physiological conformation and density of the target antigen to modulate immune responses [ ] . thiols groups also readily associate with gold surfaces. the strong interaction between sulfur and gold drives the sulfur atom to fill the free orbitals of a gold atom, creating a coordinate covalent bond [ , ] . such approach has been extensively applied to associate nucleic acids [ ] [ ] [ ] [ ] [ ] and antigens [ , ] with gold nanoparticles. amine groups, on the other hand, are present on all protein and peptide antigens, which can be linked to nanocarriers via amide bond formation, typically through carbodiimide crosslinker chemistry. bioconjugations with amine-containing antigens are commonly performed using -ethyl- -( -(dimethylamino)propyl)carbodi-imide (edc) and n-hydroxysuccinimide (nhs). nhs or its sulfonated from (sulfo-nhs) is efficiently coupled to carboxyl groups with the aid of edc to form nhs esters. the nhs esters then covalently conjugate to primary amines to form an intermediate compound that is subsequently hydrolysed to the desired conjugate [ ] . many nanoparticle vaccines have been prepared with the edc/nhs conjugation method with a high coupling yield [ , ] . polydopamine functionalization is another conjugation approach that is increasingly applied to associate proteins and peptides with nanoparticles. in alkaline ph, dopamine undergoes oxidative self-polymerization to form a layer of polydopamine that can coat almost any type of material [ ] [ ] [ ] . this mussel-like adhesive layer enables a secondary reaction with biomolecules containing thiol or amine groups [ , ] . the technique has been demonstrated on both organic and inorganic nanoparticles [ , ] . dopamine-incorporated polymers have also been employed to adsorb proteins for macrophage-targeted delivery [ ] . the versatile technique can be applied to conjugate multiple cargoes for vaccine applications [ ] . electrostatic attraction between oppositely charged antigens and nanoparticles have been exploited to prepare nanoparticle vaccines. in general, cationic nanocarriers are prepared for the association of anionic protein antigens. in the case of liposomes, cationic lipid dotap is frequently applied to render the nanocarrier positively charged. dotap-based liposomes have been shown to absorb hpv e peptides [ ] , enhancing antigen-specific cd t cell response and increasing antitumor responses. strategies have also been employed to prepare antigens with added anionic moieties via recombinant protein engineering. by expressing hpv e protein and ovalbumin with an anionic lipoprotein, shen et al. demonstrated enhanced antigen association efficiency and retention to dotap liposomes. the vaccine formulation elevated antigen-specific cellular responses and inhibited tumor growth in a mouse model [ , ] . in another work based on print (particle replication in non-wetting templates) technology, electrostatic interaction was exploited to generate nanoparticle vaccines of cylindrical shape. cationic particles were prepared by blending positively charged polymers with plga prior to the imprint lithography process. upon mixing with anionic hemagglutinin proteins of influenza viruses, a high level of antigen binding to the particle surface was achieved [ ] . immunization with the nanoparticle vaccine elicited a more potent anti-influenza antibody response compared to a commercial vaccine based on inactivated subunit influenza viruses. unlike other spherical nanoparticle vaccines, the platform offers the ability to mimic the filamentous shape that can be found among many virus species. synthetic nanoparticles have high surface energies owing to their large radii of curvature. as a result, adsorption of protein can occur spontaneously owing to a combination of weak interaction forces, leading to the formation of protein corona formation [ ] . this phenomenon was demonstrated to facilitate the assembly of synthetic virus-like nanoparticles [ ] . upon mixing gold nanoparticles with viral antigens, rapid protein adsorption took place on the nanoparticle surface. this adsorption led to colloidal stabilization of gold nanoparticles, preventing their aggregation in biological buffers. biochemical analysis showed the facile assembly method surrounded each nanoparticle with approximately coronavirus spike proteins, inducing enhanced anti-viral immune responses. the adsorption approach has been used extensively to associated antigens with nanocarriers, including nanoparticles made of gold [ ] , calcium phosphate [ , ] , aluminium hydroxide [ ] , carbon [ , ] , silica [ , ] , and organic polymers [ , ] . the range of adsorbed biomolecules vary from dna [ , , ] , adjuvant [ ] , small peptides, and protein antigens [ , , , ] . it is important to note that formation of protein corona is a dynamic process that can be strongly influenced by both nanomaterials and antigen of interest [ ] . studies have also shown that antigens can undergo conformational changes upon nanoparticle adsorption [ ] , which could have both positive and negative effects on the resulting vaccine formulations. coating nanoparticles with cellular membranes is an emerging functionalization approach that can pave ways to novel vaccine formulations with virus-mimetic features. it has been shown that following dispersion of cell membrane vesicles with nanoparticles, highly controlled membrane coating can be achieved, yielding unilamellar membrane cloaked nanoparticles retaining membrane proteins in their right-side-out orientation [ ] [ ] [ ] . these nanoparticles are structurally analogous to enveloped viruses consisting of cell-derived lipid membranes stabilized via viral capsids or matrix proteins [ ] . the membrane coating approach has been adapted for vaccine preparations against cancer and bacteria [ , [ ] [ ] [ ] . enhanced dendritic cell activation and presentation of a melanoma-associated tumor antigen (gp ) was demonstrated by fang et al. using plga nanoparticles cloaked in b -f membranes [ ] . the enhanced immune response to the membrane antigen was attributed to increased cellular delivery and colocalization with immunological adjuvant facilitated by the nanoparticles. gao et al. also showed that bacterial outer membrane vesicles (omvs) can be rendered more immunogenic following coating on gold nanoparticles. the omv-coated nanoparticles significantly increased antigen delivery to lymph nodes and elevated production of cytokines associated with bacterial containment [ ] . the membrane cloaking approach offers a titillating strategy towards future vaccine designs as the method enables coupling of membrane-anchored antigens in their native conformation with immunogenic nanocarriers. advances in nanotechnology and its adoption in vaccinology have helped push the boundaries of non-live, subunit vaccines, resulting in many exciting demonstrations of effective immune potentiation by nanoformulations. not only can nanoparticle vaccines enhance humoral responses against target antigens, they have been shown to promote cell-based immunity as well as immunological memory. these are hallmarks of good vaccine formulations that are often inherent to live attenuated viruses. an increasing number of studies have shed light on the mechanisms behind the benefits of nanoparticle vaccines, including lymph node targeting, multivalent antigen display, and coordinated delivery of antigens and adjuvants. these features can find their mechanistic analogues in the immunological processing of viruses. given the frequent semblance between nanoparticle vaccines and viruses regarding their size, morphology, antigen display, and adjuvanticity effect, nanoparticles present a compelling platform in bridging the gap between live and non-live vaccines. emerging techniques in nanoparticle functionalization also pave ways to novel formulation designs that promise controlled immune modulation. going forward, understanding of virology may assist 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biomolecular coronas provide the biological identity of nanosized materials investigation of the effects of surface chemistry and solution concentration on the conformation of adsorbed proteins using an improved circular dichroism method erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform nanoparticle biointerfacing by platelet membrane cloaking marker-of-self' functionalization of nanoscale particles through a top-down cellular membrane coating approach structural basis for membrane fusion by enveloped viruses nanoparticle-detained toxins for safe and effective vaccination nanoparticle-based antivirulence vaccine for the management of methicillin-resistant staphylococcus aureus skin infection cancer cell membrane-coated nanoparticles for anticancer vaccination and drug delivery bacterial protein toxins targeting rho gtpases the authors acknowledge support from the ministry of science and technology, r.o.c. the authors have declared that no competing interest exists. saborni chattopadhyay is a phd candidate in the institute of biomedical sciences at academia sinica, taiwan. she received her m. tech in biological sciences and bioengineering from indian institute of technology kanpur. her research interests include cancer, therapeutics and drug delivery. saborni is an avid reader and enjoys travelling.jui-yi chen is a research assistant in the institute of biomedical sciences at academia sinica, taiwan. she received her master's degree in material science and engineering from the national tsing-hua university in taiwan. her research interests include biomimetic materials and drug delivery. key: cord- -ovdhnajp authors: gyasi, razak m.; anderson, eugenia a. title: rethinking the gendered dimensions in the impacts and response to covid- pandemic date: - - journal: nan doi: . /j.puhip. . sha: doc_id: cord_uid: ovdhnajp abstract the outbreak of the novel coronavirus disease (covid- ) has demonstrated the urgency to rethink the health system quality to efficiently support and improve global health. whilst efforts are now gathering pace in response to the deleterious impacts of covid- , gendered analysis of these impacts and response to the pandemic has been far too little. a critical consideration of gendered lenses and the respective brunt of health crises of the pandemic on men and women can improve our understanding and direction of efforts toward building robust and equitable public health response. strategic plans for preparedness must be fully grounded in strong gender analysis in the remit of gender roles and norms, and strengthen the leadership and resourceful participation of women in the decision-making process toward addressing covid- outbreak. in almost all global regions and countries, covid- caused by a novel coronavirus sars-cov- is causing exceptional health, societal behavior and socioeconomic crises. international and respective national endeavors are now gathering pace in response to the deleterious impacts of covid- . , yet, the global effort to unravel the gendered impacts and perspectives in response to covid- has been far too little. there is currently a dearth of knowledge about the gendered dimension in the impact and response to covid- particularly in sub-saharan african countries. international politics and health authorities charged to convey the information on covid- such as the etiology, diffusion, and the susceptibility of different population cohorts could maximize efforts by gauging the gender implication of the pandemic. specifically, taking into account the gendered sensitivities and the respective brunt of health crises of the outbreak on men and women can improve our understanding and direction of efforts toward building robust and equitable public health response. lessons learnt from past epidemics such as ebola and zika suggest the need for critical gender analyses in response strategies to enhance the efficacy of health and policy interventions for covid- . at the moment, covid- is indiscriminately ravaging men and women of any age group. some demographic data from various geographic regions depict certain diversities in the severity of the infection. however, the evidence on gender disparities of covid- infections is highly mixed. what we do know, however, is that like adults years or over, men are more likely to die from the pandemic. covid- thrives on people with comorbidities including older people with such conditions as cardiovascular disease, chronic respiratory and pulmonary disease, and active cancers. these chronic conditions are disproportionately prevalent in men compared to women. key evidence also points to the sex-induced immunology. sex hormones differ between men and women and are involved in the way the immune system triggers an inflammatory response to pathogens. men have lower innate antiviral immune responses to a range of infections including hepatitis c and hiv. studies in mice suggest that this may also be true for coronaviruses, though specific evidence on covid- is lacking. lifestyle choices such as harmful drinking, excessive smoking as well as sociocultural attitudes, masculine norms, and stoic-induced reluctance to seek help could potentially predispose men to covid- -related vulnerabilities such as infection and mortality. it should be recognized that the curve of the outbreak is yet to be flattened. hence, it is early days yet to propose a gender-based hypothesis for covid- fatalities. estimates indicate that over % of health care workforce globally are women and are on the frontlines of the covid- response, potentially placing them at a greater risk of the infection. crucially, an average gender pay gap of about % may aggravate the vulnerability of women in times of crises such as an outbreak of an epidemic. many preventive protocols of covid- including various levels of lockdown, physical and social distancing, market closure, and travel restrictions unleash a greater burden on women who predominantly provide informal familial caregiving and support. this may limit their opportunities in resilience and economic empowerment. this is particularly problematic in low-income settings especially in africa where women dominate the informal economic sector. in many african countries such as ghana, nigeria, and kenya, there is a growing concern of heightened domestic violence against women as a result of lockdown. these may not only present serious psychological, wellbeing, and human rights implications for women but household management, support, and caregiving for older people and children could also be under siege. evidence from the zika and ebola outbreaks in east africa and west africa respectively indicate that women had limited decision making power for epidemics. , it was also evident that most women experienced unmet care needs because social and health resources were mostly diverted from routine health services such as reproductive health services and sanitary needs to emergency response deliveries. coupled with poor access to socio-economic resources, these circumstances deepened the inequalities and hardship for women. with previous experiences, would the covid- outbreak further segregate the gender inequities in impacts and response strategies? in the publication of the strengthening preparedness for health emergencies, the who executive board advocated for the inclusion and full representation of women in the decision making process at international and national levels during outbreaks. however, not much has been done to involve women in preparedness and response to covid- pandemic particularly sub-saharan africa countries which have been rated as vulnerable to exposing huge populations to covid- and a moderate capacity to control the outbreak. indeed, the capacity of sub-saharan african countries to provide sufficient infrastructure, personnel, and clinical care to support people with covid- has been questioned vehemently . global public health security is a shared responsibility that requires a collaborative and all-inclusive response. the effectiveness in the fight against the covid- outbreak will largely depend upon gaining a deeper understanding of the gendered dynamics and the mechanisms that may correlate the diversity and susceptibility to the infection of the virus and management modalities among gender groups. we propose to the global health organizations, national political forces, and policymakers to conspicuously include gendered sensitivities in the analysis of impacts and response to covid- pandemic. strategic plans for preparedness must be fully grounded in strong gender analysis in the remit of gender roles and norms, and strengthen the leadership and resourceful participation of women in the decision-making process toward addressing covid- outbreak. there is no any funding source. rmg and eaa developed the concept, wrote and revised the manuscript. all authors approved the final manuscript none bearing the brunt of covid- : older people in low and middle income countries: a global expert group on older people might be useful emergency committee regarding the outbreak of novel coronavirus (covid- ). the world health organization ebola, gender and conspicuously invisible women in global health governance fighting covid- : fear and internal conflict among older adults in ghana epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study sex hormones and gender role relate to gray matter volumes in sexually dimorphic brain areas sex drives dimorphic immune responses to viral infections prevalence and patterns of tobacco smoking among chinese adult men and women: findings of the national smoking survey explaining the gender gap in health services use in ghanaian community-dwelling older cohorts gender equity in the health work force: analysis of countries serological evidence of flavivirus circulation in human populations in northern kenya: an assessment of disease risk - a gendered human rights analysis of ebola and zika: locating gender in global health emergencies overcoming the 'tyranny of the urgent': integrating gender into disease outbreak preparedness and response executive board eb /conf/ : strengthening preparedness for health emergencies; implementation of international health regulations preparedness and vulnerability of african countries against importations of covid- : a modelling study none key: cord- - rmzaau authors: rhee, joon haeng title: current and new approaches for mucosal vaccine delivery date: - - journal: mucosal vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: rmzaau mucosal surfaces are the interface between the host’s internal milieu and the external environment, and they have dual functions, serving as physical barriers to foreign antigens and as accepting sites for vital materials. mucosal vaccines are more favored to prevent mucosal infections from the portal of entry. although mucosal vaccination has many advantages, licensed mucosal vaccines are scarce. the most widely studied mucosal routes are oral and intranasal. licensed oral and intranasal vaccines are composed mostly of whole cell killed or live attenuated microorganisms serving as both delivery systems and built-in adjuvants. future mucosal vaccines should be made with more purified antigen components, which will be relatively less immunogenic. to induce robust protective immune responses against well-purified vaccine antigens, an effective mucosal delivery system is an essential requisite. recent developments in biomaterials and nanotechnology have enabled many innovative mucosal vaccine trials. for oral vaccination, the vaccine delivery system should be able to stably carry antigens and adjuvants and resist harsh physicochemical conditions in the stomach and intestinal tract. besides many nano/microcarrier tools generated by using natural and chemical materials, the development of oral vaccine delivery systems using food materials should be more robustly researched to expand vaccine coverage of gastrointestinal infections in developing countries. for intranasal vaccination, the vaccine delivery system should survive the very active mucociliary clearance mechanisms and prove safety because of the anatomical location of nasal cavity separated by a thin barrier. future mucosal vaccine carriers, regardless of administration routes, should have certain common characteristics. they should maintain stability in given environments, be mucoadhesive, and have the ability to target specific tissues and cells. mucosal surfaces are the interface between the host's internal milieu and the external environment, and they have dual functions, serving as physical barriers to foreign bodies and pathogenic microbes and providing the foundation for crucial survival functions such as uptake of air and nutrients, reproduction, and perception of signals. the protection of mucosal surfaces is ensured by the specialized mucosal-associated lymphoid tissues (malts). mucosal vaccines, in contrast to parenteral vaccines, generally induce more efficacious protective immune reactions by inducing secretory iga responses and cell-mediated immunity in mucosal tissues and portals of entries of mucosal pathogens. for food components and inert materials in breathing air, the malt should remain tolerant so as not to cause unnecessary inflammatory responses. despite the many advantages of mucosal vaccines, there are only limited numbers of licensed mucosal vaccines. almost all licensed mucosal vaccines are composed of whole components of pathogens, either live or dead. there is no successful subunit mucosal vaccine so far. live attenuated or whole cell (wc) killed vaccines are not formulated with any specific adjuvant or delivery system. in those vaccines, pathogen-associated molecular patterns (pamps) play the role of built-in adjuvants, and cell corpuscles serve as delivery systems for protective antigens. there could be many reasons for the sluggish progress of development of mucosal vaccines. concerns about safety are the most prominent reason. mucosal surfaces are continuously exposed to environmental and food antigens and allergens, and inflammatory immune responses against mucosal vaccine antigens would result in sustained pathologic inflammation. in the case of nasal vaccination, the nasal cavity is separated from the central nervous system by a thin partition, and olfactory nerves are directly projected from the brain to cavity. the scarcity of optimal delivery systems is another reason for the slow progress. in the mucosal environment, there are many physicochemical conditions that would interfere with proper stimulation of immune cells by antigens and adjuvants. in the case of oral administration, to be taken up microfold (m) cells in the distal jejunum and ileum, antigens should be able to accommodate very low ph in the stomach and a sudden alkaline surge in the duodenum, and they should be able to resist proteolytic attacks of digestive enzymes. in contrast to the oral route, antigens delivered intranasally do not experience that dramatic fluctuation in ph, but they should be able to survive profuse mucosal secretions, mucociliary clearance, and the relative inefficiency of antigen uptake by antigen-presenting cells (apcs). for more efficient delivery of mucosal vaccines, many new delivery systems based on nanotechnology and biomaterials have been studied, but very few of them have been approved for clinical use. more vigorous clinically oriented research is needed [ , ] . in vaccine formulations currently approved or under clinical trials, nanoscale (, nm) carriers are already in use [ ] . current nanotechnology and nanocarriers on the market or in the literature are summarized in immunostimulating complexes (vlps), emulsions, liposomes, immunostimulating complexes (iscoms), polymeric and nondegradable nanoparticles (nps), and nanogels [ ] . some of the nps are able to enter apcs by diverse pathways, thereby differentially modulating downstream immune responses. moreover, the nano-based delivery systems are also able to carry antigens and specific adjuvants such as tlr ligands simultaneously in the same carriers; carriers by themselves sometimes exert adjuvant activities. the nanoscale vaccine carrier systems generally constitute three key components: an antigen, against which adaptive immune responses are induced; an adjuvant, to potentiate the interaction between innate and adaptive immune systems in reacting to the antigen(s); and a delivery or targeting system to ensure that the antigen(s) and adjuvant(s) are delivered together to the right location at right time [ ] . in this context, many effective mucosal delivery systems using the nano/microscale carriers have been very actively researched up to clinical trial levels in recent years. viral-vectored vaccines and live or killed virus vaccines by themselves are already nanocarriers with built-in pamp adjuvants. vlps and virosomes behave similarly to viruses in stimulating immune responses and carrying antigens in nanoscales. some adjuvant formulations are already composed of nanoscale structures. formulation of adjuvants with vaccine antigens became inevitable in modern vaccine development to enhance the immunogenicity of highly purified antigens that have insufficient immunostimulatory capabilities. while early adjuvants (e.g., aluminum, oil-in-water emulsions) were used empirically, rapidly increasing knowledge of how the immune system interacts with pathogens allowed better understanding figure . different micro/nanocarriers that could be applied to the development of mucosal vaccine delivery systems. iv. current and new approaches for mucosal vaccine delivery of the role of adjuvants and how the formulation of modern vaccines can be better tailored for the desired clinical benefit [ ] . of interest, currently licensed oil-in-water emulsion adjuvants such as mf , as , and as comprise nanoscale structures in the formulation. squalene is popularly incorporated in oil-inwater emulsions because of its physical and immunostimulatory properties [ ] . mf (novartis vaccines & diagnostics), as (gsk biologics), and glucopyranosyl lipid adjuvantstable emulsion (gla-se) (infectious disease research institute (idri)) have a squalene content of around %À % (w/w) with additional emulsifying agents [ ] . mf was the first oilin-water emulsion on the market produced by microfluidization and contains sorbitan trioleate and polysorbate- (ps ) as surfactants. mf has a particle size of around nm [ ] . as contains α-tocopherol and ps as a surfactant. as has a particle size of about nm [ ] . gla-se (idri) consists of a squalene emulsion in combination with gla, which is a synthetic form of monophosphoryl lipid a (mpla) and a potent immunopotentiator. gla-se has a particle size of about nm [ ] . it has been suggested that emulsions with particle sizes ranging from to nm are efficiently taken up by dendritic cells (dcs) and hence effectively stimulate immune responses against coadministered vaccine antigens [ ] . tlr ligands and immunostimulatory agents such as qs are formulated into oil-water emulsion or liposomes to make adjuvants for vaccines against diverse infectious agents and tumor immunotherapy. iscoms are approximately -nm cage-like particles produced by combining protein antigens, cholesterol, phospholipid, and the saponin adjuvant quil a [ ] . iscom is composed of matrix serving traps for protein antigens. typically, membrane antigens containing hydrophobic domains are well trapped in iscom through apolar interactions [ ] . liposomes are spherical carriers composed of one or more phospholipid membranes with aqueous core. thanks to the structure, liposomes provide a wide range of options for vaccine formulation design. proteins, peptides, dna, rna, and adjuvant components can be readily encapsulated inside the aqueous core, embedded within the lipid layer, or attached to the surface by adsorption, hydrophobic anchor insertion, or covalent fusion. liposomes by themselves are considered to be nontoxic and biodegradable when mainly phospholipids are used, since they are normal components of mammalian cell membranes and lipids are relatively nonimmunogenic. liposomes could be designed and manufactured to have the desired physicochemical characteristics optimal for inducing desired immune responses against vaccine antigens: vesicle size, lamellarity (number of lipid layers), surface charge, bilayer fluidity, and incorporation of immunostimulatory components [ a] . polymeric nps are have also been robustly studied to deliver vaccine antigens and adjuvants. polymeric nps are submicron-sized colloidal systems of natural or synthetic polymers used as delivery carriers of chemical drugs, proteins, peptides, and nucleic acids, owing to their high bioavailability, controlled release, biodegradable and biocompatible properties, and low toxic profiles [ ] . compared with liposomes, polymeric nps can more easily incorporate both hydrophilic and hydrophobic biomolecules and have better storage stability. the most commonly studied polymers are poly(d,l-lactide-co-glycolide) (plg), polylactide (pla), and poly(d,l-lactide-co-glycolide) (plga) [ , ] . these biodegradable, biocompatible polymers are well characterized and have been approved by the u.s. food and drug administration (fda) for use in humans because of their excellent safety profiles. they have been extensively studied for the formulation of vaccine antigens (proteins, peptides, dna, etc.) [ ] . in these formulations, antigens can be either entrapped or adsorbed to the surface of the particles and are protected from proteolytic degradation conferring longer half-lives in vivo. by not-so-difficult additional engineering, these particles can be regulated to degrade or to release cargos (adjuvant and/or antigens) over a wide range of rates. additionally, polymeric particles more easily pass m cells and reach to apcs in the malt after surviving harsh physicochemical conditions in many mucosal compartments [ , ] . plg polymers have been evaluated for drug delivery since the early s and have been used widely for pharmaceutical and medical device applications with excellent safety profiles in humans [ ] . generally, plg forms microparticles rather than nps. plg microparticles received attention for vaccine delivery by the world health organization (who) special program for vaccine development from the late s [ ] . triggered by this program, many antigens, such as tetanus toxoid, hepatitis b antigen, and diphtheria toxoid, were formulated with plg microparticles and evaluated in comparison with aluminum salt adjuvants [ ] . although some promising results with plg-based vaccines in small animal models were reported, challenges concerning antigen stability and insufficient immune responses compared with alum-adjuvanted vaccines prevented the use of plg microparticles in commercial vaccines [ ] . one more disadvantage of plg polymer-based vaccines is their inefficiency in translocating to lymph nodes where apcs present antigens to t lymphocytes. the particle size can influence transport to specific location and cell types in the draining lymph nodes [ , ] . nps ( À nm) drained to the lymph nodes and localized in the lymph-node-resident dcs and macrophages, whereas larger particles ( À nm) were mostly associated with dcs at the injection site. plg particles would be inefficient in presenting antigens to dcs in draining lymph nodes. in this regard, plg microparticles have handicaps to being more widely applied to the development of vaccine delivery systems. pla is a linear aliphatic polyester composed of lactic acid building blocks that are naturally occurring organic acids derived from sugarcane and cornstarch [ ] . pla's physical properties can be tuned through combining racemic mixtures of these enantiomers: poly-l-lactide and poly-dl-lactide semicrystalline and amorphous polymers, respectively [ ] . thanks to their good safety profiles, pla-based products have been approved by the fda and the european medicines agency (ema) for multiple biomedical applications. this polymer can be easily chemically modified with different ligands to improve their specificity to targeted cells [ ] . in vivo, pla is hydrolyzed into α-hydroxy acid, which is easily metabolized in the body via the krebs cycle [ ] . pla polymers can be fabricated into both microparticles and nps [ ] . despite its physicochemical and pharmaceutical advantages, pla has been less intensively applied than other polymers, such as its copolymer plga, in clinical stage vaccine developments. although a considerable number of reports have shown the usefulness of pla polymer nps as versatile vaccine carriers, the scaling-up of these laboratory methods to industrial production has faced hurdles, which are mostly related to particle size and size distribution. for further development of pla polymers as clinical-grade vaccine delivery systems, these practical problems need to be solved in advance. biodegradable nano/microparticles of plga and plga-based polymers are more widely explored as carriers for controlled delivery of macromolecular therapeutics such as proteins, peptides, vaccines, genes, antigens, and growth factors. plga is one of the most successfully developed biodegradable polymers. among the different polymers developed to formulate polymeric nps, plga has won strong attention, owing to its attractive properties: biodegradability and biocompatibility, fda and ema approval in drug delivery systems for parenteral administration, well-described formulations and methods of production adapted to various types of drugs such as hydrophilic or hydrophobic small molecules or macromolecules, protection of the drug from degradation, the possibility of sustained release, the possibility of modifying surface properties to provide stealthiness and/or better interaction with biological materials, and the possibility of targeting nps to specific organs or cells [ ] . the plga-based carriers' cargo release characteristics could be relatively well controlled by modulating encapsulation, particle size, formulation additives, molecular mass, ratio of lactide to glycolide moieties in plga, and surface morphology [ ] . the most widely used plga, with a monomer composition of : , has the fastest biodegradation rate; it completely occurs in approximately À days. the polyglycolide acid is more hydrophilic than polylactide, owing to the absence of a methyl side group [ ] . a higher glycolic acid percentage causes more water uptake and consequently faster degradation of plga polymers. plga is hydrolyzed into the original monomers lactic acid and glycolic acid, which are by-products of various metabolic pathways and are not associated with any significant toxicity except lowered ph. next to release characteristics, various other physical traits of plga particles can be manipulated, including particle size, size distribution, zeta potential, polydispersity index, encapsulation efficiency, and cargo loading [ ] . among a myriad of choices in nano/microcarrier polymers, plga has more advantages other than those listed above: plga particles can be administered via diverse routes; plga particle formulation may dampen toxicities of vaccine components; plga particles could protect the antigen from degradation and allow controlled release; plga particles could be made to target apcs and increase cross-presentation of the antigen; and plga particles allow concomitant delivery of multiple vaccine components with dose sparing. while many properties of plga polymers are favorable and controllable as vaccine delivery tools, there are drawbacks as well. plga has a negative effect on the stability of encapsulated protein antigens during preparation and storage, primarily owing to the acid-catalyzed nature of its degradation. its hydrolysis leads to the accumulation of lactic and glycolic acids in the microenvironment, which will denature encapsulated protein antigens and consequently compromise immunogenicity [ ] . in addition, processing conditions used in manufacturing plga carriers have negative effects on protein secondary structures [ ] . to overcome problems associated with protein degradation, many efforts have been made to optimize the manufacturing process and to add excipients that would protect the protein antigens being encapsulated. protein antigens adsorbed to plga particles are relatively more protected from those physicochemical insults. adsorbed antigen would offer improved stability and activity over encapsulated antigen by avoiding exposure to organic solvents used during formulation and acidic ph conditions caused by degradation of the polymer. but this may result in premature high burst release of the antigen before uptake by apcs, which can lead to deficient immune responses [ ] . natural polymers are attractive vaccine delivery vehicles, owing to their low toxicity and biocompatibility. synthetic polymers such as plga or pla are also reported to be safe and biocompatible. their degradation products affect the microenvironment by lowering ph which may be detrimental to functions of apcs and compromise immunogenicity of vaccine antigens. on the other hand, natural polymers are generally composed of biological components, making them physiologically resorbable with few to no adverse effects [ ] . the major drawback of natural polymers as vaccine delivery systems is reproducibility, which should be overcome by further technological researches. there are two major groups of natural polymers that are used to manufacture particulate carriers: peptide/proteins and polyelectrolytes including alginate, chitosan, and dextran [ ] . chitosan and chitosan derivatives are cationic polymers, which, owing to their structure, have excellent mucoadhesive and absorption-promoting properties. chitosan is manufactured by alkaline deacetylation of chitin (e.g., derived commercially from exoskeleton of crustaceans or fungi) and is a linear copolymer of β - linked monomers of d-glucosamine and n-acetyl-d-glucosamine [ ] . it is biodegradable and biocompatible. the pka of the primary amine group of chitosan is approximately . , and the nascent polymer at neural ph carries no charge; hence chitosan is insoluble in water. this solubility characteristic should prevent nascent chitosan from being able to deliver antigens that are soluble and stable at neutral ph. structural modifications have been made to chitosan to produce derivatives that are soluble at neutral ph yet retain the positive charge and unique properties of nascent chitosan. because chemical modifications make it possible to substitute both amine and hydroxyl functional groups of chitosan, various chitosan derivatives have been produced by introducing hydrophilic groups such as hydroxyalkyl, carboxyalkyl, succinyl, thiol, and sulfate or by grafting solubility enhancer polymers such as polyethylene glycol and poloxamer [ ] . of all the water-soluble derivatives, n-trimethyl and carboxymethyl derivatives of chitosan have been studied most extensively, owing to their relative ease of synthesis, ampholytic character, and ample application possibilities. soluble n-trimethyl chitosan has both mucoadhesive properties and excellent absorption-enhancing effects even at neutral ph because of its cationic charge at neutral ph [ ] . n-trimethyl chitosan is rather widely applied to the development of mucosal vaccine delivery systems, since it is mucoadhesive and has penetration-enhancing ability through the paracellular route even at neutral ph. chitosan particles can be fabricated to successfully deliver both adjuvant and antigen to dcs in the tissue or draining lymph nodes [ ] . adjuvants and antigens could be either incorporated inside or adsorbed on the surface of chitosan microparticles or nps on purpose. in addition to being a carrier, chitosan can be used to coat other polymer particles to enhance their immunogenicity, bioadhesiveness, and surface adsorption potential [ ] . n-trimethyl chitosan is freely soluble over a wide ph range as compared to other chitosan derivatives and bears positive charges, independently of the environmental ph. methylcarboxy chitosan is a polyampholytic polymer that is able to form viscoelastic gels in aqueous environments or with anionic macromolecules at neutral ph values. on the basis of these characteristics, the complexation of two chitosan derivatives without using any cross-linker could generate a vaccine delivery carrier that has high loading efficiency and can maintain integrity of a protein antigen [ ] . alginate is a linear, anionic polysaccharide found in the cell walls of brown algae. it has a high affinity for water and forms an inert and highly aqueous environment within the particle, which limits its ability to carry hydrophobic vaccine antigens and adjuvants. it is also biocompatible, biodegradable, and easily eliminated from the body [ , ] . as with chitosan particles, adsorption of adjuvant onto the surface of microspheres allows differential release of the antigen and adjuvant for temporally controlled stimulation of immune cells [ ] . alginate itself, besides its role as a vaccine carrier, has immune-stimulatory activities though stimulating nf-κb signaling pathway [ ] . dextran is a polysaccharide composed of repeating branched glucose molecules. its most commonly used form is dextran sulfate [ ] , which is biocompatible and hydrophilic and decomposes into natural byproducts. anionic dextran sulfate is often fabricated with cationic poly-l-arginine to make layer-by-layer antigen-adjuvant carriers [ ] . because this type of particle is assembled layer by layer, multiple antigens and adjuvants can be incorporated in multiple layers to maximize targeting and activation of immune cells. self-assembled peptides have been reported to be useful candidates for future vaccine delivery systems [ ] . peptide molecules can be rationally designed to self-assemble into specific nanoarchitectures in response to changes in their assembly environment, including ph, temperature, ionic strength, and interactions between host and guest molecules. they could be manufactured in the forms of nanomicelles, nanovesicles, nanofibers, nanotubes, nanoribbons, and hydrogels and would have a diverse range of mechanical and physicochemical properties [ ] . peptide delivery systems may have potential advantages over liposome or nps, since they can be composed of amphiphilic molecules with high loading efficiency, low antigen leakage, biodegradability, and high permeability to biomembranes of target cells. these molecules can be designed for cellspecific targeting by including adhesion ligands, receptor recognition ligands, or peptide-based antigens in their design, often in a multivalent display [ ] . these molecules can also act as intracellular transporters and respond to changes in the physiological environment. generally, self-assembling peptides are nonimmunogenic, serving as built-in adjuvants for fused antigenic peptides [ ] . the adjuvant activity is closely related to nanostructures, since the mutation of key amino acid residues in the self-assembling domain demolishes the immunogenicity of the self-assembled peptide vaccines [ ] . the adjuvanticity in a nanofiber self-assembled peptide vaccine was reported to be t-cell-and myd -dependent, but specific interactions with tlr and tlr as well as nalp were not noted, suggesting a novel immunomodulating mechanism. although peptide nanofiber vaccines are more efficiently taken up by dcs and subsequently activate them, these vaccines do not cause reactogenicity and nonspecific inflammatory reactions at the administration site [ ] . nanogels became more prominent recently as a vaccine delivery system. the term "nanogel" defines refers to nanoscale particles (, nm in diameter) composed of physically or chemically cross-linked bifunctional networks having good swelling capacity in aqueous environments [ ] . nanogels have a high cargo loading capacity, biocompatibility, and biodegradability. cationic nanogels are adhesive to epithelial cell surface and serve as artificial chaperones protecting antigens from aggregation and denaturation. loaded antigens are subsequently released in native forms and captured by appropriate apcs nearby [ ] . the surfaces of nanogels are relatively easy to modify by specific ligands, enabling targeted delivery to specific cells or tissues. nanogel vaccine formulations can be delivered via a wide range of routes, such as parenteral, oral, nasal, pulmonary, or ocular administration [ ] . nanogels can be formulated by various polysaccharides such as chitosan, mannan, hyaluronic acid, dextrin, cycloamylose, pullulan, and enzymatically synthesized glucogen [ ] . in recent years, pullulan has played a critical role in the development of nanogel systems for vaccine and drug delivery [ ] . pullulan is an aqueous polysaccharide synthesized by the yeast-like fungus aureobasidium pullulans. it consists of hundreds of repeated units of a maltotriose trimer. pullulan is widely used in diverse biomedical industries because it is easily modified by rather simple chemical reactions that are nontoxic, nonmutagenic, noncarcinogenic, and, most important, nonimmunogenic [ , ] . pullulan hydrophobized by cholesterol becomes amphiphilic and forms self-aggregates [ ] . the cholesterol-bearing pullulan (chp) can form complex nps with various protein antigens. chp can self-assemble in water into the nps and encapsulate protein antigens in the internal space through hydrophobic interactions. the complex np protects internal protein antigens against physicochemical or enzymatic degradation, serves as an ideal delivery vehicle, and releases payloads in a controlled fashion [ ] . the most valuable characteristic of chp nanogels is its artificial molecular chaperon activity [ ] . protein antigens are captured in denatured form in the chp nanogel under reversible denaturation temperature or in the presence of reversible denaturation reagents [ ] . in the nanomatrix, the nanogel protects denatured protein antigen as an artificial molecular chaperone and helps in proper refolding after release [ ] . another advantage of chp is its targeting ability to apcs. the chp nanogels and protein antigens could form colloidally stable nps nm in diameter, which is a relevant size allowing effective uptake by epithelial cells and apcs [ ] . moreover, chp nanogels can be modified to have cationic charge (cchp) by adding amine groups to the chp nanogels [ ] . the cchp nanogels could be well formulated with protein antigens and effectively carry vaccine antigen to the negatively charged nasal epithelium after intranasal administration [ ] . the positive charge of cchp nanogel provides more efficient adhesion to negatively charged nasal mucus and epithelia, leading to higher level and more sustained delivery of antigens to dcs inhabiting underneath mucosa. more important, the cchp vaccine formulation could induce significantly higher immune responses without adjuvant addition, while the cchp nanogel itself could not activate immature dcs, suggesting no biologically active adjuvant-like activity [ , ] . the reason why cchp, having no direct stimulatory effect on innate immune cells, could significantly enhance the immunogenicity of cargo antigens was thought to be the improved antigen residence time in the nasal cavity, which leads to better antigen transport to the nasal dcs. the -nm cchp nanogels carrying clostridium botulinum type a neurotoxin heavychain c-terminus (bohc/a) were bound by nasal epithelial cells and subsequently endocytosed. the bohc/a antigen was separated from the nanogel by protein exchange and sustainably released by exocytosis, which was subsequently taken up by cd c dcs in the mucosa [ ] . epithelial cells served as a reservoir for the cargo antigen, while no overt cytotoxicity was observed. neither the cchp nanogel nor cargo bohc/a antigen was taken up by the olfactory bulb or brain tissue, suggesting that the cchp nanogel system is safe for nasal administration (chapter : nanodelivery for mucosal vaccines). past, present and future gastrointestinal (gi) infection is a significant global health challenge, especially in developing countries. most gi infections are spread via the fecalÀoral route, primarily through contaminated water and food due to poor sanitation and social infrastructure. an efficacious vaccination policy is the most economical way of solving gi infection problems from a public health perspective. the oral vaccination is generally the best way to induce secretory immunoglobulin a (siga) in the gi tract and igg antibody responses in the systemic compartment. in fact, the only oral vaccine that has been widely used globally in infants and children in a national immunization program is the oral polio vaccine (opv) developed by albert sabin in the s. since sabin's opv vaccination, several oral vaccines against rotavirus, salmonella typhi, and vibrio cholerae have been licensed and marketed [ ] . those vaccines are made of live attenuated organism or killed microbial cells. there is as yet no licensed subunit oral vaccine on the market. there have been continuous efforts to develop oral vaccines because of the advantages of oral vaccination. as was noted during the haitian cholera epidemic, oral vaccination is a faster way of containing circulating infections and prevention of further outbreaks [ ] . after the september , , terrorist attacks, the threat of biological warfare became highlighted worldwide. the potential bioterrorism agents are likely to be disseminated by either aerosol or in food or water supplies targeting the wide mucosal surfaces in the respiratory or gi tracts, respectively. considering that the bioterrorism agents invade from the gi mucosa, oral vaccines, inducing protective immune responses at the route of entry, have generated the most interest as a frontline tool in biodefense [ ] . oral vaccination has several advantages, such as better patient compliance, mass immunization capability, easy administration or selfdelivery, simplified production and storage, lower production cost, and no needleassociated risks such as injuries and carryover infections (table . ) [ ] . the most important virtues of oral vaccination are its needle-free painless administration and that there is no need for trained personnel for administration. two major mucosal vaccination routes, oral and intranasal, are compared in table . . the most widely used oral vaccine is sabin's opv, which contributed enormously to the eradication of poliomyelitis worldwide. but recently, despite its efficacy, opv has been replaced by injectable poliovirus vaccine (ipv) in developed countries. opv is likely to be replaced by ipv globally over several coming years. live poliovirus was discovered in the stool of opv vaccines, possibly spreading infectious material in the environment [ ] . another serious concern about opv is the rare event of reversion to virulent strains in vivo, which could cause a serous iatrogenic vaccine-associated paralytic poliomyelitis [ ] . the same concerns apply to other mucosal vaccines using live attenuated organisms. but generally, oral vaccines are regarded as a better choice than injectable parenteral vaccines from production, economic, and regulatory perspectives [ ] . oral vaccines have better compliance and fewer adverse reactions. oral vaccines are better for large-scale production and mass vaccination campaigns in developing countries, since no needles are required and self-administration is possible. thermostabilization technologies would enable successful cold-chain-free vaccination of killed as well as live attenuated formulations in resourcepoor settings such as developing countries [ ] . thanks to the many virtues of oral vaccines and the success of opv, research into oral vaccines has rather a long history, but only a very limited number of oral vaccine products have become available. many oral vaccines that proved to be efficacious in preclinical studies have failed in clinical trials. live-attenuated vaccines against rotavirus and s. typhi have been successfully introduced into the market. in the case of cholera, two types of oral cholera vaccines (ocvs) are currently available: wc-rbs, which are killed wc monovalent (o ) vaccines with a recombinant b subunit (rbs) of cholera toxin (ct) (dukoral), and killed modified wc bivalent (o and o ) vaccines without the b subunit (shanchol, euvichol, and morcvax) (who cholera vaccine position paper-august at www.who.int) (chapter : cholera immunity and development and use of oral cholera vaccines for disease control). the three wc vaccines are based on the same cholera strains and dosage. although the wc killed cholera vaccines listed proved to be efficacious in multiple clinical trials, many other prototype killed and subunit vaccines could not be put on the market because of suboptimal immunogenicity. oral vaccines should have strong immune-stimulatory adjuvants and optimal delivery strategies to drive effective innate and adaptive immune responses against vaccine antigens [ ] . to achieve optimal immunogenicity, dukoral has a huge number of killed emulsions such as mf and as are extensively tested for many vaccines and immunization routes. however, in the literature, it is rather rare to find successful test results for oral vaccines against infectious diseases employing an oil-in-water or water-in-oil emulsion formulation or adjuvant system. a successful oral tumor vaccine study showed that an antigen complex (melanoma antigen mage , heat shock protein , and staphylococcal enterotoxin a) incorporated in a nanoemulsion with a small size of À nm induced efficacious protective immune responses comparable to those of subcutaneous administration [ , ] . vaccine antigen can be delivered inside the core or attached on the outside to the shell of micelles, depending on the electrochemical properties of the vaccine formulation [ ] . oral immunization of pla-peg-pla and plga-peg-plga copolymer micelles loaded with dna encoding hcv multiple epitope antigen could induce satisfactory immune responses [ ] . the copolymers showed an innate adjuvant activity and caused no significant adverse reactions [ ] . micelles can be synthesized as nanocarriers and engineered to penetrate mucus and be taken up by mucosal apcs [ ] . however, micelles may have a propensity to dissociate when diluted, leading to a loss of loaded antigen. while most clinical trials and delivery system developments employing liposomes have focused on the parenteral routes, there are still continued efforts to develop liposome-based oral delivery tools [ ] . conventional liposomes are vulnerable to acidic gastric juice and are easily digested by pancreatic lipase [ ] . also, intestinal bile salts can destroy the phospholipid membrane integrity and lyse the liposomes, resulting in the premature release of vaccine antigens [ ] . to tackle these problems, researchers have investigated different lipid moieties such as archaeal lipids or bile salts in the liposomal membrane [ , ] . an important determinant of the adjuvanticity of liposomes is the surface charge. positively charged (cationic) liposomes have been demonstrated to possess the strongest adjuvanticity compared to neutral and negatively charged liposomes [ ] . mucoadhesion is promoted by the cationic surface charge that would have a stronger interaction with negatively charged gi mucus. cationic liposomes also better adhere to negatively charged membranes of m cells and enterocytes, limiting flushing by peristalsis and providing a better chance to be internalized [ ] . however, the greater toxicity of cationic than anionic liposomes is of concern. recently, a study reported that cationic liposomes induce necrosis to release damageassociated molecular patterns and cause inflammation in vivo [ ] . to extend the clinical applications of liposomes as carriers of oral vaccines by improving stability and sustainability in gi tract mucosa, the surface modification of liposomes has been rigorously investigated [ À ]. the addition of cholesterol and quil a saponin to liposomes results in the generation of self-assembling pentagonal dodecahedrons that have intrinsic adjuvant properties and thus earned the name iscoms. two types of iscoms exist. the first, "classical" type was manufactured to entrap protein antigens, making them act as both a vaccine antigen delivery and an adjuvant system. the second type, referred as iscom matrices, contains no entrapped antigen and serves as a codelivered adjuvant, which is later formulated with vaccine antigens. early iscom formulations could not be evaluated in humans and were limited to veterinary use, owing to the reactogenicity of quil a. quil a was later replaced with qs , and iscoms could be then given clinical trials. qs is more purified in nature and shows an improved safety profile while remaining active as an adjuvant [ ] . while iscoms were widely tried with very diverse antigens for parenteral and intranasal vaccination studies, trials with oral vaccines have been relatively scarce [ , ] . one major challenge in working with iscoms is the difficulty associated with antigen incorporation. in this context, many trials used iscom matrices purely as adjuvants and formulated with vaccine antigens rather than incorporating them, which elicited immune responses as beneficial as those elicited by classical iscoms [ ] . the use of unmodified iscoms as adjuvants would significantly simplify production; however, the benefit of antigen encapsulation, which is an important key to success for the oral route, might be lost. for this reason, iscom matrix adjuvants in combination with enteric vaccine antigens are used for boosting through intranasal or parenteral routes after oral priming with antigens only or antigens with other adjuvants [ , À ] . to use iscoms for the induction of efficacious immune responses in the gi tract, the immunization protocol employing intelligent oral or injectable prime-boost regimens and incorporating additional coatings or adjuvants should be tried. vlps imitate the three-dimensional conformation of real virus and mimic infection by authentic viruses. moreover, vlps can be engineered to express additional antigens and target epitopes in repeated array. vlps have been studied as oral vaccines against virus or tumor antigens [ À ]. those vlp oral vaccines induced humoral and cellular immune responses in both systemic and mucosal compartments. significant antigen-specific siga responses were also observed. vlps could also be expressed in plant tissue successfully [ , , ] and could be purified from plant tissue at lower cost. on the other hand, rather crude freeze-dried plant tissue containing vlps could be directly administered orally to induce protective immune responses in animals, which suggests the possible development of human edible vaccines using vlps expressed in plant tissues such as tomato and potato [ , À ] . synthetic particles can serve as the most versatile oral delivery tools in which vaccine antigens and adjuvant could be loaded by encapsulation, conjugation, or adsorption. with advances in polymer material chemistry and formulation technology, different types of natural and synthetic nps are being actively tested for possible use for effective oral vaccine delivery. these recent technologies enable overcoming previous barriers in oral vaccination and allow better targeting of antigens and adjuvants to the desired tissue location and cells. particles can be engineered to release antigens and adjuvants upon degradation, swelling, and diffusion from the polymer, or change in electrostatic interactions. the production of particles in defined sizes, architectures, and chemical properties would enable oral delivery, which is the most difficult vaccination route in terms of targeted delivery, thanks to the major development in nanotechnology and biomaterial science. depending upon the polymer choices, the delivery systems by themselves provide adjuvant activity along with biocompatibility and biodegradability. the most extensively studied biodegradable polymers for the development of oral vaccine nanocarriers are pla, plga, β-glucans, alginate, and chitosan [ , ] . in the case of oral delivery, the greater surface area of nps, owing to their small size, would allow increased absorption across the intestinal epithelium, which will furnish reduced dosage and administration volume [ ] . hydrophilic nps are generally transported through enterocytes, whereas hydrophobic polymeric nps are better transported through m cells [ , , , ] . orally administered pla nps ( À nm) reached the peyer's patches (pps) through a three-step process. most particles are first entrapped in the mucus. then crossing of the epithelial barrier takes place exclusively through m cells, leading to an accumulation in pps. finally, the nps interact with underlying b cells and dcs in the pp tissue. all three steps can occur within minutes. furthermore, dcs engulfing nps were induced of tlr expression [ ] . to be licensed and clinically used for mucosal vaccine delivery, nanocarriers should be able to protect the payload from degradation, to penetrate the mucus barriers, and to control the release of both antigens and adjuvants at targeted sites. in principle, these properties could be tuned by altering their particle size, surface chemistry, and three-dimensional architecture [ ] . it is widely accepted that particulate antigens are more efficiently trafficked across the mucosa and delivered to mucosal apcs than are soluble antigens [ ] . uptake of particles in the gi tract occurs primarily via m cells. despite some controversies, particles with a diameter smaller than μm are thought to have a better chance of being taken up by m cells [ ] . to meet requisites for delivering diverse vaccine antigens and adjuvants to a specific mucosal target, plga surface characteristics have been extensively modified by coating with ionic surfactants or polymers such as polyethylene glycol (peg), sodium dodecyl sulfate, aminodextran, chitosan, polyethylene imine, poly-llysine, protamine, or cetyltrimethylammonium [ , À ] . the surface chemistry of plga particles can also be altered to increase diffusion through mucus and uptake by m cells through oral delivery [ , ] . other methods, such as coating antigen/adjuvant-loaded plga nps with methacrylate-based polymer eudragit fs d, produced gastric-acid-resistant microparticles ($ μm) that released payloads from the terminal ileum, where the ph level reaches . or higher [ ] . the ph-sensitive polymers derived from methyl methacrylate, methacrylate, methacrylic acid, acrylate, and/or dimethacrylate have been blended with plga for the protection of payloads in the np against enzymatic attacks in the stomach and small intestine [ ] . the antigen encapsulated by plga nps sequentially coated with phase-transitional shielding layer, poly[(methyl methacrylate)-co-(methyl acrylate)-co-(methacrylic acid)]Àpoly(d, l-lactide-co-glycolide) (pmmma-plga), was found to protect antigens in the gi tract and achieve targeted vaccination in the large intestine [ ] . hydroxypropyl methylcellulose phthalate (hpmcp), another enteric coating agent, was shown to make acid-resistant b nm nps with plga carrying helicobacter pylori antigen effectively induce th /th protective immune responses [ ] . to induce enhanced protective immune responses against antigens delivered by plga carriers, plga particles are functionalized by m-cell-targeting ligands in combination with stabilizing agents. incorporation of m-celltargeting lectins such as uea or lta into plga nps would enhance antigen-specific immune responses [ , ] . arginine-glycine-aspartate (rgd) ligand binding to β integrin can also target m cells. pegylated plga nps grafted with rgd or rgd peptidomimetic ligand showed significantly increased uptake by m cells and enhanced specific igg responses [ , ] . claudin , one a member of the integral membrane protein family expressed primarily in tight junctions, also serves as a target for developing m-cell-binding nanocarriers [ ] . in one study, an antigen-loaded porous plga microparticle was successfully coated with water-soluble chitosan conjugated with an m-cell-targeting peptide (cks ). the resulting microparticles effectively reached pps through m cell transcytosis to induce balanced th /th protective immune responses [ , ] . the phsensitive polymeric delivery systems employing hydroxypropyl methylcellulose phthalate (hpmcp) could be attuned by adding thiol groups to be selectively released under ileal ph condition. by formulating m-cell-homing peptide (cksthplsc) conjugated bmpb antigen with attuned hpmcp, delivery to pps and subsequent adaptive immune responses could be significantly enhanced [ , ] . recent research into oral vaccine delivery of nps has been directed toward the incorporation of mucoadhesive polymers. the mucoadhesive polymers prolong retention time of the particles in the mucus by steric or adhesive interactions. coating of nonbioadhesive nanospheres with poly(butadiene-maleic anhydrideco-l-dopa) increased the particle uptake by -fold in the small intestine [ ] . conjugation of immunostimulatory ligands to bioadhesive polymers should induce longer-lasting mucosal and systemic immune responses against entrapped antigens. bioadhesive poly(anhydride) nps ( À nm) coated with mannose or salmonella flagellin induced more potent and balanced th /th immune responses compared with noncoated particles [ ] . chitosan and its derivatives have been tried for the oral delivery of protein and dna vaccines [ À ] . the limited solubility of chitosan at alkaline and neutral ph has been circumvented by fabrication of chitosan by graft copolymerization with acyl, alkyl, monomeric, and polymeric moieties. modifications through quarterization, thiolation, acylation, and grafting resulted in copolymers with higher mucoadhesion strength, increased hydrophobic interactions (advantageous in hydrophobic antigen entrapment), and increased solubility in alkaline ph, higher solubility, and controlled/ extended release profiles, which consequently confer wider application of chitosan derivatives for oral vaccine delivery [ , ] . chitosan and its derivatives are mucoadhesive and have the ability to stimulate immune cells either by directly interacting with the m cells or by opening the tight junctions between the epithelial cells [ ] . because of the advantages mentioned above, chitosan has been applied to the manufacture of orally deliverable nps or coating of micro/nanocarriers made of other synthetic or natural biopolymers [ , ] . alginate has been used to make oral vaccine carriers utilizing its acid resistance and immunostimulatory properties [ ] . in order to overcome chitosan's instability in low-ph environments, cationic chitosan nps can be coated with acid-resistant alginate to make composite carriers [ , ] . alginate encapsulation of chitosan nps entrapping protein antigens was proved to protect payload protein antigens and dna from acidic attack in the stomach after oral administration. owing to its acid resistance property, alginate is also used to encapsulate bacterial cells to develop oral vaccines. a single oral dose of alginateencapsulated bcg elicited effective long-lasting mucosal and systemic immune responses [ ] . cold-chain-free ocv could be developed by encapsulating heat-inactivated bacterial cells with alginate [ ] . oral vaccines against edwardsiella, brucella, and aeromonas infections were also developed by encapsulation with alginate [ À ]. glucan particles are porous -to -micron cell wall shells manufactured by treating baker's yeast (saccharomyces cerevisiae) with a series of alkaline, acid, and solvent extractions [ ] . by in situ layer-by-layer synthesis through electrostatic interactions, dna could be encapsulated at high density [ ] . protein antigens could be encapsulated in glucan particles through hydration and lyophilization and could induce significant intestinal siga and th /th cellular responses to encapsulated antigens following oral vaccination [ ] . glucan microparticles target enterocytes and m cells for uptake and activate them to secrete and express cytokines and β-glucan receptors [ À ]. β-glucans, the major component of glucan microparticles, are fungal pamps, signaling through receptors such as dectin- and complement receptor expressed on dcs, monocytes, and neutrophils [ , ] . although the efficacy of glucan particles as an oral vaccine carrier was well proved with model antigens such as ova or bsa, the application to pathogenic microorganisms has been relatively rare [ ] . one reason for the limited use of glucan particles is that their manufacture is currently limited to liquid formulations, which require cold-chain storage and therefore are not optimal for the use in poorer regions [ ] . plant-based oral vaccines have advantages over the traditional vaccines in cost, safety, and scalability. since , researchers have manufactured edible plant-based vaccines in carrot, soybean, tomato, rice, potato, and tobacco against microbial pathogen antigens such as the heat-labile toxin b subunit (ltb) of enterotoxigenic escherichia coli, cholera toxin b subunit (ctb), and antigens from yersinia pestis and viruses, such as hepatitis b virus, rotavirus, and norwalk virus [ ] . many conventional vaccines are not widely distributed in developing countries where those vaccines are urgently needed, because of high production costs and the requirement of better infrastructure. one more problem standing in the way of wider distribution of desperately needed vaccines is that the conventional cell fermentation systems for producing recombinant protein vaccine antigens are often expensive and are not easily scalable [ ] . another emerging infectious disease field is one health, dealing with zoonotic diseases spreading in both animals and humans. a solution to this may be the use of plants or plant cells as bioreactors. molecular farming has become well established for the production of vaccines, and many proofs of principle and important proofs of efficacy are accumulating continuously [ ] . mucorice should be one of the most innovative approaches for oral vaccine delivery using edible rice as a carrier (chapter : plant-based mucosal vaccine delivery systems). rice seeds have stability and resistance to digestion in the stomach, making mucorice an attractive oral vaccine delivery system. in , it was first reported that cholera toxin b subunit (ctb) could be expressed in the rice seed. as much as μg of ctb per seed was stored in the storage organelle protein body. when orally ingested, rice seeds expressing ctb induced ctb-specific serum igg and mucosal iga antibodies with neutralizing activity, while no rice storage-specific immune response was noted. when expressed in rice, ctb was protected from pepsin digestion in vitro [ ] . rice-expressed ctb also remained stable and thus maintained immunogenicity at room temperature for more than years, and it provided more than months of protection against ct-or lt-induced diarrhea after primary immunization [ ] . these results show that the mucorice vaccine could be stockpiled longer at room temperature and could be widely used for oral vaccination without cold-chain management. rice-based oral vaccine developments are under way against many infectious diseases and noninfectious diseases such as allergy, autoimmunity, and alzheimer's disease [ ] . the most widely used form of whole bacterial cell vaccines for cholera and typhoid fever was liquid suspension. because of the lack of shelf stability, the liquid format is unsuitable for storage and distribution in developing countries. in this regard, a stable solid dosage vaccine platform is required for those vaccines. formulation in tablets or capsules would provide more stability and ease of handling. in comparison to microparticles and nps, capsules are significantly larger in size and could serve reservoir for multiple vaccine/ adjuvant formulations [ ] . while no subunit or wc killed oral vaccine is currently delivered by capsule or tablet, the live attenuated salmonella vaccine vivotif is routinely delivered in an enteric-coated format [ ] . capsules could be manufactured in appropriate physical sizes (the average size of capsules and tablets ranges from to mm) suitable for administration to target populations. with enteric coatings, tablets and capsules could be protected from gastric acid and endowed with controlled release properties, which will provide facilitated delivery to discrete locations in the intestine. in principle, capsules allow the incorporation of many previously introduced delivery technologies in one primary delivery format. recently, a tablet-based oral avian influenza vaccine was shown to elicit strong antiviral antibody and ifnγ t-cell responses. this approach utilized a nonreplicating adenovirus type vector expressing avian flu hemagglutinin antigens together with a dsrna tlr agonist [ ] . among the choices of mucosal routes for vaccine administration, nasal delivery has been the most widely employed for innovative research because of the ease of approach and less harsh physicochemical conditions in the nasal cavity compared with the gi tract. furthermore, nasal vaccines could be administered without professional training. there are at least three nasal vaccines licensed worldwide. the fda approved flumist, a live attenuated trivalent/quadrivalent influenza vaccine. fluenz is an ema-approved quadrivalent influenza vaccine. these two intranasal vaccines are manufactured by the same company, medimmune. the serum institute of india licensed the monovalent nasovac against pandemic a/california/ / h n influenza (chapter : nasal influenza vaccines). besides influenza vaccines, the nasal route has been widely studied for development of many prophylactic and therapeutic vaccines against other infectious diseases, such as allergy, cancer, alzheimer's disease, and lifestyle-related diseases [ À ]. the human nasal cavity is an attractive route of mucosal immunization, having a total surface area of cm with a volume of À ml [ , , ] . the nasal cavity is divided into five anatomical and functional regions: the nasal vestibule, the atrium, the respiratory region, the olfactory region, and the nasopharynx [ ] . the respiratory region is where nasal delivery of drugs and vaccines occurs, since it is the most permeable region, having a large surface area and a rich vascular bed [ ] . the respiratory region is covered by a pseudostratified epithelium composed of columnar cells interspersed with goblet cells, which are interconnected by tight junctions (zonae occludens). the tight junctions are relatively resistant to paracellular passages of particulate materials in the breathed air [ ] . the respiratory region is where mucus production actively takes place. the mucus layer in the nasal tract is relatively thinner ( μm) than other mucosal surfaces. the nasal cavity is equipped with nasopharyngeal-associated lymphoid tissue (nalt), which is highly similar to peyer's patches in the ileum [ ] . nalt is also covered with m cells that have active antigen sampling capacity [ ] . intraepithelial dcs project dendrites toward mucosal lumen and sample antigens. particulate antigens are preferentially sampled by m cells, and small soluble antigens have access to epithelium, where they are captured by intraepithelial dcs [ ] (chapter : anatomical uniqueness of the mucosal immune system (galt, nalt, ibalt) for the induction and regulation of mucosal immunity and tolerance). the mucociliary clearance mechanism should have negative effects on nasal vaccination. the rapid turnover of mucus ( À minutes) and fast mucus flow (b mm/min) in the nasal cavity limit the length of residence of administered vaccine. continuous outward movement of cilia on the epithelial apical surface accelerates the clearance of mucus-trapped substances. to make matters worse, nasal enzymes and local ph negatively affect the stability of nasally administered vaccine antigens [ ] . this could be why only live attenuated influenza vaccines proved effective in clinical trials and were approved by the fda and ema. a live influenza virus should be able to survive in the nasal mucosa and be harnessed with built-in adjuvants. an inactivated split influenza vaccine was also tested for nasal delivery but proved ineffective without coformulation with appropriate mucosal adjuvants [ À ] . to achieve equivalent antibody responses without adjuvant, an inactivated split antigen should be given at least three times more, or an inactivated whole virus should have been immunized [ À ] . given that even an inactivated virus antigen requires potent mucosal adjuvants to achieve optimal immune responses in the systemic and mucosal compartments, protein antigens should employ even stronger mucosal adjuvants to be effective by nasal vaccination. many mucosal adjuvants are suggested as formulation partners of nasal vaccine antigens [ , ] . ct and related e. coli heat-labile toxin (lt) and their mutant derivatives are the most widely tried mucosal adjuvant in preclinical studies [ ] . although those enterotoxins served as potent adjuvants for nasal vaccination of diverse antigens in animal studies, they have seldom been adopted for the development of human nasal vaccines. the use of enterotoxins as nasal vaccine adjuvants has a very serious failure history. the subunit influenza nasal vaccine nasalflu berna adjuvanted with e. coli lt had been significantly connected with bell's palsy with an odd ratio of in an epidemiological study in switzerland. the vaccine was consequently withdrawn from the market [ ] . the adverse effect could be related with the capacity of ltb and ctb subunit to bind to gm ganglioside expressed on neuronal cell and retrograde translocation toward the brain [ , ] . since the nasal cavity and brain are separated by a thin anatomical structure and are directly connected by the olfactory nerve, binding of any vaccine component to the olfactory nerve should contribute neurotoxicity. in this context, any nasal vaccine, adjuvant, or delivery system must clear the safety concern to be introduced to the market. given the versatility and ease of nasal vaccination, numerous research groups are studying safe nasal adjuvants that could replace ct and lt. pamps are most widely studied as alternative nasal adjuvants. recently published literature shows that ligands of tlr , tlr , and tlr , sting agonist, and flt ligand could be used as effective and safe nasal adjuvants [ , À ] . vaccine antigens should remain sufficiently stable in the nasal mucosa and should be able to reach to antigen-capturing cells surviving the mucociliary clearance mechanism. to overcome those hurdles, micro/nanocarriers for nasal vaccine delivery have been actively researched. to increase the residence time at mucosal surfaces, several strategies have been developed to increase adhesiveness of antigen delivery systems to the nasal mucus [ , ] . however, the mucus is not a static barrier; it is continuously secreted and cleared from the nasal cavity by the cilia beating on columnar epithelial cells. mucoadhesion ability of delivery carrier would cause earlier removal of vaccine antigens when mucociliary clearance mechanism is intact. to cope with this problem, nasal vaccine carriers should cross the mucus layer rapidly and deliver antigens to m cells and dcs rather than strongly adhering to it [ À ] . strategies that prevent vaccine car-rierÀmucus interactions and hence allow for free diffusion by mucopenetration should be more effective in inducing efficacious immune responses [ ] . however, many reports claimed that mucoadhesive nps effectively enhanced the efficacy of mucosal vaccines [ ] . one study showed that mucoadhesive nps disrupted the protective human mucus barrier by altering its microstructure [ ] . the disrupted microstructure resulted in a % increase in mucus mesh pore size. this disrupted mucus mesh would allow increased passage of other nps to reach to the epithelium. the comparative advantages and disadvantages of intranasal vaccination are summarized in table . . the most outstanding advantage is the ease of administration, while the safety issue is the most essential problem to be resolved. nanoemulsions, owing to their stability, small droplet size, and optimal solubilization properties, have great potential in nasal drug delivery. furthermore, they may act as an active adjuvant in nasal vaccine formulations. despite the promising results of in vitro and animal studies, the application of nanoemulsions for nasal delivery in humans appears to be hindered mainly by the lack of detailed toxicology studies and the lack of extensive clinical trials [ ] . a cationic nanoemulsion formulation could have facilitated cellular uptake of model antigen ovalbumin in the nasal epithelial cell line [ ] . the intranasal vaccination of hiv gp immunogen formulated in oil-inwater nanoemulsions induced robust serum anti-gp igg and th -polarized systemic cellular immune responses [ ] . a large number of studies have investigated the potential of liposomes as a delivery system for nasal vaccination. those [ ] . peptides, proteins, and dna can be successfully carried by liposomes having neutral, negative, and positive charges. cationic liposomes were shown to interact more efficiently with epithelial cells and dcs [ , ] . induction of cell-mediated immunity is another important feature of liposomemediated adjuvanticity [ ] . intranasal administration of dna vaccine formulated with cationic liposomes, together with il- -and/or gm-csf-expressing plasmids, resulted in both high levels of hiv- -neutralizing antibodies in feces and serum and high levels of hiv-specific ctl responses [ ] . using this characteristic of the liposomal delivery system, a successful antitumor cellular immune response could be induced by intranasal immunization of dctargeting liposomes carrying a tumor antigen [ ] . similarly, intranasal immunization with liposome-encapsulated plasmid dna encoding influenza virus hemagglutinin elicited both mucosal cell-mediated and humoral (iga and igg) immune responses [ ] . intranasal chitosan solution formulations were reported to enhance protective immune responses against many antigens, including diphtheria, pertussis, and influenza [ À ] . chitosan solutions seem to induce balanced th and th responses with neutralizing antibodies [ ] . whole influenza virus formulated with trimethylated chitosan showed much closer interaction with the epithelial surface, with the potential to generate enhanced uptake and induction of immune responses with minimal local toxicity in terms of ciliary beat frequency in the nasal cavity [ ] . chitosan dry power in salt form enables a thermally stable vaccine formulation that does not require cold chains. chitosan power formulations were shown to outperform solutions in eliciting humoral responses against diphtheria, anthrax, and norovirus [ ] . chitosan microparticles and nps are being robustly studied for the intranasal delivery of vaccines. chitosan particles are basically mucoadhesive and able to deliver adjuvants and antigen cargos to efficiently promote humoral and cellular immune responses. protein and peptide antigen-loaded chitosan particles are taken up by apcs in the administration site and eventually trafficked to draining lymph nodes, where t-cell activation occurs [ ] . to be used for better intranasal delivery, chitosan should be chemically modified for better solubility, stability, mucoadhesiveness, safety, and resilience against degradation [ ] . chitosan itself shows strong adhesion to mucosal surfaces, providing a longer retention time at the nasal mucosa, and disrupts the tight junctions between nasal epithelial cells, which leads to enhanced paracellular transport of antigens [ ] . the paracellular transport of the vaccine formulation into the nasal mucosa would lead to enhanced antigen uptake and presentation by apcs, with consequently augmented adaptive immunity [ ] . this was demonstrated in a subunit influenza vaccine study showing protection against a heterologous viral challenge [ ] . ntrimethyl chitosan nasal vaccine formulation was superior to plga nps in inducing nasal iga responses [ ] . improved mucosal (iga) and humoral (igg) antibody responses are generally observed in mice as well as in other animal models such as rat and rabbit [ ] . the cationic chitosan enhanced th and th responses as well as dc maturation through type i interferon induction by the cgas-sting pathway, suggesting the involvement of multiple immune components [ ] . modified chitosan particles were generated to improve delivery efficiency and targeting. pegylation improved water solubility and stability of conjugated antigens [ ] . chitosan coated poly-(ε-caprolactone) nps induced enhanced mucosal immune responses against coformulated influenza antigen [ ] . protein antigen-loaded pluronic f- /chitosan microparticles showed improved antigen release and induced higher antigen-specific secretory iga responses after intranasal vaccination [ ] . ova-loaded trimethyl chitosanÀhyaluronic acid nps demonstrated superior immunogenicity after intranasal immunization [ ] . mannosylation of chitosan particles enhanced macrophage targeting and antigen-specific secretory iga responses in mucosal secretions after intranasal immunization [ ] . chitosan application to intranasal vaccination has already reached clinical trial stages (phases and ) in the form of nps [ ] and antigen-conjugates [ ] . norovirus vlp formulated with mpla was administered intranasally twice to healthy volunteers, inducing specific iga responses in % of vaccinated individuals [ ] . a clinical study testing intranasal vaccination of neisseria meningitidis serogroup c polysaccharide (mcp)-crm conjugate antigen mixed with chitosan showed specific iga responses in nasal washes and balanced igg /igg responses in serum [ ] . despite several clinical studies with promising results, no chitosan-based product for intranasal vaccination has yet reached the market. influenza viral antigens encapsulated within bioadhesive starch and propylacrylic acid mixtures induced significant systemic antigen-specific igg responses but not mucosal iga after intranasal delivery of the influenza vaccine in rabbit [ ] . inactivated influenza antigens in positively charged nps have been tested in a phase clinical study. significant mucosal iga antibodies were produced in individuals who received two-dose nasal immunizations [ ] . a cationic maltodextrin np (cationic surface with an anionic lipid core) showed longer nasal residence time after nasal administration than liposomes and plga nps [ ] . the plga nps are the most used synthetic polymer for nasal vaccine delivery studies. cationic modification of plga enhanced residence time in the mucosa and resulted in better immune responses with higher serum antibody and siga levels [ ] . the surface modification of plga carriers with chitosan can increase mucoadhesion through a change of zeta potential from negative to positive without affecting particle size and dispersion. moreover, the clearance rate in the nasal cavity was reduced, resulting in enhanced systemic and mucosal antibody responses [ ] . many antigens encapsulated in plga nano/microparticles were immunized through the nasal route to show enhanced immune responses in both systemic and mucosal immune compartments; they were ovalbumin, bovine serum albumin, bovine parainfluenza virus, bovine syncytial virus, hbsag, malaria, swine fever virus, y. pestis, and streptococcus equi [ ] . targeting efficiency to m cells could also be achieved by functionalization of the particle surface [ ] . although many types of nanogels were tested as vaccine delivery systems, the cholesteryl group-bearing pullulan (chp) is the most extensively studied one for mucosal vaccine delivery [ ] . the cationic chp (cchp) nanogel binds better to epithelial cells and is subsequently taken up with high efficiency into the cells. the cchp nanogel itself did not activate dcs and did not have biologically active adjuvant activity. however, vigorous neutralizing serum igg and siga responses were noted without coadministration of mucosal adjuvants. the cchp nanogel was suggested as a universal protein-based antigen delivery vehicle for adjuvant-free intranasal vaccinations [ ] (chapter : nanodelivery vehicles for mucosal vaccines). to further potentiate immune responses against cargo antigens, cytokines or adjuvant could be coencapsulated in the chp nanogel. tnfα encapsulated in the nanogel acted as a vaccine adjuvant for a nasal influenza vaccine [ ] . in an antiobesity vaccine study, cchp was engineered to carry the adjuvant cyclic di-gmp along with self-origin antigen ghrelin peptide hormone conjugated to a carrier protein pspa [ , ] . the cchp-based nasal vaccines were successfully tested for use against several infectious diseases and lifestyle-related diseases: influenza, streptococcus pneumoniae, c. botulinum, obesity, and hypertension [ , , , , , ] . another very promising virtue of the cchp nanogel nasal delivery system is safety. it was shown reiteratively that protein antigens carried by the nanogel did not accumulate in the olfactory bulb and brain, thus excluding the risk of neurotoxicity or brain damage [ , ] . when all the physicochemical, biological, and immunological characteristics are considered, the cchp nanogel platform seems to be the most promising nasal delivery system to be translated into more aggressive clinical applications. although mucosal vaccination has many advantages, a very limited number of mucosal vaccines have been licensed. the most widely tested vaccination routes are oral and intranasal. currently licensed oral and intranasal vaccines are composed predominantly of wc killed or live attenuated microorganisms, where cell bodies serve as delivery systems and whose cell components act as built-in adjuvants. future mucosal vaccines should be made with more purified antigen components, which will require safe and efficacious adjuvants and delivery systems. recent developments in biomaterials and nanotechnology have enabled many innovative mucosal vaccine trials. for oral vaccination, the vaccine delivery system should be able to stably carry antigens and adjuvants and resist the harsh physicochemical conditions in the stomach and intestinal tract. besides many nano/microcarrier tools generated by using natural and chemical materials, the development of oral vaccine delivery systems using food materials should be more robustly researched to expand vaccine coverage of gi infections in developing countries. for intranasal vaccination, the vaccine delivery system should survive the very active mucociliary clearance mechanisms and provide safety, given the anatomical location of the nasal cavity, which is separated from the central nervous system by a thin barrier. future mucosal vaccine carriers, regardless of administration routes, should share common characteristics. they should maintain stability in given environments, be mucoadhesive, and have targeting ability to specific 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residence time and uptake of porous and cationic maltodextrin-based nanoparticles in the nasal mucosa: comparison with anionic and cationic nanoparticles strong systemic and mucosal immune responses to surface-modified plga microspheres containing recombinant hepatitis b antigen administered intranasally intranasal and oral vaccination with protein-based antigens: advantages, challenges and formulation strategies cholesteryl pullulan encapsulated tnf-alpha nanoparticles are an effective mucosal vaccine adjuvant against influenza virus cationic pullulan nanogel as a safe and effective nasal vaccine delivery system for respiratory infectious diseases mucosal or systemic administration of re glycoprotein antigen loaded plga microspheres nasal and pulmonary vaccine delivery using particulate carriers transient facial nerve paralysis (bell's palsy) following intranasal delivery of a genetically detoxified mutant of escherichia coli heat labile toxin intranasal delivery of nanoparticles encapsulating bpi v proteins induces an early humoral immune response in mice induction, function and regulation of il- -producing t cells induction of mucosal immune responses and protection of cattle against direct-contact challenge by intranasal delivery with foot-and-mouth disease virus antigen mediated by nanoparticles synergistic action of cholera toxin b subunit (and escherichia coli heat-labile toxin b subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues key: cord- -nrodyagi authors: schutzer, steven e. title: the use of host factors in microbial forensics date: - - journal: microbial forensics doi: . /b - - - - . - sha: doc_id: cord_uid: nrodyagi advances have been made in the forensic analysis of microbes and toxins. an underdeveloped and underutilized area in microbial forensics is how the host interacts with microorganisms in a way that provides unique signatures for forensic use. for forensic purposes, an immediate goal is to distinguish a potential victim and innocent person from a perpetrator, and to distinguish between a naturally acquired or intentional infection. principal methods that are sufficiently developed are characterization of the humoral immune response to microbial antigens including vaccine-induced immunity and detection of antibiotics that may be present in a possible perpetrator. this chapter presents central elements of the host response in a simplified fashion and describes a representative example, which, in the appropriate context, has a high potential of providing evidence that may aid an investigation to distinguish a perpetrator from a victim. this chapter also presents information about the immune system so that the interested reader can have a fuller understanding of the immune response in general. considerable advances have been made in the forensic analysis of microbes and toxins. these advances include sequencing, genomics, and microscopy. an underdeveloped and underutilized area in microbial forensics is how the host interacts with microorganisms in a way that provides unique signatures for forensic use. for forensic purposes, an immediate goal is to distinguish a potential victim and innocent person from a perpetrator, and to distinguish between a naturally acquired or intentional infection. two principal methods that are sufficiently developed are characterization of the humoral immune response and identification of vaccine-induced immunity or antibiotics that may be present in a possible perpetrator. this chapter presents central elements of the host response in a simplified fashion and describes a few representative examples, which, in the appropriate context, have a high potential of providing evidence that may aid an investigation to distinguish a perpetrator from a victim who has been exposed to a particular microbe or by-product, such as a toxin. this chapter also presents nonmicrobial forensicedirected information about the immune system so that the interested reader can have a fuller understanding of the immune response in general. the primary aims of a microbial forensics are to identify the biological agent, its source, and the individuals responsible for the event (budowle et al., ) . analytic approaches differ when the suspected biothreat agent is encountered in a container or the environment, as opposed to in vivo in a human, animal, or plant. analyses of trace elements, pollens, growth media, latent fingerprints, and microbial and nonmicrobial nucleic acids are all applicable to the container and environmental sample (states et al., ) . however, once the microorganism or its toxin is in the living host, it is no longer possible to analyze the preceding items except the microbial nucleic acid. however, the host's response to the biological agent may be available for analysis for clues. this is akin to other forensic studies where physical traces of bite marks, scratches, wound trajectories, and sizes of wounds are often surrogate evidence of the teeth, fingernails, and bullets (averill and odontology, ) . while the forensic pathologist is familiar with evidence related to determining the manner of death including the host response, those involved with healthcare alone are more familiar with the host response. in the context of microbial forensics, it is important to integrate all of these with intelligence information so that they may be included in the analytical data and attribution picture. the physician and other healthcare providers may be among the first to realize that a patient is a victim of a biocrime. in the case of a covert attack, it may be the physician or medical examiner who first recognizes the index case. these healthcare workers are in key positions to preserve critical evidence and, thereby, contribute to the investigation (schutzer et al., ) . there are a number of steps that should be followed when the possibility of a biological attack arises, either with the consent of the patient or because individuals are compelled by law to interact with public health and law enforcement. a joint statement by the fbi, the cdc, and the dhs advises calling the fbi and public health authorities if a suspicious situation arises (investigation et al.) . some guidelines on the procedure(s) to report of suspicions of biocrimes are provided by the centers for disease control and prevention (cdc; http://www.cdc.gov), the federal bureau of investigation (fbi; http://www.fbi.gov), and the department of homeland security (dhs; http://www.dhs. gov) and detailed in previous article (schutzer et al., ) . the host response to a microorganism or other foreign substance is often a wellorchestrated series of events, which may protect the individual from harm (zabriskie, ). at the same time, these host responses may provide clues as to identity of the offending microorganism or toxin as well as a rough chronology of when it occurred and for how long it has been persisting. emerging technologies such as transcriptional arrays and bioinformatic analysis will eventually be refined and methods validated to provide even greater help in delineating more of the pathways and components of the host response to an infectious agent (sala et al., ; popper et al., ; ko et al., ) . other technologies are sufficiently mature to be of use today. the immune system and its components are a mainstay of our protection against infections and malignancies (zabriskie, ; paul, ; murphy and weaver, ) . inflammation is often a side effect as the immune system contains and eradicates a microorganism or eliminates foreign tissue. specific arms of the immune system can be used as markers in support of or against the presence of an infection. the humoral or antibody response to an invading microorganism is one example of a specific arm that can have forensic value. some of the antibodies produced may have a protective role together with other parts of the immune system by eradicating the pathogen or neutralizing a toxin. other antibodies may not be as effective in this role. however, in their ability to recognize unique and specific microbial antigens, they can serve as indicators that a specific microorganism was recently present or was present in the past. in the case of a vaccine, specific antibodies may recognize highly specific epitopes of one microbe versus those of a related microbe (e.g., influenza virus). this is especially so with different recombinant vaccines and could have forensic importance. substances such as antibiotics, which can rapidly kill a pathogen, may modify the immune response by removing or reducing the infectious driving force for a full-scale response. as noted above, in clinical and veterinary medicine, measurement of the immune response helps the diagnostician decide what infection was present and how recently. in these situations, the intent is to provide treatment. the forensic scientist may exploit parts of the immune response to discover who is likely a victim of an attack and who might be responsible. this chapter will discuss the basics of the host immune response in a simplified manner that can have utility in a forensic sense. examples will provide a sense of what information is achievable and what is . the use of host factors in microbial forensics not likely to provide clues with a high degree of certainty. in response to a new exposure to a microbe, the innate immune system may be the first line of defense. then the immune system starts to activate the antibody system. typically, a cell known as a macrophage ingests and degrades some of the invading pathogens. it then presents part (antigens) of the microorganism to a helper t cell (a lymphocyte), which then directs other lymphocytes known as b cells to produce antibodies to those antigens of that particular microbe that were presented. it usually takes at least days before any microbe-specific antibody can be detected (parslow, ) . antibodies are a specific form of the proteins known as immunoglobulins (igs). igm, igg, iga, and secretory iga are the principal classes of immunoglobulins with prime relevance to this chapter and will be discussed in more detail. in an infection, immunoglobulins usually appear in the order of igm, igg, and iga. b cells first begin to produce igm, and then some b cells undergo an irreversible switch to those that produce igg. later, some of these b cells undergo a switch to become iga-secreting b cells. immunoglobulins persist for varying times; for example, the half-life of particular igm antibodies is approximately days, while that of igg can be as long as e days (table . ) (paul, ; murphy and weaver, ) . in certain circumstances of ruling in or ruling out a suspect, the specific ige may be of value. those individuals unfortunate to have allergies have problems due to ige against allergens (such as ragweed, peanut, or cat dander). in this case, the ige molecules sit on the surface of mast cells and basophils. these cells can release histamine and other allergic mediators when the offending allergen bridges two ige molecules. similar to the effect from an infection with a live microbe, vaccines are often designed to provoke an antibody response. the vaccine can be composed of a live or attenuated microbe, a whole nonproliferating microbe, or an antigenic (recombinant) part of the microbe or a toxoid. regardless, the intent of immunization with a vaccine is to engender protection, often by the generation of protective neutralizing antibodies. although the half-life of an individual igg molecule is less than a month, a population of antibodies of the igg isotype form may persist for life. memory b cells can sustain these antibodies and retain the ability to quickly generate the appropriate antibodies when challenged. when the immune system encounters another infection or is subjected to a revaccination (booster), the result is an accelerated production of the particular antibody and increase in the levels of antibodies that circulate in the blood ( fig. . ) . perhaps, the pattern of antibody response which has the most forensic value, by providing a timeframe, is the appearance of igm first, followed by a b-cell switch to the longer-lasting igg. during the early phase of exposure, igm predominates, as time goes on, igg may wax and wane and igm is no longer found ( fig. . ) . the antibody response to a particular agent may be directed to different antigens at different times, that is, early or later after the initial exposure. that response often involves igm at the early stage and igg later. late in the course of (bennett et al., ) , a virus known to cause mononucleosis. during acute early disease, it is common to find high levels of antibody of the igm isotype to the viral early antigen (ea) and viral capsid antigen (vca). it is rare to find igg antibody to the vca or to epsteinebarr nuclear acid (ebna) in anything but low titers (levels). as the patient recovers from their first infection with ebv, it is rare to find anything but low levels of igm to ea or vca, but igg to vca in higher or increasing levels is common. antibodies to ebna are often very low during this stage. several months after clinical recovery, igm to ea and vca remain at low levels, whereas igg to vca and ebna are present at high levels, often for years. a controlled experiment or normal clinical event illustrates what happens when the immune system responds to the infectious agent or a vaccine again. the controlled experiment may be in a laboratory animal or a patient illustrative concepts receiving a booster vaccine. the uncontrolled but normal clinical event occurs when the patient is reexposed to the infectious agent. consider a generic antigen exposure. the first time the immune system encounters antigen x (agx), it responds as shown in figs. . and . . initially, antibodies to agx are barely discernible; then levels rise and later fall to a plateau. if a simultaneous exposure were to occur with agx and a new agy from another microorganism, the immune system would quickly mount a brisk response with high levels of ab to agx, while the course of ab to agy would be slow and delayed, just as it was in the response to the first exposure to agx. this phenomenon is termed immunological memory or an amnestic response. this can be useful when the symptoms and signs of exposure to either x or y are similar. this is the case with the early flu-like symptoms of pulmonary anthrax (raymond et al., ; waterer and robertson, ; bush et al., ) and with the influenza virus itself (meltzer et al., ; cao et al., ; lessler et al., ) . another example common to all of us is repetitive exposure to different strains of flu viruses (meltzer et al., ; janeway, ) . as illustrated in table . , a person infected for the first time with one strain of the influenza virus has a response to most of its antigens (as a theoretical example, ag , , , , , ). three years later, the same individual exposed to a partially similar influenza virus responds preferentially to those antigens that were also present on the original influenza virus. the person also makes a smaller initial antibody response to new antigens, that is, those not shared with the first virus. ten or years later, during a new flu season and exposure to a third strain of influenza, the most brisk responses would be to antigens previously recognized by the immune system. this is the scientific basis for giving the flu vaccine, which contains a variety of possible antigens common to multiple strains of the flu virus so that a rapid and protective antibody response will occur. utility of serologic analysis of people exposed to anthrax: strengths and limitations our knowledge of the humoral response to infection with biothreat microbes is limited compared with our knowledge of the kinetic response to common human infections. nevertheless, in the appropriate context and with sufficient background information, detection of antibodies to a particular microbe and its antigens can have important value for a microbial forensic investigation. this information may have critical probative value or it can guide investigative leads. the absence of a specific antibody response may also have value in a particular investigation. certainly, its importance is increased in the context of information of what organism could be involved, when the exposure was likely to have occurred, the route of exposure, what symptoms and signs are manifesting in the host, and other data such as presence of antigens and microbial nucleic acids (jackson et al., ) . other information such as how many hosts (people or animals) have had this infection in the geographic region, what is the incidence, and background prevalence of antibody titer to the organism in question or a related organism, in the population being studied, is also important. vaccination responses can have forensic value. the current protective antigen (pa) vaccine has small amounts of ef and lethal factor (lf), which are responsible for some of the side effects, so one might expect to see antibodies against these antigens as well as to pa. the recombinant pa is just pa so anti-lf and anti-ef would be absent in an immunized individual. the anthrax-letter attacks raised multiple questions for every person infected, possibly exposed, vaccinated, or treated. some of these questions included how these persons were infected by spores, if at all; that is, through breaks in the skin (cutaneous anthrax); by inhalation of spores (pulmonary anthrax (bennett et al., ) ), or by ingestion (gastrointestinal anthrax (bravata et al., ; tutrone et al., ) ). or, were they among the "worried well"? consider the situation where a close associate comes down with symptoms compatible with inhalational anthrax after receiving a letter containing powder and that material is no longer available. until this is shown not to be anthrax, great worry will ensue. in several cases of documented exposure, there was not enough time for the patient to develop antibody to a specific anthrax antigen, at least as probed for igg. serial serum samples obtained on november , , , and of were tested for igg antibody to the pa component of the anthrax toxins by enzymelinked immunosorbent assay (elisa); all samples were nonreactive. serial tests for serum igg antibody to the pa toxin of anthrax were performed on workplace-exposed persons. all but one test was negative. most of the specimens were collected on october and (traeger et al., ) . it is instructive to look at the positive antibody case in the context of the nature and utility of serologic analysis of people exposed to anthrax: strengths and limitations duration of that individual's symptoms when he developed a positive test. ernesto blanco, a -year-old mailroom clerk (case ), experienced fatigue on september . he worked in the mailroom of the ami building and delivered mail to the index case. on september , he developed a nonproductive cough, intermittent fever, runny nose, and conjunctivitis. these signs worsened through october when he was hospitalized. in addition, he had shortness of breath with exertion, sweats, mild abdominal pain and vomiting, and episodes of confusion. his temperature was elevated to . c ( . f), heart rate was rapid at /min, respiratory rate was slightly fast at /min, and blood pressure was / mm hg. he had bilateral conjunctival injection and bilateral pulmonary rhonchi. at the time of admission, his neurologic exam was normal. no skin lesions were observed. the only laboratory abnormalities were low albumin, elevated liver transaminases, borderline low serum sodium, increased creatinine, and low oxygen content in the blood. blood cultures were negative on hospital day , after antibiotics had been started. the chest x-ray showed a leftsided pneumonia and a small left pleural effusion but no "classical" mediastinal widening (dewan et al., ) . the patient was initially given intravenous azithromycin; cefotaxime and ciprofloxacin were subsequently added. a nasal swab obtained on october grew bacillus anthracis on culture. computed tomography (ct) of the chest showed bilateral effusions and multilobar pulmonary consolidation but still no significant mediastinal lymphadenopathy. pleural fluid aspiration was positive for b. anthracis dna by pcr. bacterial cultures of bronchial washings and pleural fluid were negative. immunohistochemical staining of a transbronchial biopsy demonstrated the presence of b. anthracis capsule and cell wall antigens. during hospitalization, his white blood count rose to , /mm , and fluid from a second thoracentesis was positive for b. anthracis dna by pcr. immunohistochemical staining of both pleural fluid cells and pleural biopsy tissue demonstrated the presence of b. anthracis capsule and cell wall antigens. serial serum samples demonstrated > fourfold rise in serum igg antibody to the pa component of the anthrax toxins by an elisa assay. the patient was able to leave the hospital on october on oral ciprofloxacin. table . illustrates both the clinical and microbial forensic approach and context in which to analyze such a patient. it is likely to be common to most situations where a biocrime ( ). these questions include was the infection acquired naturally or was it an intentional action that led to the infection; how did the particular individual acquire it if it was not a natural infectiondwas he the target or a bystander. an alternative possibility in the right circumstances is a laboratory-acquired infection. this case also demonstrates that cultures may be negative at different times from different fluids and tissues because of early administration of antibiotics. however, the remnants of the infection, even dead organisms, can be found by probing for antigens and dna. this patient's response demonstrated a classic principle of infectious disease, a rising antibody titer over time. in this case, it was igg to a particular antigenic component of the anthrax toxins (friedlander and little, ; cunningham et al., ) . the subject's antibody response may have been detected earlier if igm to this component or to other antigens of anthrax had been sought. the case also points out the utility of integrating the presence of antibody with other indications of an anthrax infection such as culture, pcr, and antigen detection. these take on their greatest significance during clinical illness in someone who was possibly exposed. early administration of antibiotics can prevent or interfere with the isolation of a pathogen by culture (kaeberlein et al., ) . of the first pulmonary anthrax cases associated with the letter attacks, three patients had no isolate of b. anthracis from any clinical samples; however, culture was attempted after initiation of antibiotic therapy. history of exposure in conjunction with compatible symptoms and signs of disease and objective laboratory findings were the basis for the diagnosis. b. anthracis was identified in pleural fluid, pleural biopsy, or transbronchial biopsy specimens by reactivity with b. anthracis-specific cell wall and capsular antibodies or by the detection of dna in pleural fluid or blood by pcr (jernigan et al., ) . it is important to understand the limitations of any assay used in medicine or forensics (budowle et al., ; schutzer et al., ). an igg-based elisa for anti-pa illustrates the importance of understanding the limitations of an assay. the elisa was developed at the us army medical research institute of infectious disease (usamriid) and put into operation after optimization and internal validation at the cdc for functional sensitivity and specificity in detecting an antibody response to b. anthracis infection. its major limitation was that only one antigen was used and only igg was measured. therefore, a negative result shortly after exposure may, in effect, be a false-negative result. a gap such as this may be filled by development of an assay for antigenspecific igm, and by probing for other b. anthracis antigens or epitopes yet to be characterized. the assay may be very useful in its present form to screen asymptomatic people with possible exposure. the study by dewan et al. (dewan et al., ) provide a contemporary background database on a group of postal workers who may have been exposed to b. anthracis. beginning on october , , postal employees and others who had been to the washington d.c. postal facility went to the d.c. general hospital for antibiotics in addition to those people whose treatment began on october , . serum samples were also obtained from the individuals who had been to the washington d.c. postal facility during the precious weeks. all were negative for specific anti-pa igg, including three individuals who reported a remote history of anthrax vaccination. the consistent negative findings may be explained by the fact that antibiotic therapy was initiated before serum testing and that there were no baseline serum samples available for testing. in addition, the time period from exposure to sampling was very short. among individuals in the capitol region with culture-positive nasal swabs who received prophylactic antibiotics immediately, none had a positive culture from a nasal swab repeated days later, and none developed igg to pa antigen days after exposure. this again emphasizes the limitation and interpretation of a test in someone who had early antibiotic treatment. it does raise forensic utility considerations. even with these easily disseminated spores, an antibody response may be aborted or modified with antibiotics by early eradication. furthermore, antibiotics taken before exposure would likely be effective in preventing laboratory and clinical signs of an infection. detection of microbial dna, antigen, or the organism itself on a person's body, clothing, or possessions should raise a red flag for exposure. the route of infection is important in interpreting results and the limitations of the assay used. the example of cutaneous anthrax in paraguay illustrates this notion, as well as the need to search for other antigens as markers of exposure (harrison et al., ) . in an analysis of an outbreak of cases of cutaneous anthrax that followed contact with raw meat from a sick cow, sera from cases and colony and noncolony controls were examined by western blot for antibodies to pa and lf weeks after the outbreak. an elisa was used to probe for antibodies to the poly-d-glutamic acid capsule. of the cases, had antibody to pa, for a sensitivity of . %; none of the controls was positive. only of cases had antibody to lf; all controls were negative. anticapsule antibodies were positive in of but were also positive in of controls. the results of this study demonstrate the need to consider other antigens. some of the principles discussed above are highlighted by a report on severe acute respiratory syndrome (sars). the appearance alone of this coronavirus responsible for this disease evoked concern of a possible terrorist origin at the onset. a report in the morbidity and mortality weekly report (mmwr (cfdcap, ) ) on the "prevalence of igg antibody to sars-associated coronavirus in animal traders" discussed the need to validate and interpret tests in appropriate populations. also discussed was the inability to date the time of infection by the igg assay, and the possibility of assay crossreactivity to a near neighbor that might be unknown. in a promed bulletin, dr. steve berger looked at the same data from a different perspective and reported "this week's study in mmwr indicates that animal contact may indeed promote infection; however, the most striking finding seems to have eluded the authors: . percent to . percent of individuals in a healthy control group of adults were also found to be seropositive! the population of guangdong province is . million ( ), of whom . million are adults over age . if we assume that the seropositivity rates among controls is representative of the province as a whole, , to , , adults in guangdong have at some time been infected by the sars virus. these figures are -to -fold the total number ( ) of sars patients reported worldwide to date!" this comparison is a good illustration of the advantage of open dissemination and discussion of information as well as the need to question the methodology of acquisition of data before accepting their application in formulas or for analyses for forensics and epidemiology. it is also of value to remember that many infections include many with sars coronavirus have been asymptomatic or mildly symptomatic. plague, is a zoonotic infection caused by yersinia pestis, which occurs in the western united states with regularity and has an animal reservoir (bennett et al., ) . the situation with the naturally occurring yersinia is in contrast to the appearance of a case of smallpox which would raise an immediate red flag for a bioterrorist event. cases need to be approached from an epidemiologic standpoint first to determine whether it is a naturally acquired case or whether the facts point to a deliberate introduction of the organism. analytic techniques could include genomic analysis of an isolated organism and immunological response of the host. in the new era of rapid and deep sequencing, our capacity to investigate the genomics is growing (mardis, ; stavnsbjerg et al., ) . in consideration of animal reservoirs, elisa assays were compared with other tests for detection of plague antibody and antigen in multimammate mice (mastomys coucha and. mastomys natalensis) (shepherd et al., ) , which were experimentally infected and then sacrificed at daily intervals. igg elisa was equivalent in sensitivity to passive hemagglutination and more sensitive than the igm elisa and complement fixation. antibody was detectable by day after infection using all four tests. igm elisa titers fell to undetectable levels after weeks. plague fraction antigen was detected in of bacteremic sera from m. coucha and m. natalensis. this antibody pattern comparison shows that the principle of igm versus igg to this pathogen works to temporally situate the infection as an early versus late or past event. it also shows that when the information is combined with antigen detection, it engenders more confidence in the results. it should be noted that conclusions from this older reference has been substantiated with more defined antigens and assay technologies. melioidosis is caused by burkholderia pseudomallei (ashdown, ) . key clinical signs and laboratory results may raise the possibility of an infection with this pathogen. whether it is an acute, persistent, or past infection can be determined by assessing several host responses. often a simple indicator such as erythrocyte sedimentation rate or c-reactive protein (crp) can raise a clinical suspicion of an infection. in a study of patients with clinical melioidosis, ( culture-positive and culturenegative) had relatively uneventful disease courses. initially, they had elevated serum crp that decreased with antibiotic therapy and returned to normal as the disease resolved. in another series of patients, igm and igg were measured by elisa in sera from septicemic cases and sera from cases with localized melioidosis (chenthamarakshan et al., ) . sixty-five sera from culture-negative cases seronegative for other endemic infections but suspected of melioidosis were also examined. other controls included serum from nonmelioidosis cases, high-exposure risk cases, and healthy individuals. the igg-elisa was % sensitive and % specific. all sera from cases with septicemic and localized infections and of sera from clinically suspected melioidosis cases were positive for igg antibody. the sensitivity and specificity of the igm elisa were % and %, respectively. a geometric antibody index for igm antibody in the sera of the melioidosis cases was significantly higher in cases compared with that of the noncase controls. in another study by some of the same authors, a rapid test for igg and igm was shown to have clinical utility (cuzzubbo et al., ) . a study with the intent of evaluating the utility of an igg assay compared with other assays illustrates how the clinical and temporal context must be integrated for interpretation (dharakul et al., ) . it also illustrates how there is room for technical improvement in tests but the best setting is often the endemic area itself or at least using samples from that area in which the infections are occurring. these tests were evaluated in the actual clinical setting in an area endemic for melioidosis. specificity of igg ( . %) and igm ( . %) assays was significantly better than that of an indirect hemagglutination test (iha) ( . %). the sensitivity of the igg assay ( . %) was higher than that of the iha test ( . %) and the igm test ( . %). specific igg was found in septicemic cases ( . %) and localized infections ( . %). the igg test was also better than the igm test and the iha test in identifying acute melioidosis cases in the first days after admission. igg antibody to a b. pseudomallei antigen remained high for longer than years in recovered, disease-free patients. because this is a disease that may have an incubation of days to years, an acute case may very well be detected by a rise in specific igm if it were a matter of days from infection. although endemic for southeast asia, if b. pseudomallei was used as a biothreat agent in a different environment, its course and manifestations may not be recognized due to unfamiliarity with the disease. the above example also points out how the context in which a test is used determine is valuable. the concept of predictive value is instructive in determining how useful a test may be. in terms of disease detection, a high positive predictive value indicates the test is useful in determining that the disease is present. a high negativity predictive value would indicate that the test is useful in excluding the presence of the disease. another zoonotic agent is rift valley fever virus (rvfv), which can be transmitted via aerosols (clark et al., ) . one study with the intent at looking for improved tests showed the utility of igm to determine an early exposure to rvfv (niklasson et al., ) . two elisa igm tests detected specific igm antibodies to rvfv during the first weeks after vaccination. three inactivated vaccine doses were given on days , to , and to . elisa serum igm on days e were negative or in the lower range of detection; on days e the serum antibody values were strongly positive; on days e , they were waning and in later collected samples were negative. the plaque reduction neutralization test was negative on days e and became positive in later samples. similar to the examples shown above, these data suggest that three doses of rvfv vaccine induced a prolonged primary antibody response. the authors of that study concluded that the elisa igm may be useful for early diagnosis of acute human infection. good correlation of a neutralization test and elisa igg would indicate a later infection. taken together, these examples illustrate that an ideal test or analysis for both clinical and forensic use would incorporate endemic and incident area controls, historical contextual information, knowledge of the route of exposure, background incidence, and kinetics of transmission. each of these scenarios must take into account multiple factors and the limitations of any analytic process to be applied. on one extreme is the situation that occurred with the onset of acquired immunodeficiency syndrome (aids) from the human immune deficiency virus (hiv) in the united states. initially, there were no cases, and therefore a precise highly sensitive and specific test with excellent positive and negative predictive values (such as exists now when a combination of tests are used) would not likely yield a positive result in an area where there was little hiv infection and disease at the onset such as kansas. a positive test by today's methodologies from a serum sample from kansas would be considered a probable falsepositive and warrant further investigation. today, several viral and nucleic acid assays are available that would provide a definitive diagnosis in a short period of time (bennett et al., ) . however the same sample tested at the beginning of hiv testing could have been positive if the person had adult t-cell leukemia, which is caused by human t-cell leukemia virus- (htlv- ) because the original tests for what became known as aids involved whole viral lysates in which up to % of the htlv- sera cross-reacted. questions regarding the interpretation of the test results could be raised by knowledge of different presentations of the infection. for example, htlv- can actually be used in the laboratory to immortalize cells. in the patient, it actually increases the t-cell count, as is the nature with leukemia, instead of decreasing them, as with hiv infections. other laboratory indicators such as hypercalcemia would now raise the leukemia as a consideration. interpretation of a positive laboratory test must also take into account the health status of the person being tested. this is important for the practice of medicine and can have relevance when extended to forensic analysis (schutzer et al., ) . the following examples illustrate this concept. individuals who have syphilis, a treponemal bacterial infection, can typically have a positive fluorescent treponemal antibody test result for years, even after successful treatment. however, while infected they would have a positive venereal disease research laboratory (vdrl) test, which reverts to negative following successful antibiotic therapy. the vdrl test detects nonspecific anticardiolipid antibodies and can produce false-positive results with other conditions (e.g., pregnancy). there are some notable exceptions related to crossreactive epitopes or autoimmune diseases. these are readily distinguishable by history and clinical information. similarly, individuals infected with active tuberculosis will likely have a positive skin test (mantoux) or a positive interferon-gamma release assay (dewan et al., ; ota and kato, ) , whereas the uninfected healthy person will be negative. in certain instances, a sick person with a cell-mediated immune deficiency will be anergic, that is, he/she will be negative to multiple skin tests including common antigens such as candida. the key difference here is that there is a great difference between the healthy person being tested and an ill or immunocompromised individual being subjected to the same test. tests may also discriminate between the length of the infection (i.e., acute or chronic); limitations of these tests may lead to different interpretations unless one is familiar with those limitations. an example of this occurred with the bacterial infection of borrelia burgdorferi, which causes lyme disease. antibiotics can abrogate the antibody response because elisa results were negative in % of patients with known disease who were treated early (dattwyler et al., ) . in early cases, reactivity to a unique antigen, ospa, was also negative in serological assays despite a demonstrable t-cell possible scenarios of bioterrorism attacks: distinguishing victims from perpetrators response (krause et al., ) . analysis of these same sera found that there was antibody to b. burgdorferi, but it was below the threshold of detection by conventional assays. it was detectable in its bound form, in immune complexes (schutzer et al., ; schutzer and coyle, ) . anthrax can be used as an example where investigatory leads can be generated by considering a scenario in toto. the elderly woman who died in connecticut from inhalation of anthrax clearly had no occupational exposure nor was she known to have had contact with anyone who had anthrax. it was possible that she had contact with cross-contaminated mail. however, if this case had occurred as the index case or out of context of the mail attacks, it would have been reasonable to question her travel history, what her work if any was, or if she received or used spore-contaminated products from an anthrax endemic area. similarly, the vietnamese woman who died of inhalation anthrax in new york city would also have had these questions investigated. it would have been useful to search for direct or indirect evidence of anthrax by physical examinations of her contacts or close neighbors. inspection and cultures from her workplace, apartment, and apartment complex (especially contiguous neighbors) are important for detecting the presence of b. anthracis. coworkers, friends, neighbors, and other contacts could have had their serum analyzed for antibody to antigens of b. anthracis. these samples could have been frozen so that if one were positive it would be available for a comparison study in the future. at a minimum, these types of studies could serve as future control data for the geographic region. with molecular methods, even trace amounts might be detectable (lasken and egholm, ) although parallel investigation as to background control would be necessary. although hypothetical, several results could have occurred, and each will be considered separately. first example, a close contact is positive for igm to one of the b. anthracis antigens, such as pa. this finding would suggest that this person had recent exposure and, if nothing else, should be treated. this individual could conceivably be the one who knowingly or unknowingly passed the spores to the patient. given the october onset of illness, which is late in the mailing sequence, it would be less likely that this individual was a perpetrator but rather a recent victim. however, if this person were igg-positive, then there are several other possibilities. perhaps, this person had past exposure in an endemic region and was treated (e.g., haiti, where anthrax is known as "charcoal disease"). or this person could have been vaccinated for bona fide reasons such as a researcher who received it for occupational exposure. or this person could have obtained the vaccine originally for legitimate or illegal purposes but was nevertheless vaccinated. the vaccine usage may have been for a clinical trial or animal experimentation. animal vaccines may be more obtainable without strict record keeping. this person could have loaded the mail with relative impunity if there was protective antibody generated from the vaccination. situations similar to this one will require intelligence information regarding access, ability, and motive. in an area where recombinant vaccines are being developed or used antibody response would be different between someone using one type of recombinant vaccine as compared with someone using another type of vaccine. nevertheless, finding igg to one or more antigens of b. anthracis could point investigators toward such a seropositive individual, whereas an igm finding could justify critical therapy. where information points to a particular individual, investigation could be extended to search for ingestion or injection of antibiotics as illustrated below in the ciprofloxacin example. questions would be raised regarding access to antibiotics, recent ingestion/injection of them, half-life of the antibiotic, half-life of the metabolites of the antibiotics, and in which body fluids or tissues can the residual be found. as illustrated from . the use of host factors in microbial forensics the data in the earlier sections, someone with antibiotics in their system may be protected following exposure to a potential pathogen. this person would be antibody-negative and likely antigen-and microbial dna/rnanegative, because the infection would have been eradicated before the organism can proliferate in any significant quantity. the widespread prophylactic use of ciprofloxacin during the period following the anthrax mailing attacks is illustrative of an understudied area. ciprofloxacin has been increasingly associated with tendonitis and ruptured achilles tendons (akali and niranjan, ; palin and gough, ; godoy-santos et al., ) . in the future, better methodology to follow the pharmacokinetics of an antiinfective compound may have forensic implications. in the last example, someone who takes an antibiotic prophylactically while manipulating a lethal microbe may exhibit side effects that in the proper context of an investigation may add to the picture of possible culpability. this area is far from established at this point in time. strategies can be employed to examine suspicious but possible accidental transmission of infections. this approach is illustrated by a recent study of avian influenza using a multitude of assays. tools to determine person-to-person spread as the mode of transmission included viral culture, serologic analysis, immunohistochemical assay, reverse transcriptasee polymerase chain reaction (rt-pcr) analysis, and genetic sequencing (ungchusak et al., ; meinel et al., ) . it is likely that future understanding of the immune system and evolving technologies will bring new analytic power to the field, but in the interim we can make good use of proven principles for forensic purposes. serial serum c-reactive protein levels as an aid to the management of melioidosis management of bilateral achilles tendon rupture associated with ciprofloxacin: a review and case presentation manual of forensic odontology mandell, douglas, and bennett's principles and practice of infectious diseases inhalational, gastrointestinal, and cutaneous anthrax in children: a systematic review of cases index case of fatal inhalational anthrax due to bioterrorism in the united states clinical features of the initial cases of pandemic influenza a (h n ) virus infection in china detection of immunoglobulins m and g using culture filtrate antigen of burkholderia pseudomallei systematic literature review of rift valley fever virus seroprevalence in livestock, wildlife and humans in africa from mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen evaluation of a new 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with ciprofloxacin medical immunology, tenth ed. lange medical books/mcgraw-hill medical pub. division fundamental immunology, sixth ed. wolters kluwer health gene transcript abundance profiles distinguish kawasaki disease from adenovirus infection specific, sensitive, and quantitative enzyme-linked immunosorbent assay for anthrax lethal toxin impairs il- expression in epithelial cells through inhibition of histone h modification dissecting regulatory networks in host-pathogen interaction using chip-onchip technology sequestration of antibody to borrelia burgdorferi in immune complexes in seronegative lyme disease biocrimes, microbial forensics, and the physician use of forensic methods under exigent circumstances without full validation comparative tests for detection of plague antigen and antibody in experimentally infected wild rodents handbook of forensic services comparison of two commercial broad-range pcr and sequencing assays for identification of bacteria in culture-negative clinical samples first case of bioterrorism-related inhalational anthrax in the united states cutaneous anthrax: a concise review probable person-to-person transmission of avian influenza a (h n ) bioterrorism for the respiratory physician essential clinical immunology key: cord- - wj gr authors: katze, michael g.; fornek, jamie l.; palermo, robert e.; walters, kathie-anne; korth, marcus j. title: innate immune modulation by rna viruses: emerging insights from functional genomics date: journal: nat rev immunol doi: . /nri sha: doc_id: cord_uid: wj gr although often encoding fewer than a dozen genes, rna viruses can overcome host antiviral responses and wreak havoc on the cells they infect. some manage to evade host antiviral defences, whereas others elicit an aberrant or disproportional immune response. both scenarios can result in the disruption of intracellular signalling pathways and significant pathology in the host. systems-biology approaches are increasingly being used to study the processes of viral triggering and regulation of host immune responses. by providing a global and integrated view of cellular events, these approaches are beginning to unravel some of the complexities of virus–host interactions and provide new insights into how rna viruses cause disease. viruses can have a devastating effect despite their small genomes. all rna viruses encode proteins that are essential for structural components and replication, and most encode proteins that function to circumvent host antiviral responses [ ] [ ] [ ] . this limited number of proteins is sufficient to ensure the entry, replication and subsequent spread of the virus. however, viruses do not self-propagate and depend on various host-cell functions to complete their life cycle. the processes of viral entry, the triggering and regulation of the host antiviral response and subsequent viral replication together result in an intricate series of interactions between virus and host. much can be learnt about the nature and complexities of these interactions by global profiling of the transcriptional changes in host cells that occur during viral infection (box ) . in this review, we discuss how functional genomic and systems-biology approaches are contributing to our understanding of interactions between rna viruses and the host, of viral pathogenesis and of host immunity to infection. rather than providing a comprehensive literature review, we present examples of how these approaches are providing insight into the interaction of viruses with innate immune defence mechanisms, the evaluation of therapeutics that target these pathways and the crucial balance between protective immune responses and immunopathology. in addition, we describe how genomic approaches are being applied to vaccine evaluation and design, and how these approaches can be combined with other high-throughput technologies to provide an improved and integrated systems-biology view of virus infection. although genomic approaches are being used to study a wide variety of viruses, we highlight the current literature through discussion of a select few. among these is influenza virus, for which the looming threat of a new pandemic and concerns regarding therapeutic and vaccine preparedness have stimulated exciting new research efforts. we also review findings relating to hepatitis c virus (hcv) infection, for which genomic analyses are being used to shed light on the response of patients to treatment with type i interferons (ifns) and the relationship between hcv replication and liver disease. in addition, we highlight studies of west nile virus, severe acute respiratory syndrome-associated coronavirus (sars-cov) and ebola virus, all of which have revealed previously undescribed strategies used by these viruses to regulate innate immunity. finally, we discuss how genomic approaches are being applied to vaccine evaluation and how genomics is being combined with other high-throughput approaches to provide a systems-biology view of virus-host interactions. viruses and innate immunity a variety of cellular signalling networks have evolved in host cells to detect and respond to viral infection. one area in which genomics-based analyses are being put to abstract | although often encoding fewer than a dozen genes, rna viruses can overcome host antiviral responses and wreak havoc on the cells they infect. some manage to evade host antiviral defences, whereas others elicit an aberrant or disproportional immune response. both scenarios can result in the disruption of intracellular signalling pathways and significant pathology in the host. systems-biology approaches are increasingly being used to study the processes of viral triggering and regulation of host immune responses. by providing a global and integrated view of cellular events, these approaches are beginning to unravel some of the complexities of virus-host interactions and provide new insights into how rna viruses cause disease. these genes contain interferon (ifn)-responsive promoters and are responsible for the antiviral, antiproliferative and immunomodulatory properties of ifn. over such genes have been identified by microarray analysis. some, such as protein kinase r, ribonuclease l, mx (myxovirus resistance ) and isg (ifn-stimulated protein of kda), have well documented antiviral activities, but the precise biological function of the majority of these genes is unknown. particularly good use is in shedding new light on the components of innate antiviral defence mechanisms and the viral strategies used to overcome them. in this section, we review recent studies in which genomic approaches have been used to provide new information on how viruses trigger and regulate innate immune pathways, and to evaluate the use of type i ifn-based therapy as a means to enhance the innate immune response to hcv. mammalian cells have specialized proteins that are responsible for the recognition of virus infection, and other proteins that elicit responses to combat the invading virus. the antiviral response is triggered when host pathogen-recognition receptors (prrs) are engaged by pathogen-associated molecular patterns (pamps) in viral proteins and nucleic acids (reviewed in refs , ) . prrs that function in virus recognition include the cytosolic double-stranded rna helicases retinoic-acid-inducible gene i (rig-i) and mda (melanoma differentiation-associated gene ) and certain toll-like receptors (tlrs) that are present on the cell surface or in endosomal membranes. after binding to viral pamps, prrs initiate intracellular signalling cascades that result in the activation of transcription factors, including ifn-regulatory factors (irfs) and nuclear factor-κb (nf-κb). these transcription factors in turn regulate the expression of hundreds of genes, such as ifns and ifn-stimulated genes (isgs) , , and pro-inflammatory cytokines and chemokines that are involved in the orchestration of the adaptive immune response (fig. ) . one way in which gene-expression profiling has been used to examine this aspect of the antiviral response is through the use of mouse embryonic fibroblasts deficient in rig-i or mda . a recent study demonstrated that west nile virus infection of wild-type cells led to the induction of irf target genes and isgs, including several subtypes of ifnα (ref. ) . this was followed by a second phase of ifn-dependent antiviral gene expression that occurred at a later stage of infection. by contrast, cells lacking rig-i had delayed or inhibited initial and secondary gene-expression responses to the virus, indicating that rig-i has an essential but not exclusive role in initiating innate immune responses to west nile virus (fig. ) . the additional deletion of mda in these cells was found to further block their ability to respond to infection, indicating that the host immune response to west nile virus also involves mda . this is a noteworthy finding, as previous studies suggested that rig-i and mda recognized a specific subset of viruses, rather than acting cooperatively as found in the response to west nile virus . the role of rig-i in the response to influenza virus infection has also been assessed . similar to west nile virus, genomic analysis of influenza virus-infected wild-type and rig-i-deficient mouse embryonic fibroblasts revealed that rig-i is necessary for the type i ifn response to this virus (fig. ) . in rig-i-deficient cells, influenza virus fails to elicit the expression of ifnβ and of many isgs, including key antiviral mediators such as irf , stat (signal transducer and activator of transcription ), ifit (ifn-induced protein with tetratricopeptide repeats ; also known as isg ) and isg (also known as ifit ). this study also showed that, unlike during infection with west nile virus, mda does not function as a secondary mediator of the response to infection with influenza virus . important next steps in these studies will be to compare the profiles of genes induced by each of these viruses -and to determine whether some genes are specific for rig-i or mda signalling -and to begin to define the involvement of these genes in innate immunity. although this biological validation process will be necessary to follow-up genomic analyses, few studies so far have included such experiments. functional genomic analyses have also been helpful in elucidating the complex transcriptional events triggered following tlr signalling. tlrs are expressed by various immune cells, including macrophages, dendritic cells and lymphocytes, and a subset of these receptors are involved in viral recognition. so far, genomic studies have largely focused on the analysis of macrophages treated with tlr ligands, such as lipopolysaccharide (lps; a component of the cell wall of gram-negative bacteria) or polyinosinic-polycytidylic acid (a synthetic mimic of viral double-stranded rna, dsrna) [ ] [ ] [ ] . to obtain a comprehensive view of the transcriptional programmes that are induced by tlr activation, elkon et al. used a computational approach to analyse geneexpression data sets derived from four studies in which human or mouse macrophages were stimulated with pathogen-mimetic agents that engage various tlrs . this analysis identified one transcriptional profile that is universally activated by all tlrs and a second profile that is specific to both tlr (which specializes in the recognition of viral dsrna) and tlr (which recognizes genomics is broadly defined as the study of genomes. the term was first adopted nearly years ago to describe the emerging discipline of using nucleotide sequencing, gene mapping and computational biology to define the structure and organization of a genome . as ever increasing amounts of nucleotide sequence information have become available, the focus of genomics has expanded to include gene function . the human genome project was a driving force in advancing both structural and functional genomics, and the nucleotide sequence information generated by this project has fuelled tremendous advances in our understanding of human health and disease. one way in which this has occurred is through the convergence of comprehensive genome sequence information with advances in high-throughput technology. today, the standard technology in functional genomics is the oligonucleotide microarray [ ] [ ] [ ] . several alternative platforms are available, with the most common being microarrays for which thousands of oligonucleotide 'probes', each corresponding to an mrna transcript, are synthesized in situ directly on a glass slide. such microarrays enable researchers to simultaneously measure the expression of virtually all genes in a genome. for 'target' preparation, mrna is extracted from experimental samples and labelled with fluorescent dyes by reverse transcription. the labelled target is then hybridized with the microarray, and the fluorescence of the features is determined using an array scanner. following image analysis, the data are subjected to a variety of bioinformatic processes to identify statistically significant changes in gene expression between samples. because each comparison yields tens of thousands of data points, mining the data for biological meaning is a formidable challenge. a variety of sophisticated commercial and open-source analysis tools are therefore used to find relationships between differentially expressed genes, to identify networks or signalling pathways that are activated or repressed and to compare gene-expression profiles between experimental samples. envelope components of viruses and cell-surface components of bacteria (such as lps)). a computational analysis of promoter sequences identified nf-κb as the key regulator of the universal response, which occurs early after tlr stimulation, and the ifn-stimulated response element (isre) as the key component of the tlr and tlr response, which is induced after the nf-κb response. this computational approach provided additional knowledge regarding the kinetics of the tlr and tlr response, the regulatory circuitry involved and the identity of the genes figure | stimulation of interferon-stimulated gene expression and initiation of antiviral activity. pathogenassociated molecular patterns (pamps) in viral proteins and nucleic acids are recognized by cellular pathogen-recognition receptors (prrs) that include rig-i (retinoic-acid-inducible gene i), mda (melanoma differentiation-associated gene ) and certain toll-like receptors (tlrs). prr-pamp interactions trigger signalling cascades that result in the activation of transcription factors, including interferon (ifn)-regulatory factor (irf ) and nuclear factor-κb (nf-κb), which induce the production of type i ifns, ifn-stimulated genes (isgs) and pro-inflammatory cytokines and chemokines. the specific process differs between antigen-presenting cells, in which both the tlr pathway and the rig-i or mda pathway are operative, and other cell types, in which only the rig-i or mda pathway is present. activation of prr signalling induces an antiviral state in all cell types, and in antigen-presenting cells it can also induce the production of pro-inflammatory cytokines and chemokines. this normally results in an innate antiviral response that controls infection until it is resolved by the adaptive immune response. however, some viruses, such as the pandemic influenza virus, elicit an aberrant or disproportional response that results in immunopathology. alternatively, viruses that suppress the type i ifn response can subvert the mechanisms of innate surveillance and diminish the potential adaptive immune response, resulting in a chronic infection. for vaccine strategies, the best induction of a broad adaptive immune response might require some degree of type i ifn response in the initial stages of infection. dcs, dendritic cells; dsrna, double-stranded rna; ifnar, ifnα receptor; il, interleukin; ips , ifnb-promoter stimulator ; oas, ′, ′-oligoadenylate synthetase; pkr, protein kinase r; ssrna, single-stranded rna; stat, signal transducer and activator of transcription; tap , transporter associated with antigen processing ; tnf, tumour-necrosis factor. chimeric scid-alb/upa mouse model a chimeric mouse model of severe combined immunodeficient (scid) mice that contain a urokinase plasminogen activator transgene driven by an albumin promoter (alb/upa). these mice can be transplanted with human hepatocytes to generate chimeric mousehuman livers, providing the only small-animal infection model for hepatitis c virus infection. activated in both the universal and tlr -and tlr mediated responses. although these studies have provided considerable information regarding the genes activated downstream of tlr activation, it will be advantageous to extend genomic analyses in the context of viral infection using cells lacking the expression of specific tlrs. the ability of a virus to establish an infection depends, at least to some extent, on its ability to block the host innate immune response or to modulate the activity of antiviral effector proteins. hcv is one example of a virus that has devised a means to block the initial triggering of the host innate immune response. several studies have shown that the hcv ns -ns a serine protease blocks the tlr -dependent activation of irf (refs , ) . this is achieved by ns -ns a-mediated cleavage of trif (toll/interleukin- (il- ) receptor-domain-containing adaptor protein inducing ifnβ), an adaptor protein that links tlr to kinases that are responsible for activating irf and nf-κb , . hcv also inhibits the ability of rig-i to activate irf (refs , , , ), which is achieved through ns -ns a-mediated cleavage of ips (ifnb-promoter stimulator ; also known as visa, cardif, mavs), a recently identified rig-i adaptor protein [ ] [ ] [ ] [ ] [ ] . in light of these findings, it is both perplexing and paradoxical that virtually all gene-expression profiling carried out using hcv-infected tissue shows the induction of isg expression, including irf target genes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the induction of isg expression is observed in liver tissue from hcv-infected patients , , and during the initial host response in acutely infected chimpanzees , , and is a major part of the transcriptional response to hcv infection in the chimeric scid-alb/upa mouse model . this poses an interesting question about the source of both type i ifns and isg expression. it is possible that isgs are mainly expressed in uninfected hepatocytes and are induced in response to exogenous type i ifn released from adjacent hcv-infected cells. alternatively, it has been suggested that t cells and plasmacytoid dendritic cells that infiltrate the liver are a possible source of hepatic type i ifns . although this is possible, it is relevant to note that hcv infection in the scid-alb/upa mouse model is also associated with the induction of hepatic isg expression in the absence of these immune cell types . other genomic studies have revealed examples of highly virulent viruses that are relatively successful at inhibiting isg expression. perhaps the best example is a characterization of the host transcriptional response of human liver cells infected with filoviruses . this study demonstrated the marked suppression of genes in key innate antiviral pathways, including those mediated by irf . interestingly, this study also suggested a correlation between the antagonism of the type i ifn response and filovirus virulence. highly virulent viruses, such as zaire ebola virus and marburgvirus, inhibit the expression of most isgs that are induced in uninfected ifn-treated cells. by contrast, the relatively non-pathogenic reston ebola virus is less inhibitory and induces the expression of more than % of these genes. the suppression of the type i ifn response by the pathogenic viruses is associated with more rapid viral spread and higher rate of viral replication than that observed during reston ebola virus infection. a comparable trend was seen in a study evaluating the host transcriptional response and inflammation in the brains of mice infected with rabies virus . this study revealed that infection with an attenuated virus results in both inflammation and the induction of expression of key isgs. however, these events are either absent or diminished during infection with a highly pathogenic rabies virus. on the basis of results with filoviruses, it would follow that attenuation of the type i ifn response would be associated with higher viral replication and spread in the case of pathogenic infection with rabies virus; however, this was not measured in the study. similarly, infection with highly virulent pseudorabies virus suppressed the induction of a subset of isgs, even in type i ifn-treated cells . together, these data suggest that the virulence of acute, highly pathogenic viruses is at least partially related to their ability to suppress the host antiviral response, which seems to allow higher levels of viral replication. genomic analyses using cells that lack rig-i (retinoic-acid-inducible gene i) show the requirement for this pathogen-recognition receptor in the induction of interferonregulatory factor (irf ) target genes and interferon-stimulated genes (isgs) by west nile virus and influenza virus. a | the infection of rig-i-deficient cells by west nile virus results in the delay and partial inhibition of isg expression. deletion of mda (melanoma differentiation-associated gene ) further blocks the response to infection (not shown), indicating that the response to west nile virus also involves mda . b | by contrast, the infection of rig-i-deficient cells by influenza virus results in a near complete inhibition of isg expression that is not further blocked by the absence of mda , suggesting that mda does not mediate influenza virus-induced gene-expression changes. pamps, pathogen-associated molecular patterns. images generated from data in refs , . suppression of innate immunity and persistent infection. evidence discussed in this review suggests that suppression of elements of the innate immune response enables extensive viral replication and increased pathogenesis. does the converse hold true for a virus such as hcv, which typically establishes a persistent infection characterized by mild (or slowly progressing) disease? some evidence suggests that this might be the case; for example, studies using the chimeric scid-alb/upa mouse model indicate that an attenuated type i ifn response is associated with higher levels of intrahepatic hcv replication together with a greater induction of lipid metabolism and oxidative-stress genes, which have the potential to cause cytopathic effects . similarly, gene-expression profiling of serial liver biopsies obtained from patients that had received an hcv-infected liver transplant shows that rapid progression of fibrosis following transplantation is associated with the suppression of genes involved in the type i ifn response, antigen presentation and the cytotoxic t-cell response . although in these studies the apparent defect in the host antiviral response is probably related to host genetics rather than viral factors, the concept that a defective innate immune response correlates with enhanced pathogenesis is still evident. it is possible that the selective pressures on persistent viruses never resulted in a need for a complete subversion of host innate antiviral responses, so such viruses use these responses to limit their replication to a level that does not significantly affect the normal functions of the host cell. conversely, acute viruses, such as filoviruses, highly pathogenic influenza virus and rabies virus, seem to have evolved to antagonize these responses following cell entry to allow immediate, high levels of replication, which subsequently facilitate virus spread and transmission. given the importance of the innate immune response in regulating virus infection, there is considerable interest in enhancing or modulating this response for therapeutic benefit. one role for genomics in this area is assisting in the evaluation of type i ifn treatment of hcv infection. combination therapy with ifnα and the antiviral drug ribavirin results in virus clearance in only ~ % of individuals infected with hcv genotype and ~ % of individuals infected with hcv genotypes or (refs - ). as ifnα is the only approved treatment for chronic hcv, there is strong interest in improving this therapy, in understanding the molecular mechanisms that underlie treatment failure and in identifying markers to accurately predict a patient's response to treatment (that is, responders or non-responders). several groups that have used transcriptional profiling of patient hepatic tissue to address these issues have found that higher levels of expression of isgs before treatment are associated with treatment failure. for example, chen et al. carried out microarray experiments on pretreatment liver tissue obtained from a cohort of patients with chronic hcv infection who subsequently underwent ifnα and ribavirin therapy . this analysis identified a set of genes, many of which are known isgs; in general these genes were more highly induced in the livers of patients that did not respond to therapy. although the authors suggest that this set of genes could therefore be used to predict the response to therapy, it remains to be determined whether they can be used to accurately predict the response in other patient cohorts. similarly, feld et al. showed that non-responders have significantly higher intrahepatic pretreatment expression levels of isgs than patients who respond to type i ifn therapy . although these studies are intriguing, it is still unclear whether there is a causal relationship between higher pretreatment levels of isgs and therapy failure. other factors, such as viral quasispecies diversity, may also be important. owing to the technical and ethical issues of obtaining sufficient liver material for gene-expression studies, investigators have also used peripheral-blood mononuclear cells (pbmcs) to evaluate the response to treatment , . an example is virahepc, a multicentre study designed to define the differences in response rates among caucasian and african americans and to identify host and viral parameters associated with a lack of response to treatment . overall, this study showed that, during the first days of treatment, a lower level of induction of known isgs is associated with non-responsiveness to type i ifn treatment. however, in many cases, these differences are not strikingly dissimilar between responders and non-responders. the implication of such minor differences with respect to antiviral function is uncertain and the feasibility of using them for predicting a patient's response is questionable. in addition, analyses using pbmcs should be interpreted with caution, as a recent study showed that the transcriptional response to type i ifn treatment is significantly different in the blood and the liver of hcv-infected chimpanzees, presumably owing to the absence of hcv replication in pbmcs . although it has not yet been evaluated, this will almost certainly hold true for humans as well. an alternative mechanism of a failed response to type i ifn treatment could involve the induction of genes associated with ifn inhibitory pathways . walsh et al. found significantly increased intrahepatic expression of the gene encoding suppressor of cytokine signalling (socs ) in patients who did not respond to type i ifn treatment . enhanced intrahepatic socs expression is also thought to contribute to the non-responsiveness of hcv-infected chimpanzees to type i ifn therapy . however, a separate evaluation of patients for intrahepatic socs mrna expression before antiviral therapy actually found higher levels of expression in those patients who went on to respond successfully to type i ifn treatment . therefore, the relationship between treatment failure and induction of type i ifn inhibitory pathways is currently less clear than that between higher pretreatment levels of expression of isgs and treatment failure. there are still surprisingly few answers to the fundamental question of how virus infection results in disease pathology. although the mechanisms are certain to be different for each virus, a common theme is that there is abarrently high and sustained nature reviews | immunology infection resolves immunopathology a crucial balance between protective immune responses and immunopathology , . although the innate immune response is designed to target and eliminate invading pathogens, genomic analyses have indicated that some viruses, such as the highly virulent influenza virus that was responsible for the pandemic, elicit aberrant or disproportional innate immune responses that may also harm the host. the influenza virus pandemic (known as the spanish flu) killed as many as million people worldwide , and several studies have begun to provide clues to what made this virus so deadly (reviewed in . although genomic analyses have previously been carried out using engineered viruses containing one or more genes from the pandemic virus , , a major advance in the ability to study this virus came from its reconstruction based on nucleotide sequence information . genomic analyses of lung or bronchial tissue derived from mice or macaques that were infected with the reconstructed virus indicate how the beneficial role of the innate immune response can be tipped towards immunopathology. mice infected with the reconstituted influenza virus show severe pulmonary pathology and an increased and accelerated transcriptional activation of immuneresponse genes . this includes a marked activation of genes associated with pro-inflammatory and cell-death pathways by hours after infection (fig. ) , which remain unabated until the death of the animals. this response is in contrast to the less dramatic and delayed host immune responses (and less severe disease pathology) in mice that were infected with influenza viruses containing only subsets of genes from the virus, including the haemagglutinin (ha) and non-structural protein (ns) genes, or the ha, neuraminidase (na), matrix (m) and nucleoprotein (np) genes. these findings suggest that enhanced pro-inflammatory and cell-death responses can contribute to severe immunopathology. an additional study that evaluated the host response to the influenza virus using a cynomologus macaque (macaca fascicularis) infection model produced similar results . in macaques, the virus replicates to high levels and spreads rapidly throughout the respiratory tract of infected animals, causing severe lung damage and the massive infiltration of immune cells throughout the course of infection. functional genomic analyses of bronchial tissue revealed that the virus triggers the aberrantly high and sustained expression of numerous genes involved in the innate immune response, including pro-inflammatory cytokines and chemokines. although the timing of the response is somewhat different, the increased and sustained host response in macaques that were infected with the virus is similar to that observed in mice. these studies reveal similarities and differences in the host response to contemporary and pandemic influenza virus infection. first, contemporary and viruses each trigger an innate immune response that includes the expression of nf-κb and irf target genes, which is expected to occur if the virus triggers the rig-i pathway in infected respiratory cells. second, both viruses trigger a robust cytokine response that probably attracts immune-cell infiltration to infected tissues. unlike contemporary virus strains, in which the early response to infection is resolved, the innate immune response triggered by the virus is characterized by a strong and sustained induction that is associated with massive tissue damage and death of the infected animal. however, in preliminary genomic analyses carried out with lung tissue from macaques that were infected with avian h n viruses, we have found that there are significant differences in the regulation of antiviral responses by the pandemic and h n viruses (j. c. kash and m.g.k., unpublished observations). therefore, there may be differences in the ways in which highly pathogenic influenza viruses regulate the innate immune response and cause disease. the enhanced pathogenicity of the and h n influenza viruses might be attributed to distinct components of their genomes. although much emphasis has been placed on the ns protein of the virus acting as an inhibitor of the type i ifn response, recent evidence suggests that the viral proteins pb (a polymerase), ha and na contribute to its pathogenicity . likewise, the polymerases of h n viruses have been linked to increased viral pathogenesis , suggesting that the increased pathogenesis of these viruses may be related to their replicative fitness. another respiratory virus, sars-cov, has emerged recently and has caused great concern among the public health and research communities. it has been suggested that disease pathology associated with sars-cov is caused by a disproportional immune response, illustrated by increased levels of pro-inflammatory cytokines and chemokines [ ] [ ] [ ] . studies carried out in our laboratory have combined the use of functional genomics with a cynomologus macaque infection model to study the host response to this virus . we observed that sars-cov-infected macaques show a strong increase in the expression of innate immune response genes early after infection and that this response wanes after days. conversely, genes that are induced later in infection tend to be involved in the cell cycle and in cell repair. none of the animals used in this study succumbed to infection, and sars-cov-induced pathology in these macaques resembled the pathological changes seen in the majority of human patients with sars who recover from the disease . unlike the findings of the pandemic influenza virus study, these data suggest that early immune responses to sars-cov infection are productive and enable the host to properly fight the virus, allowing a return to cellular homeostasis. however, in the % of human infections in which sars-cov infection is fatal (mostly in the elderly), it is possible that the timing or magnitude of the response results in immunopathology. studies using aged macaques might help to address this possibility. viruses such as sars-cov, h n influenza virus and influenza virus are all zoonotic infections, in which a virus that was adapted to another host was transferred to humans. because the type i ifn response is somewhat different in different hosts, it is possible that these viruses, which have adapted to their normal animal hosts, elicit an aberrant response when infecting a human host in which adaptation has not occurred, resulting in immunopathology. this possibility also raises the question of how appropriate the various animal infection models (such as mice and macaques) are for the understanding of human pathogenesis. as reviewed elsewhere , there are both advantages and disadvantages associated with different animal models, and it is important to keep in mind that responses observed using an animal model may not always accurately reflect the response in humans. genomics in vaccine evaluation and design genomic information and high-throughput technologies are beginning to have an impact on the field of vaccine development, but the main focus has been directed towards identifying important conserved features of pathogens that could serve as immunogens and characterizing host genotypes associated with strong protective responses [ ] [ ] [ ] . in recent years, it has become evident that the type i ifn response has a significant role in the development of the adaptive immune response. this commences with the influence of type i ifns on the activation, maturation and migration of dendritic cells , . the development of the antibody response is also enhanced by type i ifns through the direct effect of ifn on b cells and on the priming or function of cd + t helper cells . there is now also evidence that type i ifns act directly on cd + t cells to promote clonal expansion and indirectly by stimulating cross-priming by antigen-presenting cells that have engulfed infected cells to acquire antigen [ ] [ ] [ ] . so, viruses that suppress the type i ifn response not only subvert the mechanisms of innate surveillance, but also diminish the potential adaptive immune response that could mediate viral clearance or establish a quiescent, non-pathogenic state. for vaccine strategies, the implication is then that the best induction of a broad adaptive immune response will require some degree of type i ifn response in the initial stages. just as dna microarray technology spurred the development of functional genomics, the development of immunomic microarray technology is driving the emerging field of functional immunomics (reviewed in ref. ) . the goal of immunomics is to provide a detailed understanding of host immunological responses to foreign antigens through the use of high-throughput technologies and computational methods. the technologies that are central to this effort include antibody microarrays (consisting of antibodies as probes and antigens as targets), peptide microarrays (consisting of antigen peptides as probes and serum antibodies as targets) and more recently peptide-mhc microarrays (consisting of recombinant peptide-mhc complexes and co-stimulatory molecules as probes and populations of t cells as targets). antibody microarrays are used to measure the concentration of specific antigens (such as cancer antigens), whereas peptide-mhc microarrays can map mhc-restricted t-cell epitopes which are involved in helper and regulatory functions of the immune system. peptide microarrays are used in various applications, including b-cell epitope mapping and detection and diagnostic assays. peptide microarrays are also being used in vaccine studies for mapping epitopes associated with effective immune responses and for testing the ability of experimental vaccines to generate specific antibody responses against those epitopes after immunization and challenge . studies of immune responses that are associated with different clinical outcomes, such as those of patients who are hiv positive and who rapidly progress to aids and those of infected long-term survivors, can also provide direction for the development of vaccines . it is probable that immunomics will become an increasingly integral part of a systems-biology approach to vaccine development and of obtaining a better understanding of host immunity to virus infection. animal models. we have used functional genomics to evaluate a live influenza virus vaccine in a macaque model, in which attenuation of the virus was accomplished by truncation of the gene encoding ns . this modification eliminates or reduces the ability of the ns protein to antagonize type i ifn production and, in mouse and swine models, such attenuated live viruses are immunogenic and protective , . gene-expression profiling of tracheal and bronchial epithelial cells from macaques immunized with the ns -truncated virus show clear evidence of a robust type i ifn response. compared with immunization with a traditional killedvirus vaccine, the attenuated live-virus-vaccine group had higher antibody titres before and after challenge and a broader range of influenza virus-specific t-cell responses. following challenge with infective virus, the protection afforded by the attenuated live-virus vaccine was evident by the limited viral replication and minor pathology observed in treated animals. in addition, gene-expression profiles of lung tissue from animals that received the attenuated live-virus vaccine show less upregulation of innate and pro-inflammatory response genes compared with animals immunized with the killed-virus vaccine or untreated animals. at the same time, the transcriptional profiles for the attenuated live-virus-vaccine animals showed a stronger induction of genes that are associated with b-cell and t-cell responses. the general picture overall is that the truncated-ns containing influenza virus vaccine undergoes minimal replication but induces sufficient type i ifns to galvanize the adaptive immune response, leaving the host in a state of adaptive preparedness after just one immunization. the early induction of type i ifns in response to the truncated-ns -containing vaccine might be especially important in the local b-cell response that is crucial for viral clearance. a relevant observation in this regard is that early stimulation of the respiratory-tract b cells (within hours of influenza virus infection) was shown to be strongly driven by virus-induced type i ifns , . human studies. at present, there are only limited examples in which gene-expression profiling applied to vaccine design supports a picture consistent with that described above for the influenza virus model. the standards for prevention of measles and yellow fever are immunizations with attenuated live-virus vaccines. to assess the impact of infection on primary target cells, gene-expression profiling was carried out in tissue-culture systems comparing wild-type and vaccine strains. for both measles and yellow fever, it was clear that the attenuated vaccine strains led to a greater induction of the type i ifn response than the pathogenic wild-type virus , . although in the case of measles virus this disparity in the ifn response has previously been shown by serological techniques , expression analysis indicated that the antagonism of the response by the wild-type virus originated at the level of transcription. this early induction of the type i ifn response was also evident in microarray studies examining chimeras of the yellow fever vaccine strain that were devised as attenuated live-virus vaccines against other flaviviruses such as dengue virus . this contrasted with the low-level induction of type i ifns by dengue virus infection as seen by expression profiling using infection of primary cells or macaque disease models , . it is interesting to note that the measles and yellow fever vaccine strains are attenuated by passage in cells from other species. therefore, with suitable molecular understanding, the ability of some viruses to induce type i ifns might be optimized by directed molecular techniques, as was done for the truncated-ns influenza virus strain. as an alternative, one might consider using recombinant type i ifns as vaccine adjuvants instead of inducing them with the vaccine constituents , but at our present level of understanding, these approaches have yet to prove clinically tenable . functional genomics for the evaluation of immunological memory. functional genomic studies have been more equivocal in assessing the significance of type i ifn production during the immunological memory response. in the aforementioned macaque influenza virus study, animals receiving the attenuated live-virus vaccine showed upregulation of type i ifn pathways in tracheobronchial cells days after challenge, and this coincided with the development of a strong memory response . this type i ifn induction seems to be weaker than that observed at the corresponding time after the primary exposure to the vaccine, but is far lower than the type i ifn induction observed after challenge of animals receiving the killed-virus vaccine or of naive animals. this would suggest some role of this innate pathway in stimulating immunological recall. in contrast to this, examination of transcriptional profiles observed shortly after rechallenge of human pbmcs from individuals previously immunized against influenza virus are more in accord with early production of ifnγ, possibly arising from antigenic stimulation of memory cells . dhiman et al. also did not see evidence of a type i ifn response in a microarray study of whole blood taken from individuals immunized with measles virus after rechallenge with an attenuated live-virus vaccine strain, although genes associated with lymphocyte activation and survival were upregulated . it could be considered that technical issues might hamper the relevance of these studies in assessing the role of type i ifns in the memory response. in the case of the first study , pbmcs are not a primary target of influenza virus, so virus internalization might have been inefficient and a type i ifn response might have been poor. in the measles study , the earliest time point examined was days after rechallenge rather than early, when the type i ifn response would be expected to be strongest. therefore, further functional genomic experiments, with appropriately designed models, are required to address whether an early innate immune response is a key stage in triggering immunological memory. functional genomics has proven to be a highly efficient method for providing broad views of the host response in studies of virus-host interactions. as we have discussed, these techniques have revealed the activation or nature reviews | immunology repression of innate immune signalling pathways, crosstalk between pathways, the timing and magnitude of the immune response and, depending on the experimental system, the degree to which the immune response varies among individuals. conversely, functional genomics has been less effective in pinpointing the role of specific host genes in the antiviral response or, somewhat surprisingly, in identifying previously undiscovered genes and pathways that are important in the infection process, despite this being one of its early goals . indeed, the early assumption that functional genomics would provide quick answers to the complexities of virus-host interactions has proved naive. how then can greater benefits be gained from using functional genomics to study virus-host interactions? rather than being used as a singular approach, the future of functional genomics in virology will be in the integration of genomic data with data derived from other high-throughput technologies (fig. ) . the obvious complementary approach to functional genomics is proteomics, which will provide much needed information regarding the correlation of gene expression with protein abundance [ ] [ ] [ ] . our group has begun to integrate genomic and proteomic data to better understand the host response to influenza virus infection . other possibilities for data integration are also beginning to unfold. for example, micrornas, which regulate both transcription and translation, might have an important role in mediating virus-host interactions . the discovery of micrornas in certain large dna viruses, such as herpesvirus, suggests that some viruses may encode micrornas to regulate cellular functions . in addition, immunomic strategies (box ) will provide additional opportunities to interrogate the host immune response; screens using small interfering rnas are currently being combined with genomic data to identify specific cellular proteins that are used by viruses during infection [ ] [ ] [ ] . together with virology, clinical and pathology data, this integrated set of information might provide the systems-biology view that will be needed to clearly understand the role of specific host genes and pathways involved in the development of immunity or disease after virus infection. another use for genomics that will no doubt expand is expression quantitative trait loci (eqtl) mapping . the combination of global gene-expression data with eqtl mapping provides greater power in elucidating complex genetic traits in addition to providing insights into specific genes or mutations that might be responsible for the trait in question. this approach is currently being used to better understand the genetic basis for various disease conditions in mice [ ] [ ] [ ] , and it is likely that it will also be useful in increasing our understanding of virus-host interactions. for example, using recombinant inbred strains of mice derived from parental strains that react differently to infection with a given virus, it should be possible to use eqtl mapping to determine chromosomal locations for potential traitcontributing factors and highlight genes of interest for the trait. with this increased level of complexity, however, it will be important to work closely with the bioinformatics and computational-modelling communities, and to make best use of the sophisticated bioinformatics tools, data-mining schemes and mathematical-modelling strategies that are continually being developed , . it might also be necessary to take a step back to simpler experimental systems (such as cell-culture models) to dissect cellular events before moving on to more complex in vivo models. the use of combined computational approaches that can account for gene-regulatory networks and cell-to-cell interactions will also facilitate the move to whole animal physiological modelling. functional genomics is clearly providing advances in our understanding of virus-host interactions, and the evolution to an integrated systems-biology approach holds even greater promise for the field. in addition to providing new insights into viral pathogenesis and host immunity, this approach provides a host-oriented antiviral discovery paradigm with the potential for discovering the benefits of functional genomics will be further enhanced by integrating genomic data with data derived from other high-throughput technologies. the potential information and biological insights provided by these technologies are shown. together, these approaches will help to provide a systems-biology view of virus-host interactions that spans the flow of biological information from dna (genetics) to mrna (genomics) to protein (proteomics) to protein function (immunomics). new targets for broad-spectrum antiviral therapies and for improving vaccine evaluation and design. we are optimistic about continuing advancements in the technologies and computational methods used to study virus-host interactions and in improved capabilities to identify, characterize and circumvent the strategies used by viruses to outsmart their long-suffering hosts. recent studies have shown that rig-i preferentially recognizes single-stranded rna (ssrna) with polyu motifs, whereas mda recognizes long dsrna molecules. these differences might help to explain the differential recognition and innate immune signalling induced by different rna viruses [ ] [ ] [ ] . pathogenic viruses: smart manipulators of the interferon system pathogen subversion of cell-intrinsic innate immunity viruses and interferon: a fight for supremacy principles of 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critical role for pb , ha, and na in the high virulence of the pandemic influenza virus molecular basis for high virulence of hong kong h n influenza a viruses interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome expression profile of immune response genes in patients with severe acute respiratory syndrome an interferon-γ-related cytokine storm in sars patients functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov-infected macaques immunomics: discovering new targets for vaccines and therapeutics heterogeneity in vaccine immune response: the role of immunogenetics and the emerging field of vaccinomics bridging the knowledge gaps in vaccine design these authors show the importance of type i ifns in plasmacytoid dendritic-cell activation and migration using an elegant series of in vivo mouse models that exploited ifnα-receptor-deficient mice and synthetic tlr ligands type i ifns enhance the terminal differentiation of dendritic cells cutting edge: enhancement of antibody responses through direct stimulation of b and t cells by type i ifn type i interferons act directly on cd t cells to allow clonal expansion and memory formation in response to viral infection innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd t cells for clonal expansion and memory formation a role for the transcription factor relb in ifn-α production and in ifn-α-stimulated crosspriming functional genomic and serological analysis of the protective immune response resulting from vaccination of macaques with an ns -truncated influenza virus influenza virus evades innate and adaptive immunity via the ns protein immunogenicity and protection efficacy of replication-deficient influenza a viruses with altered ns genes efficacy of intranasal administration of a truncated ns modified live influenza virus vaccine in swine type i ifn receptor signals directly stimulate local b cells early following influenza virus infection influenza virus infection causes global respiratory tract b cell response modulation via innate immune signals measles virus-induced modulation of host-cell gene expression despite a limited technical capacity to measure only , genes host-cell interaction of attenuated and wild-type strains of yellow fever virus can be differentiated at early stages of hepatocyte infection evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of α/β interferon production innate immune responses in human dendritic cells upon infection by chimeric yellow-fever dengue vaccine serotypes - . am transcriptional activation of interferon-stimulated genes but not of cytokine genes after primary infection of rhesus macaques with dengue virus type type i ifn as a vaccine adjuvant for both systemic and mucosal vaccination against influenza virus the relevance of cytokines for development of protective immunity and rational design of vaccines transcriptional analysis of human peripheral blood mononuclear cells after influenza immunization immune activation at effector and gene expression levels after measles vaccination in healthy individuals: a pilot study virology in the st century: finding function with functional genomics hepatoproteomics: applying proteomic technologies to the study of liver function and disease viral proteomics: global evaluation of viruses and their interaction with the host integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics microarray analysis shows that some micrornas downregulate large numbers of target mrnas identification of micrornas of the herpesvirus family identification of host proteins required for hiv infection through a functional genomic screen copi activity coupled with fatty acid biosynthesis is required for viral replication identification of host genes involved in hepatitis c virus replication by small interfering rna technology genetical genomics: combining genetics with gene expression analysis an integrative genomics strategy for systematic characterization of genetic loci modulating phenotypes integrating genetic and gene expression data: application to cardiovascular and metabolic traits in mice uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics' computational methodologies for modelling, analysis and simulation of signalling networks the model organism as a system: integrating 'omics' data sets systems biology and the host response to viral infection a new discipline, a new name microarray analysis: basic strategies for successful experiments dna microarray technology for the microbiologist: an overview chips with everything: dna microarrays in infectious diseases from functional genomics to functional immunomics: new challenges, old problems, big rewards microarray profiling of antibody responses against simian-human immunodeficiency virus: postchallenge convergence of reactivities independent of host histocompatibility type and vaccine regimen microarray profiling of antiviral antibodies for the development of diagnostics, vaccines, and therapeutics nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses innate immunity induced by compositiondependent rig-i recognition of hepatitis c virus rna the length-dependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiationassociated gene we thank b. paeper and s. proll for discussions and assistance with preparation of the original figures. research in the authors' laboratory is supported by public health service grants (r ai , r hl , r ai , r r r , p a i , p a i , p da and p rr ) from the national institutes of health, usa. this study uses gene-expression profiling of serial liver-biopsy samples from patients that had received a liver transplant to demonstrate that rapidly progressive fibrosis is associated with an impaired immune response, as indicated by a lack of induction of genes associated with the ifnmediated antiviral response, antigen presentation and cytotoxic t-cell response. key: cord- -ntbvmssh authors: nan title: immunogenicity date: - - journal: j cell biochem doi: . /jcb. sha: doc_id: cord_uid: ntbvmssh nan ia moyecules with respect to their roles as peptide receptors and target structures for tcr interaction. particular attention has been paid to distinguishing between local and distant effects of amino acid substitutions on ia function and to determining which residues interact with peptide antigen and which (if any) with the tcr. this ex erimental approach has led to the identification of several regions of the pofvorphic amino-terminal domains of the a and p chains as playing critical roles in chain-chain association and quaternary ia conformation. the a and p l putative helical regions have been found to have distinct degrees of structural lability, with the a helix showing much greater susceptibility to conformational change due to allelic variation in other re ions of the molecule. allelically polymor hic residues in the a and p domainstave been shown to play important roles in &e activity of the assembly/folding control regions, and hence, analysis of local binding roles of specific residues in ia molecules must take this additional effect of substitutions at these positions into account. by controlling for large scale conformational effects, individual residues in the p chain have been assigned to desetopic ( eptide interaction) and histotopic (tcr interaction) roles. in the cytochrome c molel, a putative peptide bindin "pocket" involvin residues from both the postulated p l a helix and also the p-stran% floor has been defined, residues controlling both the extent of binding and the orientation of the bound peptide have been identified, and at least one residue with tcr interaction potential without obvious peptide binding properties has been localized. combining these data with those of other investigators leads us to propose a general model of class i mhc structure-function relationships. we have shown previously that memory b cells transferred into k-allotype distinct congenic rats in the absence of any priming antigen are deleted from the adoptive host within a matter of weeks (half-life of - weeks). in contrast co-injection of antigen with the cells facilitates their survival and the maintenance of a donor response for periods in excess of one year. in the experiments reported here we ask if the persistence of t cell memory is also dependent on antigen. . carrier (klh) primed t cells were transferred in the presence or absence of antigen into irradiated, k-allotype distinct adoptive host. a t various times after transfer these rats were injected with x ' hapten-carrier (dnp-klh) primed b cells together with ig of soluble dnp-klh. this limiting number of b cells makes a secondary type response only if carrier-specific memory t cells survive in the adoptive host. we found that already at weeks following transfer without antigen, no memory t cell help was available for these b cells. in contrast t cells transferred together with ~g klh provided help for secondary type donor responses at and weeks after transfer. we conclude that longterm memory at both the t and b cell levels does not reside with small, very long-lived, resting cells but. with active clones that are maintained by small amounts of antigen that may persist for long periods. once antigen is lost from lymphoid tissues both t and b cell memory wanes within a relatively short time. t cells recognize antigen in the form of short peptides associated to class i or class i mhc molecules. each mhc molecule has the ability to bind a large number of peptides and peptides with unrelated sequences can compete for binding to the same mhc molecule, as well as in vitro. in vivo competition strictly correlates with the capacity of the competitor peptide to bind to the mhc molecule presenting the antigenic peptide and its extent dependes on the molar ratio between antigen and competitor. competition among different peptides derived by processing of hen egg-white lysozyme (hel) appears to exert a major influence on the immunodominance of antigenic determinants recognized by t cells. thus, the h l peptides - and - are both generated by hel processing and are both able to bind to the i-e molecule but only - becomes immunodominant because it has th ability to compete in vivq with other hel peptides, such as - , for the available sites on the i-e molecule. however, two immunodominant t cell epitopes, such as those in hel peptides - and - , both interacting with i-ak molecules, do not compete with each other when injected together at equimolar concentrations. such a coexistance is anticipated between peptides that bind with relatively high affinity to the presenting molecule and thus have both the chance to occupy a number of binding sites sufficient for t cell activation. r v s e iii xen ic tr lantation. in v i m lnvesti$ation uslng mocloml a n t m i e s r e e -t e x e y skin gracs-val on m i c e w a s significantl pr:lrd l g anti-antihdy trea-t but n o t a b anti-antibody: w i d e r the saue animals but a n t i c d antibody did prolong minor a n t w -d i allqrdts. in v i m studies r m that primary proliferation a n f z . prcdwtion & -f i cells in response to mmkey stimulators was weak conpared to allogeneic reqonses. secondary responses t o xenogeneic stinulation were strong after in v i m priming but required the presence of responder nc's. assays for c y g t s t cell effectors in m i c e which had rejected monkey skin revealed few such cells. zhese results est that widely d i a t e xencgeneic processing and presentation, since xenogeneic antigens require that such presentation be in association with the.= antigens of regponder apc's, the xenogeneic r a f t s have a functional similarity t o aff leted allografts. shoved t h a t f e t a l r e n a l and f e t a l and p o s t n a t a l testis a l l o g r a f t s survived longer than corresponding a d u l t t i s s u e i n non-immunosuppressed outbred r a t hosts. the c u r r e n t study a s k s v h e t h e r t h e d i f f e r e n c e i n s u r v i v a l betveen r e n a l and t e s t i c u l a r g r a f t s and between g r a f t s of d i f f e r e n t ages is r e l a t e d t o d i f f e r e n t i a l t i s s u e expression of class i and class i mrna t r a n s c r i p t s or s u r f a c e antigens. and i f t h e s e p a t t e r n s change w i t h t r a n s p l a n t a t i o n . congeneic mice w e found t h a t prolonged s u r v i v a l of c bl/ f e t a l r e n a l (n= ; p< . ) and f e t a l (n= ; ~( . ) and p o s t n a t a l (n= ; ~( . ) t e s t i s mouse a l l o g r a f t s t r a n s p l a n t e d beneath t h e r e n a l c a p s u l e of a d u l t r e c i p i e n t bio.a mice and t h i s s u r v i v a l c o r r e l a t e s i n v e r s e l y w i t h t h e expression of class i and class i mrna (northern a n a l y s i s ) and p r o t e i n s (immunohistochemistryy) and t h a t both p r o t e i n and mrna increased throughout ontogeny f o r both t h e testis and kidney. after t r a n s p l a n t a t i o n t h e r e vas a marked i n d u c t i o n of mhc mrna t r a n s c r i p t s f o r both testis (n= ) and kidney (n= ). implanted f e t a l kidney t i s s u e t h a t survives. however. f a i l e d t o express d e t e c t a b l e mhc p r o t e i n , i n d i c a t i n g t h a t some p o s t -t r a n s c r i p t i o n a l modification i n t h i s t i s s u e occurs. t o a f f o r d it p r o t e c t i o n from r e j e c t i o n . implanted testis shoved i n d u c t i o n of both mrna and p r o t e i n v e l l above i t s much lower baseline. i n d i c a t i n g t h a t i t s r e g u l a t i o n , i n c o n t r a s t t o t h e kidney may be t r a n s c r i p t i o n a l . thus t h e f e t u s may lower t h e mhc burden as a s t r a t e g y t o escape r e j e c t i o n e i t h e r by p o s t t r a n s c r i p t i o n a l modification of p r o t e i n expression a s i n t h e kidney or by t r a n s c r i p t i o n a l modification of mrna as i n t h e testis. culture of thymus tissue in -deoxyguanosine ( dgua) is thought to reduce tissue immunogenicity by selectively depleting highly immunogenic, thymic immigrants of bone marrow origin. in the mouse dgua treated thymus tissue survival is markedly enhanced compared to untreated tissue when transplanted under the kidney capsule of allogeneic recipients. these experiments were repeated in the rat. as expected, strain da neonatal thymus tissue was rejected when transplanted under the kidney capsule of normal allogeneic strain pvg rats. surprisingly. acute rejection occurred even when the tissue was cultured for days in mm pdgua ( x the effective dose in mice). by in vitro criteria this dose was very effective in destroying thymocytes. to test whether residual marrow derived cells that escaped pdgua treatment were responsible for inducing rejection we "parked" the dgua treated da tissue in t cell depleted pvg rats. our working hypothesis was that the few remaining donor derived cells of marrow origin would be overgrown by host type cells. when pdgua-treated da thymus tissue was transplanted into t cell depleted pvg recipients graft rejection did not occur. however da pdgua treated thymus tissue, parked for as long as days in t cell depleted pvg rats, was acutely rejected when retransplanted into normal pvg recipients. we interpret these results to suggest that rat thymic epithelium devoid of marrow derived cells is innately immunogenic. c corinne amiel, violaine gugrin, thierry may, philippe canton, gilbert c faure, laboratoire d'immunologie and maladies infectieuses, chu de nancy, facult de mgdecine, vandoeuvre les nancy, france. lfal is a dimeric membrane molecule composed of a specific alpha chain (cdlla) and a beta chain (cd ) common to three members of the lfa family. lfal is physiologically expressed on all white blood cells, while other molecules of the lfa family (with cdllb and cdllc alpha chains) are restricted to cells of myeloid lineage. a defective expression of lfal has been described in some congenital immune deficiency and in aids. we investigated the lfal defect on peripheral blood lymphocytes from hiv+ patients. three different monoclonal antibodies were used, respectively directed to chain-specific epitopes of cdlla (spvl , sanbio) and cd (iot , immunotech) and to a conformational epitope involving both chains (iot , immunotech). cell suspensions were stained in indirect immunofluorescence and a flow cytometer (epics profile, coultronics) was used to assess the percentages of stained cell, the fluorescence intensity and the shape of fluorescent peaks. our data suggest that lfal expression is impaired in hiv+ patients both through the quantitative expression of each chain and through conformational alterations. the adhesion molecule lfa- is known to be important in antigen presentation. we have previously shown that both monocyte and t cell lfa- play a role in the interaction between these two cells (eji d; , ) . antibody to icam- (known to act as a ligand for lfa- ) also inhibits antigen presentation, although icam- is not thought to be expressed on resting t cells (eji : , ) . we have looked at the expression of icam- on t cells after incubation with cytokines and found that only il- consistently effects an increase in both the percentage of icam- positive cells and in the level of expression. in addition we have found that a proportion of resting t cells express very low levels of icam- . double labelling experiments have shown that these cells are part of the memory t cell population as defined by antibodies to uchli, lfa- and lfa- , and furthermore that icam- negative cells are unable to respond to to antigens such as ppd and flu but are able to respond to pha. this suggests that icam- represents an additional marker on the memory t cell population which more precisely defines the subset able to respond to recall antigens icam- expression on t cells, anne-marie buckle and nancy hogg, macrophage lab. icrf, lincolns inn fields, london, wc a px, u.k. immunization, francis r. carbone and michael j. bevan, department of immunology, research institute of scripps clinic, la jolla, ca . ctl recognize peptide forms of processed, foreign antigens in association with class i molecules of the mhc and are usually directed against endogenously synthesized "cellular antigens" such as those expressed by virusinfected cells. in vifro studies have shown that small exogenous peptides can directly associate with class i molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. we have recently generated ova-specific, h- kb-restricted ctl by immunizing c bl/ mice with a syngeneic tumor line transfected with the ova cdna. the ctl recognize the ova transfectant eg -ova and the synthetic peptide ova but fail to recognize the native protein. we reasoned that given the potential for direct peptide/class ?'&$%ation observed in vifro, ova s,- ra may induce ctl after in vivo priming. however, we found that this is not the case. ova,,,-,,, and peptides of increasing lengths up to which are all able to form the target complex in vitro, are inefficient at priming ~% -%~~ specific ?h%sponses following intravenous injection. this is also true for both native and denatured ova. in contrast to these results, the synthetic peptide ova g:z corresponding to a peptide in a partial tryptic digestion of ova can efficiently prime c bl/ mice in vrvo following intravenous injection. this peptide elicits ctl which appear identical to those derived from animals immunized with syngeneic cells producing ova endogenously. it is now well established that human t lymphocytes can be activated via the t cell specific cd antigen. in order to determine if a factor@) other than the single cd polypeptide is involved in cd mediated signal transduction, we have stamy transfected murine l cells with the human cd cdna. we report that such transfectants expressed hah levels of cd at the cell surface. formed sheep erythrocyte rosettes and expressed the three cd epitqm previously defined on human t lymphocytes, including the "activation associated' t i epitope. the latter observation unequivocally demonstrating that expression of the ti epitope, in contrast to a previous report, is entirely independent of t cell specific factors. combinations of cd mabs that are potent stimulators of human t cells. however, failed to elicit either an increase in the concentration of intracellular free calcium or augment [ h]-thymidine inmrporatbn in the transfectants. these results provide both formal identification of the cd cdna and dearly demonstrate that the single cd polypeptide expressed in an heterokgous cell system devoid of t cell specific factors, cannot alone transduce intracellular signals in response to stimulatory combinations of cd mabs. the results are therefore consistent with the notion. that the functional cd antigen expressed in human t lymphocytes, requlres the association of another, as yet, undefined factor@). this conclusion was based on several lines of evid nce incl ding the observation that mabs specific for the class i a domain of either h-zl or l d b interfered with t e generation of cd -dependent (low substitution at position in the a domain are not lysed by cd -dependent primary ctl but are lysed by secondary cd -independent (high affinity) ctl generated in the presence of antibody to the a domain. populations of ctl. we have isolated and characterized a c d w , cd -da-specific cpl line. this line is cd -independent and is capable of lysing the addition, we are currently generating clones from primary $-specific ctl cultures to obtain cd -dependent (low affinity) ctl. directed rnutagenesis are being tested with the cd -dependent and cd -independent clones to define additional residues important for cd recognition. the comparison of ctl clones with different cd dependencies will allow us to more precisely define the role of cd in t cell recognition. percolle from the buffy coat of one unit ot blood. these cells (= x ) are introduced into a curame elutriation centrifuge (rotor speed of rpm; loading flow of ml/min). nine fractions can be obtained. the first three containing > % lymphocytes; fraction ( rpm- ml/min) and fraction ( rpm- ml/min! contain both lymphocytes and monocytes and the next three fractions contain > % monocytes; the finat fraction (rotor off) contains monocytes + granulocytes. cells from each fraction ( x /well) are incubatee for five days with tetanus toxoid ( . lf/well) and an enriched population of t cells ( x /well). quadriplicate samples are then pulsed for hours with 'h methyl thymidine. maximum apc activity is found in fractions and representing to % of the mononuclear cells. apc activity for these two fractions can be further purified by selective absorption of the cells onto gelatin coated surfaces that have been preincubated with plasma. the non adherent lymphocytes are rgmoved after two hours. after overnight incubation spontaneously released cells ( - x ) can be harvested which have a higher apc activity than cells rotated by elutriation alone. these methods are now highly reproducible in our laboratory, so we can now begin to characterize and study these cells. the male s p e c i f i c h-y a n t i g e n h a s been shown t o behave as a minor histocompatibility a n t i g e n in man and mouse. i n t r a n s p l a n t a t i o n , male t i s s u e may t r i g g e r t h e c l o n a l expansion of h-y reactive hhc r e s t r i c t e d effector cells of female o r i g i n . although male epidermal cells (ec) can induce an anti-h-y t cell response in female mice, so far in v i t r o techniques have f a i l e d t o i d e n t i f y t h e cell-defined h-y a n t i g e n on murine ec ( ). here w e developed a cr release assay t o use human c u l t u r e d k e r a t i n o c y t e s (k) as t a r g e t cells for hla-a specific and ma-a r e s t r i c t e d h-y s p e c i f i c t cell clones. hla-a + but n o t h l a -a t k were l y s e d by anti-ma-a ctls i n a dose dependent manner. low but d e t e c t a b l e l e v e l s of anti-h-y k i l l i n g were found a g a i n s t ma-a + male k b u t n o t a g a i n s t h l a -a t male or hla-a + female k. both l e v e l s of a l l o r e a c t i v e and h-y s p e c i f i c l y s i s were d r a m a t i c a l l y enhanced after exposure of k t o ifn gamma. these r e s u l t s s t r o n g l y suggest t h a t h m a n male s k i n cells are d i r e c t l y s u s c e p t i b l e for h-y d i r e c t e d t c e l l k i l l i n g through t h e expression of f u n c t i o n a l h-y/hla complexes on t h e i r cell s u r f a c e . i n view of t h e s e f i n d i n g s , t o g e t h e r w i t h our r e c e n t s t u d i e s on t h e expression of h-y ctl determinants on h m a n hematopoietic p r o g e n i t o r c e l l s ( , t h e r o l e of h-y a s a t a r g e t s t r u c t u r e f o r c e l l mediated immunity i n o l i n i c a l t r a n s p l a n t a t i o n should be s e r i o u s l y taken i n t o account. . steinmuller d. and burlingham w.j. t r a n s p l a n t a t i o n @ , , , . . voogt p.j., goulmy e., fibbe w.e., e t a l . j . clin. invest. sept. . c diphteria toxoid (dt) presentation by hla dr transfected murine fibroblasts bismuth, laboratory of c e l l u l a r and t i s s u l a r immunology, chu p i t i e s a l p b t r i b r e , p a r i s , france and veterans medical c e n t e r , iowa c i t y , usa. l t r a n s f e c t a n t s e x p r e s s i n g s i n g l e type of human mhc c l a s s i molecules produced by dna conjugate formation has been studied with cloned t cell lines and a b cell hybridoma and with t cells and b cells from normal mice. resting t cells and b cells do not form appreciable numbers of conjugates but conjugates are formed between t cells stimulated with alloantigen for four days and b cells activated by hour culture with lps. irrelevant lymphocytes do not affect the rate of specific conjugate formation in suspensions of cells agitated by gentle rocking but impair conjugate formation when cells are allowed to settle in round bottom tubes. in further experiments, it was shown that the conditions for the induction of lymphokine secretion by the t cell were not indentical to the conditions for conjugate formation.the significance of these and other observations for the interaction of t cells and b cells in vivo will be discussed. of the primary mixed leukocyte reaction (mlr) and that this reaction occurs in multicellular dendritic cell-cd + t cell clusters [cellular immunology , - ( ) dendritic cells are able to contact, cluster, and retain allogeneic t cells and induce these alloreactive cells to proliferate and divide. tions labeled with a vital flvorescent dye, we show that only dendritic cells efficiently form stable clusters. labeled monocytes and b cells do not form clusters with t cells. when labeled monocytes and unlabeled dendritic cells are used to stimulate t cells, unlabeled clusters form. labeled monocytes do not move into the clusters until the third day of the mlr. significant levels of il- and a-ifn appear in the culture supernatant by the first or second day. blast transformation by the second day of the mlr as demonstrated by giemsa staining of cluster cytopreps. also been studied by immunoperoxidase staining. it is known that human peripheral blood dendritic cells are potent stimulators using purified dendritic cell popula- the distribution of certain adhesion molecules within clusters has c m microbiology and immunology, emory university, atlanta, ga . immunization of sjl/j mice with myelin basic protein (mbp) induces the t cell-mediated autoimmune central nervous system disease, experimental allergic encephalomyelitis. response against a dominant epitope (residues - ) leads to disease. lymph node t cells from mbp-immune mice react against several epitopes in addition to - indicating that the i-as molecule is able to form immunogenic complexes with several mbp peptides. the question asked in these studies was whether subdominant epitopes from the same molecule would compete with the dominant epitope for binding sites on the i-as molecule. to address this question two t cell clones, one specific for - (sp . ) and a second specific for a second epitope present in peptide - (sp . ) were tested for responsiveness when cultured with the dominant epitope alone or with mixtures of peptides containing dominant and subdominant epitopes. reactivity of sp . against peptide - was inhibited by peptides - and - . reactivity of sp . against peptide - was not inhibited by peptide - although peptides - and - were inhibitory. controls indicated that inhibitory reactivity was not due to toxicity at high concentrations of peptides. these findings imply that subdominant epitopes are able to compete with dominant epitopes of mbp for binding sites on i-as molecules. linda r. gooding, frances c . rawle, david i . kusher, w i l l i a m s . m. wold+ and barbara knowles*. department of microbiology and immunology, emory university school of medicine, atlanta, ga , 'institute f o r molecular virology, s t . louis university school of medicine, s t . louis, mo and *the wistar i n s t i t u t e , philadelphia, pa . i n several v i r u s systems e a r l y non-structural proteins localized predominantly i n the nucleus of infected cells are major t a r g e t antigens f o r cytotoxic t lymphocytes (ctl). whether early synthesis o r nuclear l o c a l i z a t i o n are important factors i n immunodominance is not known. w e have recently developed a system f o r studying the ctl response t o human group c adenoviruses i n mice. by us ng both transfected t a r g e t s and virus deletion mutants w e have shown t h a t , response t o wild type ad . there are two e a t r a n s c r i p t s , s and s . which both encode major e a r l y nuclear antigens d i f f e r i n g by a amino a c i d insertion: both antigens are recognized equally w e l l by ctl. the e encoded k glycoprotein (gpl k) of ad binds t o mhc c l a s s i antigens i n the endoplasmic reticulum preventing t h e i r translocation t o the c e l l surface and strongly inhibiting l y s i s by ad specific ctl. however, the presence of gpl k i n the priming v i r u s does not a f f e c t the s p e c i f i c i t y of the ctl generated f o r e l a , so the immunodominance of t h i s protein cannot be due t o the fact t h a t i t is the only major protein synthesized before gpl k i n the course of infection. using virus deletion mutants we are investigating whether ctl s p e c i f i c f o r other ad antigens can be induced i n the absence of ela, and whether e a is also the dominant antigen recognized i n mice of other mhc haplotypes. respond to antigens present on non-replicative virions. in contrast, we have obtained balc/c i-erestricted t hybridomas specific for the neuraminidase (na) glycoprotein of a/pr influenza which recognize infectious, but not non-replicative virus, closely resembling recognition requirements observed for most class i mhc-restricted responses to influenza. recognition correlated with the rte nova synthesis of viral na within antigen-presenting cells, but did not depend strictly upon the amount of na present in cultures, since high na concentrations could be achieved by addition of non-replicative virus without being stimulatory for na-specific t cells. recognition of a neo-antigen was ruled out, since, in high concentration, na isolated from purified virions, even if reduced and alkylated, was recognized by the t hybridoma clone. isolated na was recognized when added to pre-fixed apc, suggesting that this form of antigen was able to bypass the usual processing pathway of exogenous proteins. this suggests that endogenously-synthesized antigen may use different pathways to achieve class -associated presentation. t lymphocyte activation is a complex event which is influenced by a variety of distinct cell surface molecules. in order to determine the role of individual molecules in the activation process, we have developed an efficient methodology for generating cell variants in which expression of molecules is selectively inhibited by expression of anti-sense rna from an epstein-barr virus episomal replicon. in a previous study, we reported that marked inhibition of cd cell surface expression could be achieved in a human t cell clone using this approach. we have now extended this strategy to another t cell surface molecule, cd , as a first ste towards ascertaining its role in t cell activation. to this end, we s nthesized a &-mer oli onucleotide corresponding to a sequence in. the : end of the c d i n g re ion of human cd'i and inserted it in an anti-sense orientation into this replicon. this a-c% /rep construct was electroporated into jurkat cells. analysis of stable a-cd irep transfectants by immunofluorescence staining and flow cytometry demonstrated complete and selective inhibition of cd expression. in contrast to the nontransfected arent, this cd -variant demonstrated a partial loss in its ability to form conjugates a n to secrete interleukin when stimulated with anti-cd monoclonal antibodies. however, stimulation of the cd -variant with a and pma did result in interleukin secretion. several observations suggest that cd functions not only as an adhesion molecule recognising mhc class i on the adjacent cells but also potentiate the transducting capacity of the tcr/cdg complex. comparison of the mouse ly protein sequence with the homolog rat ox and human t sequences revealed most highly conserved regions in the membrane and cytoplasmic part of the molecule. the conservation of the transmembrane and cytoplasmic sequences in different species may be significant for the function of the cd molecule. in order to initiate the functional dissection of the cd -molecule we constructed mutations in different parts of the molecule. by transfecting the a and b chain genes donated by a cd dependent cytotoxic t cell clone(kb c ) into the mhc class i restricted agd cd t cell hybridoma do- . we were able to reconstitute the ability to respond to k only if the transfer was done with the ly molecules (gabert et al., . cell, . - ) . in this system surface expression of mutated and non mutated ly- molecules were checked by facs-analysis and the molecuar size of the proteins were analysed by immunoprecipitation with the anti-ly- monoclonal antibody /lj . finally functional effects of the mutations were investigated in response towards the k alloantigen. we have simulated graft versus host and host versus graft reactivity in vitro by studying primary anti-minor h responses in a limiting dilution culture system. the ability of bmm and peripheral blood mononuclear cells (pbm) to stimulate and respond in this system were compared by estimating the number of proliferating cells. in gvh-direction the combination of donor-bmm (d-beim) and host-pbm (h-pbm) was to times more effective in stimulating proliferation than any other combination; the same applied to the combination h-pbm/d-bmm in hvg-direction.-using these combinations the median frequency of proliferating cells in gvh-direction was / (range / c- / ) in pairs, in hvg-direction ( pairs) / (range / - / ). % of the proliferating cells had the phenotype of mature t-cells.-using the same combination of responder/stimulator cells we have also estimated the number of cytotoxic cells specific for the hla-identical target cell. in gvh-direction the median estimate (n= ) was / (range / o- / ), in hvg-direction (n= ) / (range / - / ). by split well analysis similar or higher frequencies of cytotoxic cells with specificity for nk-targets were detected (gvhr: / , hvgr: / ). it was however possible to identify a significant number of minor h-specific clones by segregation analysis; their specificity could be confirmed after clonal expansion. the clones had the phenotype of typical cytotoxic t-cells.-the relevance of the two cytotoxic subpopulations described above to clinical events such as gvhd, graft rejection and relapse needs to be clarified.- molecular cloning of murine icah- , k.j. horley, b. baker, and f. takei, terry pathology, university of british columbia, vancouver, b.c., canada. we have previously reported a novel cell surface antigen expressed on activated and proliferating murine lymphocytes. the antigen, termed hala- , is absent or present at low densities on thymocytes, lymph node cells, and fibroblast cell lines, indicating it is not a universal proliferation antigen. some cells of the spleen and bone marrow express mala- at a high density possibly representing in vivo proliferation in these tissues. apparent molecular weight of - kd under both reducing and nonreducing conditions, and is susceptible to endo f digestion. the monoclonal antibody yn / . that reacts with this antigen, profoundly inhibits mlr. a xgtlo cdna library was constructed from ns- cells that express a high level of mala- , and screened with synthetic oligonucleotides resulting in the isolation of a full length cdna clone (- . kb). the cdna sequence has high homology with the human icau- sequence, indicating that hala- may be the murine homologue of this characterized protein. hines, trudeau i n s t i t u t e , inc., p.o. box , saranac lake, ny a tumor c e l l l i n e , et- , has been derived from an apparent fibrosarcoma t h a t arose i n a c bl/ male mouse. antigens. mice t h a t have r e j e c t e d et- become imnune t o these minor h antigens, judged by accelerated s k i n g r a f t r e j e c t i o n , and t h i s imnunity can be t r a n s f e r r e d t o imnunod e f i c i e n t mice w i t h lymphoid c e l l s . however, spleen c e l l s from mice t h a t have r e j e c t e d according to the widely accepted view, cd (t , sheep erythrocyte receptor) is the first t cell-specific antigen to appear on differentiating thymocytes during ontogeny. it follows that cd should be expressed on all immature and mature t cells. using two-color cytofluorometry i have here identified subsets of cd -cd + t cells both in fetal human thymus or spleen and in adult peripheral blood. cd -cd + t cells constitute - % of fetal thymocytes and . - . % of peripheral blood t cells. il- -dependent longterm clones of cdi-cdj+ cells do not react with a panel of monoclonal antibodies (mab) directed against the t ll, tlll or t epitopes of cd and do not transcribe cd mrna. fetal tissue-derived clones react with the tigammaa mab and thus express a functional tcr gamma chain, while cd -cd + clones from peripheral blood are bha + and express a full-length . kb tcr c s .ria. the clones established here are currently being characterized with respect to functional capacities. i conclude that expression of cd is not an absolute prerequisite for the expression of the cd /tcr molecular complex on human t cells. if they are added after hours. these interactions are bidirectional. since both cdlla and cd . and t h e i r ligand i-cam . are expressed on the presenting c e l l s as well as the t cells. however, a l l such e a r l y adhesion related events are not bidirectional since anti-cdz and anti-lfa- . which are expressed d i f f e r e n t i a l l y on t c e l l s and presenting c e l l s respectively are also effective as inhibitors. antibodies, a n t i cd and a n t i cd antibodies do not i n h i b i t clustering but do i n h i b i t p r o l i f e r a t i o n , and t h i s i s seen irrespective o f when the antibodies are added i n t o the assay. our findings suggest t h a t there are two mechanisms involved i n dendritic c e l l -t c e l l interaction, f i r s t l y an inrnediate cell-cell adhesion step and l a t e r a secondary signal transduction process possibly mediated v i a cytokines. the q u a l i t a t i v e d i fferences between dendritic c e l l and b c e l l induced i m n o g e n l c i t y may thus l i e i n e i t h e r o f these two steps. king, department o f eathology, the bland-sutton i n s t i t u t e . university college and middlesex school i n contrast, a n t i class i m c lmmunogenicity c cultvred tissue is capable of stimulating an rggwwse when l " s p l m l e d ~e n e i c w y , robert j. ketchum and orion d. hegre, dept. of cell biology and neumanatoay, university of uinnesota, minneapolis hn . neonatal rat islets derived by culture-isolation have teen shown to k free of mlc class + cells, and are immunologically silent when transplanted to either syngeneic or allogeneic hosts. allogeneic transplantation of cultured neonatal non-islet pancreatic tissue, which is known to contain class + cells, results in rapid allograft rejection. unexpectedly, m i c transplantation of cultured non-islet ductal tissue also resulted in lononuclear lm,me cell (hnc) infiltration of the graft in % of grafts examined. highly purified syngeneic islets and ductal elements grafted syngeneically at remote sites display an i u n e response in the ductal element graft, while the islet graft is free of any imnme cell infiltrate. this syngeneic imune response does not result from the use of xenogeneic serum in the medium, since cultures carried out using syngeneic rat serum supplemented medium yielded identical results. uncultured neonatal pancreatic tissue grafted syngeneically does not result in iqk: infiltrate, thi i.rmne response to a syngeneic stimulus correlates with the presence of class + (antigen presenting) cells. in grafts free of class + cells (culture-isolated islet grafts) no i.rmne responrrc to syngeneic stimulus was observed, while a response was present when syngeneic ductal elements, known to include class + cells, were grafted. this indicates a need for cells capable of antigen presentation to stimulate this syngeneic rerrponne, and suggests that either a modified self antigen or a nomally sequestered antigen is being presented. this syngeneic imune response demonstrates many of the same characteristics of, and may be analagous to, the in vitro syngeneic, or autologous, mixed lymphocyte reactions. indicating this response is not to developnental antigen. c the presence of "self" mhc class i (ma-dr) antigens determines whether blood transfusions ihmunise or suppress. el lagaaij, a termijtelen. e goulmy, & jj van rood, leiden university hospital, the netherlands. blood transfusions can immunise the recipient, as well as induce prolonged allograft survival. it is not known what makes that some transfusions inrrmnise the recipient whereas others induce immune suppression. we investigated if certain mhc compatibilities or differences between recipient and transfusion donor and organ donor are required to induce the beneficial "transfusion effect" in man. we studied graft survival and blood transfusion induced changes in cellular and humoral immunity in different patient groups. the patients received a single blood transfusion of a randomly choosen donor. we found in all groups that to induce a beneficial "transfusion effect" compatibility for at least hla-dr antigen between recipient and transfusion donor is required. if the transfusion and recipient are mismatched for both ma-dr antigens, the recipient is immunised, resulting in an increased antibody production (p=o.ool), an increased cytoxicity (cml) (p=o.oos), an increased mixed lymfocyte reaction (mlr) (p=o.oos) and a decreased graft survival (p- . ). after a beneficial (ma-dr sharing) transfusion. the in vitro test remain unchanged or decrease. graft survival increases with the number of shared antigens between transfusion donor and organ donor (p=o.o ), suggesting that a donor specific suppression is induced. recent experiments have revealed a direct interaction between the cd molecule and hla-dr antigen. to address the nature of this interaction we have used a xenogeneic system in which a human cd cdna was expressed in the murine cd -and cd -negative hybridoma dt . . . the tcr of d . . recognizes the murine class i molecule od. a class i expressing dd-positive cell line was obtained by cotransfection of the human class i cdnas together with the murine od gene int. the murine fibroblast line dap . coculture of dt . . and dap expressing dp-dd resulted in a fold increase in il- production and in rosette formation only when both cd and dp were present on the responding t hybridoma and the presenting cell, respectively. we are using this system to map regions of the cd molecule that interact with the class i mhc ag. the cd molecule has also been shown to be the receptor for the human immunodeficiency virus (hiv) via the gp molecule. since gp and class i both interact kith cd , we have used our functional assay to verify if gp exerts an inhibitory function on cd class i interaction. recombinant gp inhibits the functional interaction and rosette formation in a concentration dependent fashion with maximal inhibition at about pg/ml.. this inhibition is specific since it can be reversed by recombinant soluble cd . the fact that recombinant. gp can inhibit the functional interaction between cd and its physiological ligand (class i ags) suggests that the use of gp on a vaccine against hiv infection could alter the immune response of such individuals. this work was supported by src, mrc and nci. t lymphocytes discern self from non-self molecules through the interaction of their antigen-specific receptors and proteins encoded by the mhc. although the nature of this association is not well-defined, a model has been proposed whereby the v-segments of the t cell receptor interact with residues along the alpha helices of the class i antigen (davis et al.; : , ) . we have recently shown that ctl generated against the class i molecule qiod crossreact on several unrelated murine class i antigens containing the shared qiod residues at amino acid positions , , and (mann et al.; j x : , ) . these residues contributed by the a- domain occur in the alpha helical portion of the class i molecule and amino acids and could interact directly with the t cell receptor. to further characterize the role of these amino acids, we are in the process of determining whether insertion of these residues by site-directed mutagenesis into a human class i molecule will allow for the antigen's recognition by anti- ctl. here the t cell repertoire becomes restricted, so that foreign antigen can be recognized only when associated with the mhc products of the host, and mature t cells are tolerized to self antigens, a process which also seems to be mhc-restricted. thus, t cells should be non-reactive to self antigens when they are associated with mhc products present on the tolerance-inducing thymic cells, whereas they may still react to the same self antigens when associated with different mhc products. to examine mhcrestricted tolerance in vivo, a model system must have: a) self antigen in the context of one mhc haplotype. and b) tolerance to both that and a second mhc haplotype. chimeras were prepared by aggregation of preimplantation embryos of two strains of mice, c bl/ (b ) and balb/c. the thymus of such chimeras should be composed of two distinct and completely intermixed populations of cells, one from each parental strain (isozyme analysis indicates no detectable fusion of cells). thus, t cells maturing in the chimeric thymus should be exposed to and tolerized to minor histocompatibility antigens (mhas) of one parental strain only in association with the mhc of that strain. for example, mice might be expected to express b mhas only with h- b (the mhc). however, our chimeras were fully tolerant to f skin grafts, which have "hybrid" combinations of mhas and mhc (e.g., b mhas with h- d). these results are most consistent with either, a) "wholesale" antigen processing and presentation of all mhas by the tolerizing thymic cells, and/or, b) functional sharing of mhc products between the parental thymic cell populations. many of the events critical to the maturation of t lymphocytes occur in the thymus. in the case of t-cell responses against viruses such response defects are associated with a marked increase in disease susceptibility as illustrated by class i mhc controlled susceptibility to lethal pneumonia induction by sendai virus. certain class i or class i mhc determined tc response defects (four out of six tested by us) can be restored by imunization in vivo and/or restimulation in vitro with dc. dc are the most effective apc. their superior apc capacity is due to ) a very high absolute number of class i and class i mhc molecules, and ) a low degree of sialylation of mhc and other surface molecules, reducing negative charge and facilitating access of the t-cell receptor to the mhc groove presenting the antigenic peptide and/or improved clustering with t cells. the more effective antigen presentation by dc allows a more prominent role for a cd + q cell independent pathway of cd + tc activation. it is postulated that the more effective direct triggering of cd + tc precursors lowers the threshold for il- production by cd + cells, reducing the requirement for il- production by the cd + cells. failure of dc to overcome certain mhc-linked specific tc response defects probably reflects complete failure of any foreign peptide derived from the processed antigen to interact efficiently with the mhc or a true tc repertoire defect. donald pious, department of pediatrics, university of washington, seattle, w mhc class i molecules bind inmunogenic peptides derivsd fro soluble antigen and the complex is recognized by specific t cells. ue have isolated eight independent mutant b -l u clones which are altered in their ability to present antigen. in standard proliferation assays using four different soluble protein antigens, the mutants are unable to stimulate the majority of t cell clones restricted to hlfl dr or dp. cllthough unable to present *hole hepatitis b surface antigen (hbdg), they effcctively present a hbdg peptide to a dprestricted t cell clone. the fact that both dr and dp restricted antigen presentation is abnormal in these mutants made it likely that the class i structurual genes are unaltered. this hypothesis is supported by the finding that dno sequences from the dr genes of one mutant are norml. however, two observations indicate that the uture class i di-expmsccd by the mutants are structurally altered. binding to the mutants with tuo polymorphic anti-dr antibodies and one anti-dp antibody is reduced, although the level of cell surface class i expression is normal. second, the class i diners from the mutants dissociate into no-rs under in vitro conditions (sds-pwe) which preserve dimers in the progenitor line. together, these functional and structural data suggest that the mutants are defective in a molecule that either associates with or post-translationally modifies class i molecules and is required for the physiologic formation of an twc/antigen complex. they do not function as restriction elements presenting foreign antigens to t-cells. to investigate the nature of this functional defect we have constructed different recombinant class i genes using dna segments from the b q (qa- ) or h- db genes. structural protein encoded by the recombinant genes is derived entirely from the q gene whereas the cis-acting transcriptional regulatory elements or the dna segment encoding the membrane anchoring domain is derived from the h- db gene. into fertilised cba/ca embryos by microinjection and transgenic lines were established. to date we have established transgenic lines. we have shown that the q (qa- ) antigen encoded by the recombinant genes behaves as a major transplantation antigen in skin grafts and provokes strong secondary cytotoxic t-cell responses in grafted animals irrespective of the tissue distribution or mode of membrane anchorage of the q antigen. at present, we are investigating whether the q antigens encoded by the recombinant genes are able to present influenza virus or mouse minor histocompatibility antigens to t-cells during immune responses and hence whether they can function as restriction elements. is a distinctive system because it permits an analysis of the activation requirements for antigen specific, resting t cells. been isolated following culture with anti-ig-sepharose and compared to dendritic cells as stimulators of cd ' t cells in the mix. i mhc products and independently stimulated the ' mlr and the production of several t derived lymphokines, including il- and - . however, the relative potencies of dendritic cells and anti-ig blasts as 'mir stimulators varied in a strain dependent fashion. times more active in stimulating hls-mismatched. mhc-matched t cells, relative to syngeneic t cells. anti-ig blasts when stimulating acroas an mhc barrier and were likewise more effective in binding iqic-disparate t cells to form the clusters in which the mix was generated. dendritic cell-t cell clustering was resistant to anti-lfa- mab, while b blast-t cell clustering was totally blocked. thus, anti-ig b lymphoblasts and dendritic cells, two cell types which differ markedly in phenotype, also differ in efficiency and mechanism for initiating responses in allogeneic t cells. only anti-ig blasts could stimulate across an mls barrier, being at least in contrast, dendritic cells were - times more potent than the as the lymphocyte f u n c t i o n antigen (lfa- ) and the i n t r a c e l l u l a r adhesion molecule (icam). we i n t e r p r e t t h i s as an increase i n the membrane expression o f these s t r u c t u r e s f o l l o w i n g incubation. the increase i s blocked by the t r a n s l a t i o n i n h i b i t o r , cycloheximide, implying t h a t p r o t e i n synthesis i s involved. helper t cell responses to soluble globular proteins require processing of the protein by ia-expressing antigen presenting cells (apc). antigen is internalized into acidic vesicles, proteolyzed, and peptides containing t ceu antigenic determinants are transported to the apc surface where they are recognized by the antigen-specific t cell in conjunction with ia. most ia-"pressing cells are competent apc, however, only b cells have antigen-specilic receptors on their surface auowing bound antigen to be processed and presented at /lw the antigen concentration required by nonspecific apc little is known about b cell antigen processing function during differentiation, or if ig-mediated apc function is altered at different maturational stages, thus allowing regulation of b cell-helper t cell interactions. neonatal acquisition of apc function was examined in mice ages day to day . splenic cells from d l to d mice process and present pigeon qochrome $, pg at - % of adult levels. by d neonatal spleen cells acquire the ability to process and present soluble pg at of adult levels. the ability to internalize antigen through ig rwptors was determined using an antigen-antibody conjugate, p$-&(ab')z. neonatal spleen cells acquire the ability to process antigen through ig simultaneously with the ability to process soluble antigen. lack of prowsing by neonatal spleen cells prior to d is not attributable to insufficient levels of surface ia. since d neonate spleen cells are able to activate t cell hybrids to % of adult levels when provided with p$ - , containing the t cell determinant. dlod neonate presentation of p@ - is indistinguishable from adult levels. b cell maturation into memory b cells was identified by the loss of the jlld differentiation marker. splenic jlld'o b cells increase from % following immunization and return to nonimmune levels after weeks. during antigen-induced b cell maturation, jllb b cells are indistinguishable from splenic b cells in the ability to present antigen introduced into the processing pathway either pinocytotically or via surface ig. p p antibody conjugates specific for mouse f(ab')z i n , igd, or igg are presented equally well by both splenic and jll@ b cells. thus, acquisition of b cell processing function appears to be developmentally regulated and may play an important role in b cell tolerance meshanisms. once b cells have acquired the ability to process antigen this function is maintained and is not regulated during maturation into memory b cells. we are currently investigating the role of ig isotype during neonatal acquisition of antigen processing. (supponed by nih grants ai- , ai- , and ai- ) as the preliminary studies suggested that carrier sc (apc) for ts vs. tcs activation might be distinct, studies were done to directly address this possibility by assessing tha ability of s coupled to various cell populations to activate ts and tcs. the results indicated that ts activation required that s be coupled to plastic adherent cells which bear both i-a and i-j determinants. these cells are nonadberent to anti-ig and nonfunctional in cyclophosphamide (cy) treated mice. i n contrast activation of tcs required coupling of s to plastic non-adherent and anti-ig adherent cells. these cells are functional in cy treated mice and bear the b cell markers jlld and i-a but not - . thus s -specific ts are activated by i-a+ i-j+ adherent cells (presumably macrophages) whereas tcs are activated when antigen is presented by b cells. (nih grant ca .) interleukin- activated killer (iak) lymphocytes (also known as lak cells) which destroy a broader spectrum of tumors invitro than nk cells have been used sucessfully in an adoptive immuno-therapy protocol for the treatment of patients with a variety of advanced cancers. the cell suface molecule(s) on tumor cells that a r e involved in specific binding to iak cells and in programming iak cells for cytolysis (iak acceptor molecules) have not been characterized. inorder to identify such acceptor molecules a crude membrane digest of the lung carcinoma cell line a was biotinylated and adsorbed to iak cells or to unstimulated human peripheral blood lymphocytes (upbl) (each from the same person). proteins from the washed solubilized cells were separated by page, western blotted and probed with streptavidin-alkaline phosphatase. several experiments demonstrated that different tumor membrane proteins bound to iak cells compared to upbl. the unstimulated cells bound one tumor membrance protein (about kd) not found on the iak-adsorded blot. the iak cells bound three tumor proteins (approximately , & kd) not found on the upbl-adsorded blot. three other proteins (about , & kd) were found to adhere equally well t o iak cells and upbl. utilizing a streptavidin affinity column, solubilized tumor membrane proteins that bound to iak cells could be separated from solubilized iak membrane proteins. the isolated tumor membrane proteins that adsorded to iak cells inhibited iak mediated lysis of a tumor cells by > %. these studies suggest that specific cellular adsorption techniques may be useful in isolating and characterizing tumor membrane proteins involved in interactions unique to cytolytic lymphocyte-tumor cell target binding and lysis. activation of human t lymphocytes occurs via the t cell receptor-cd complex but can also be induced through the non-antigen-specific cd molecule. selected combinations of mabs or the soluble cd ligand, namely lfa- and a unique anti-cdz mab (cd . ) induce human t cell activatlon. cd is an accessory molecule implicated in t h e activation of human t lymphocytes. this molecule may exert this function by increasing intercellular avidity through binding to mhc class i molecules and/or by transmitting intracellular signals. we have investigated the action of mabs directed against different epitopes on the cd molecules in the activation of human t cells via the cdz pathway. we show that anti-cd mabs inhibit cd induced t cell proliferation in an epitope-depe dent fashion. this inhibition does not appear to be linked to the lower cd mediated [ c a ' + ] response induced by anti-cd mabs, since [ca"] response is equally affected by anti-cd mabs whether or not they inhibi ed t cell proliferation. in conclusion, the partial inhibition of the cd induced [cb'] response of t cells by various anti-cd mabs suggest that : ) this inhibition does not totally account for the inhibitory effect of anti-cd mabs, ) and the proliferation induced by anti-cdz mabs may not be completely ascribed to the [ca +] response of t cells. rosenberg. and alfred singer, experimental immunology branch, nci, nih, bethesda, md . we have devised a model to study the in vivo generation of suppressor cells by using mice congenic at qa- , a class i-like molecule encoded to the right of h- d. disparate tail skin grafts (tsg) unless a second graft with additional helper determinants was also present. without any source of additional help, failed to reject their qa- graft, and were unable to reject them even upon the subsequent addition of exogenous help. thus, exposure to qa- disparate grafts, in the absence of additional help, either led to qa- specific tolerance or suppression. mice failing to reject qa- allografts revealed the presence of qa- specific suppressor cells that inhibited the in vivo activation of antigen specific effector cells capable of rejecting qa- bearing allografts. experiments using t cell subpopulations should allow for further characterization of these qa- specific suppressor cells. we found that b mice did not reject qa- however, animals engrafted with a qa- graft alone, in the thymus, the t-cell receptor genes are rearranged, the t cells learn to recognize their own major histoconpatibilitp complex "hc). and they learn to respond to foreing hec. these events seem to be linked to the interaction between t-cell precursors and the stromal cells of the thyms. thus increasing evidence points to an essential role for the thymus epithelial cells (te cells) in development of at least hec class i recognition by the t cells. to be able to study the importance of te cells in t cell maturation. we have developed a method for growing murine t cells in serum-free pediun with well defined constituents. the m d i u a allows far growth of te cells without concomitant growth of bone marrow derived cells as macrophages and fibroblasts. data obtained by en and lmmunocytochenistry showing the epithelial nature of the cultured cells, as well as autoradiographic data on the growth pattern, and characterization of tb cell supernatants will be provided in addition to results obtained from co-culture of te cells and t-cell precursors (cm-cds-thymytes). lmmunogenicity c branch, national cancer institute, bethesda, md the effector limb mediating skin allograft rejection is highly antigen specific, rejecting cells that express allogeneic mhc antigens while sparing those which fail to express allogeneic mhc determinants. disparate skin grafts are completely rejected in spite of the fact that only a small percentage of the cells within the graft express ia antigens. thus, it is possible that mhc class i disparate grafts are rejected by a mechanism that does not assess the expression of mhc determinants on each cell. we assessed the specificity of the rejection of ia disparate grafts by using allophenic skin grafts in an adoptive transfer system and concluded that skin grift rejection across an mhc class i disparity required recognition of allo-ia determinants expressed by every cell in the graft. therefore, we reasoned that mhc class i antigens must be induced on these ia negative populations. indeed, injection of mice with gamma interferon dramatically induced ia antigens on previously negative keratinocytes. we next tested whether the induction of allogeneic ia determinants on keratinocytes was necessary for graft rejection by engrafting parental strain mice with skin from f >parent bone marrow chimeras. such grafts failed to be rejected, in spite of the speciic rejection of the allogeneic langerhans cells, indicating that the failure of keratinocytes to express allogeneic class i determinants leads to graft preservation. conclusion, mhc class i disparate skin allografts are rejected in a highly antigen specific fashion, secondary to the induction of mhc class i antigens on skin cells that fail to constitutively express them. to address whether the extensive polymorphism characteristic of class i molecules influences cd binding, we have screened a panel of transfectants expressing individual class i mhc alleles. of alleles tested, only aw . did not bind. all other molecules dld bind, including a . and aw , which differ by and amino acids respectively from aw . . position in the alpha domain was identified by sitedirected mutagenesls as the critical residue differing between a . and aw . which determines binding. a mutant aw . molecule containin alanine at position bound cd , while a mutant of a . with valine at did not. alanine is found at position of all human and murine class i molecules sequenced to date except aw . and aw . , which have valine at that position. bulk cultures of a -allospecific ctl were also sensitive to this substitution, and preferenhally recognized both molecules with alanine at . this study shows that aw . differs from other class i molecules in its capacity to bind cd , and raises the possibility that aw . may not function as a restriction element as effectively as other class i alleles. t cell hybridomas derived from h-zd lnc recognked s and pres antigens in a i-ad restricted way, while t cell hybridmas from h- k lnc manifested a specificity for either pres in association w i t h i-ak or for s in association with i-!& the activation of lhese hybridomas by antigen and antigen presenting cells (af'c), as measured by il- secretion, was found to be sensitive to prostaglandines and could be completely inhibited by anti-lfa- moncclonal antihxlies. different aft populations were tested for their capacity to present spresz particles to these t cell hybridomas. various macmphage like populations such as resident, con a induced, thiiglycolate induced peritoneal exudate cells as well as splenic adherent cells were found to present efficiently the spres antigen. in conhast b cells and la+ b cell lines (ta , m . ) could not function as accessory cells in the spresz specific stimulation of these t cell hybridomas. the inability of these cells to present this antigen was not due t o inhibitory effects since these cells did not inhibit the presentation capacity of other potent apc's. funhcnnore addition of apc's of a different haplotype could not complement for the defective presentatiw of spresz by b cells and b ceu lines indicating that mhc independent accessory factors are not implicated in this process. hence it is clear that maaophage-like apc's and b cells d i f f a in their capacity to process and present spdz antigens. since spresz is a very stable particle composed of lipids and proteins it is conceivable that such antigen quires a smng degradation and swh plocessing might occur in cenain -phage-like apc's but not in b cells. recombinant human insulin biosynthetically labeled with k n d " s at several amino acids was used as an antigen and was exposed for varyillg lengths of time to ta mouse b cell apc. subcellular fractionation and hplc chromatography permitted several of the processed peptides distributed throughout the insulin molecule to be monitored. many insulin peptides localized to both the extracellular ( peptides) and intracellular ( peptides) compartments of ta cells were detected. membrane-associated peptides is in progress. many of the peptides processed by ta apc in situ co-elute with those obtained upon digestion i n vitro by the insulin-specific insulin degrading enzylg (ide). f o r the processing of -labelled human insulin suggest that insulin may be processed in b cell apc into immunogenic peptides by an enzyme(s) present on the plasma-membrane, intracellularly and extracellularly. (supported by mrc and cda) . we investigated the pathway of antigen processing itu in cell apc. hurine ctl clones specific for hia-a were generatef with the human cell line jy. four of five ctl clones were found to lyse a k transfected murine cells more effectively than a transfectants. anti-cd specific mab inhibited t lysis by these four clones, and this inhibition was more pronouncgd for a k one clone, which lysed a and a k transfectants equivalently, was shown to be insensitive to anti-cd antibody inhibition. these findings indicate that a -specific t r i n e ctl clones possess greater avidity for murine target cells expressing the a k hybrid molecule relative to those expressing the a molecule. this implies that a cd interaction with the same molecule seen by the t cell receptor is important for target cell recognition. hla class i antigens are highly polymorphic cell surface proteins involved in initiation and regulation of the immune response. allelic sequence variation primarily affects the structure of the first external domains of the a and j component chains. here we provide evidence for other types of allelic polymorphism for these genes. the sequences of two cdna clones corresponding to the hla-dqp mrnas from an hla homozygous cell line exhibit both alternative splicing and readthrough of polyadenylation. furthermore, the alternative splicing event is associated with only a subset of hla-dqb alleles, while the polyadenylation site readthrough is found in a larger subset. this suggests that polymorphic & acting elements within the hla-dqp gene control both processing steps. proteins, presumably encoded by the alternatively spliced mrnas lacking transmembrane exons, are immunoprecipitated with a monomorphic monoclonal antibody directed against hla-dq. these proteins are found in supernatants of cultured cell lines for which secretion is predicted, but not in those of cell lines which do not contain the alternatively spliced mrnas. such secretion class of i allelic products could profoundly affect interactions between effector and target cells in an immune response. departments of biology and chemistry and the cancer center, q- , university of california at san diego, la jolla, ca . we are studying the murine icam- gene and the effects of icam- on antigen recognition. using the human icam-i cdna, we have isolated cdna and genomic clones encoding the murine homologue. the murine icam- gene is a single-copy gene that consists of multiple exom spanning kb of dna and encodes a . kb mrna that is expressed at high levels in a wide variety of different cell types. sequence analysis indicates that murine icam- is % and homologous to human icam- on the protein and dna levels, respectively, and is a member of the immunoglobulin gene superfamily, consisting of several v-like domains linked tanddy. we are also studying the effects of icam- on antigen recognition. the t-cell clone d m s f ). this response is blocked with the anti lfa- antibody fd . when antigen is presented by blo.a( r) spleen cells but not when antigen is presented by dcek, a fibroblast transfected with i-ek. we are currently aansfecting the murine icam- cdna into dcek in order to determine if we can enhance the d response and to determine if the enhancement is lfa- dependent. wth an alp a beta tcr that recognizes moth the t cell differentiation anti en, cd , is expressed by mhc class i restricted t lymphocytes. mhc class i products. the association between cd expression and restriction by mhc class i rfioducts has led to the hypothesis that cd may interact with monomorphic determinants of a large body of experimental evidence suggests that cd interaction with mhc class i molecules leads to an increase in the binding avidity of t cell-stimulator cell interactions. a direct test for a functional cd -mhc class i interaction in t cell activation requires a separate evaluation of cd -ia interactions from tcr-a /ia recognition. however, a separate evaluation proves difficult since the t cell receptor and cb may interact with the same mhc class i molecule. in this report, we use a t cell activation protocol, where tcr-ag/ia recognition is replaced by tcr complex-antlcd antibodies interactions. using this activation protocol, we pave analyzed the effects of monoclonal anti-mhc class i antibodies on the activation o$a cd hybridoma in the absence of its tcr restr$tinj mhc class i molecule (ie ) but in the presence of unrelated mhc class i molecules (ie ,ia ). the data obtained clearly indicate a functional role for cd -mhc class i interactions in t cell triggering. we have targeted hen egg lysozyme (hel) to murine b cells using heterocrosslinked antibodies which specifically bind to surface igd or different mhc molecules. occurred more quickly with targeting to igd than to mhc structures as assessed by fixation and pronase stripping experiments. hel was internalized quickly into acidic compartments when targeted to igd but was detected much later when targeted to mhc molecules, as assessed by shifts of fluorescent signal of internalized fitc-hel. however, the data indicate that not all endocytosed hel entered low ph (< . ) compartments. degraded hel was released from b cells following endocytosis of -i-hel. this release was detected earlier with targeting to igd than to mhc structures. interestingly, the total amount of internal -i-hel decreased with time after endocytosis via igd, but the internal -i-hel was almost entirely whole undegraded hel at all times following endocytosis. these data and those of chloroquine and leupeptin inhibition studies indicate differences in the fate of antigen entering b cells via igd or mhc structures, and support the notion of a neutral ph storage compartment for antigen endocytosed via surface igd on normal splenic b cells. internalization and presentation of hel to hybridoma t cells laboratories, department of surgery, university o f iowa, iowa city, ia target cell lysis by cd ' ctl is a highly specific phenomenon in vitro, as we have confirmed repeatedly in reverse labelling tests by showing that admixed "third party" target cells are not lysed in the presence of specific ctl-mediated cytolysis. however, when mixtures of ctl and their specific targets are inoculated into the skin of hosts syngeneic to the ctl, host cells at the site of inoculation are destroyed, often to an extent that results in grossly observable, full -thickness necrotic lesions. we have evoked these "innocent bystander" reactions in mice with ctl directed against single and multiple non-h- antigens and tnphapten and influenza a virus-specific antigens. thus, the ability to trigger bystander tissue destruction appears to be a general characteristic of ctl-target cell interaction in vivo. our current evidence suggests that host inflamatory cells recruited and activated by factors stimulated by ctl-target cell recognition actually mediate the tissue destruction. these ctl-initiated bystander reactions may be the basis of the non-specific tissue destruction that contributes to allograft rejection and that is observed in many serious virus infections and in intense dth reactions and contact dermatitis. rong h a lin. baael i n s t i t u t e f o r i u m l o g y , baael, snitzerland. w e have investigated the b a d e for i n u n i t y or tolerance t o a m a e aezw proteinthe f i f t h caponent of c o l p l a c n t (a). i n c deficient nice t h i s protein is absent from s e r m and as a cnrwquence they are not tolerized t o cs. c deficient dca generate uy bearing t c e l l s which recognize c i n the c m t e x t of clasa . i n contrast, c s u f f i c i e n t mice i n which c protein is continuously probced d m t mtmt t c e l l reaponsea againmt u. we have tested i f t h i s self protein i s proceseed and presented with clase i i n n o m l mice and can be recognized by c specific t c e l l s i n the absence of exogenewsly added antigen. a l l clasa i bearing c e l l s fm c s u f f i c i e n t l i c e activated c specific t c e l l clonea without additional antigen. presentation was mt a cansaquence o f c secretion by macrophages i n culture but was a h t o be derived prom endqlenewaly generated cs/cl.ss i caplexea. thus t h i s self protein is e f f i c i e n t l y preaented ,inin and available f o r tolerance in&xtion. although c deficient dca cannot secrete c they s t i l l synthesize a precursor mlecule, pro-c , i n accumulating evidence from a number of models suggests that unique subsets of antigenpresenting cells a r e responsible for the induction of specific t cell-mediated responses. w e have previously described an age-dependent maturational defect in the ability of the sjl strain of mice to activate dth-inducer t cells to a wide variety of antigenic stimuli. none of the other strains tested exhibited a similar defect and all other accessory cell dependent responses were unaffected in the dth unresponsive sjl. w e have also shown that the adoptive transfer a macrophage from older dth responsive sjl or other dth responsive las strains can overcome this defect in dth responsiveness. w e have recently found that a subpopulation with the mac-i+, mac- ' and mac- -surface phenotype a r e able to transfer responsiveness. facs analysis indicate that the mac- phenotype is expressed on less than % of macrophages. titrations of the mac- ' cells isolated by facs indicate that adoptive transfer of o ly mac- ' cells can overcome the defect in dth responsiveness. by contrast, transfer of mac -or mac ' cells were unable to overcome the defect. our data suggest that the induction of cd ' antigen specific cells dth-inducer t cells is mediated by a phenotypically unique small subset of macrophage accessory cells. in our studies, we have examined the effect of administering fab' fragments of anti-l t moab (fabl-gk . ) on the inhibition of humoral immunity. treatment of klh-primed mice with . mg fabl-gk . depleted l t ' cells from lymph node tissue while leaving other lymphocyte subpopulations intact. after injection of klh in complete freund's adjuvant, these t,depleted mice were unable to produce anti-klh antibodies. long-lasting unresponsiveness against klh ( weeks) was observed despite the apparent regeneration of the t, population of the lymph node. the results obtained using either fab' or intact gk . antibody were comparable and suggest that a transient depletion of t, does not account entirely for the long-term humoral unresponsiveness. the aim of this study was to gain a more detailed insight into the molecular aspects of antigen processing during the imune response. as a first approach, endosomal vesicles were isolated from bovine alveolar macrophages and their proteolytic activity with respect to a model protein antigen, sperm whale myoglobin (mb), was characterized. during the first stage of digestion of mb by the endosomes, a limited number of fragments were preferentially released from the antigen. we have isolated and identified these fragments. the digestion of myoglobin is completely prevented by pepstatin, a specific inhibitor of aspartic proteinases, and only marginally by other proteinase inhibitors. when mb fragments preferentially released upon digestion with purified bovine cathepsin d, an aspartic proteinase abundant in macrophages, were identified, almost all coincided with the fragments released by the endosomes. to define in more detail the selectivity of cathepsin d under the mild conditions applied, other protein antigens were similarly treated with the enzyme and the peptides released were identified. the location of the preferential cleavage siteswhen related to known t-cell epitopessuggests a dominant role for cathepsin d in the processing of protein antigens to yield fragments for presentation to t-cells. possibly, the observed selectivity of the enzyme may account for the structural similarities among t-cell epitopes, noted by others. actively acquired tolerance in mice to the antigens of the mhc (h- ) is induced by exposure of the animals to allogeneic lymphocytes within hours of birth. actively acquired tolerance to the mhc in humans (hla) cannot be studied in the same way. however, we have evidence for the existence of actively acquired tolerance in humans in a study of highly sensitized patients waiting for a renal allograft. they had developed complement dependent antibodies to the hla antigens of almost all unrelated caucasoid donors. the sera of these highly sensitized patients were tested against a panel of lymphocytes that were mismatched for only one hla class i antigen. we found for these patients hla class i antigens that, although different from those present in the recipient, did not lead to a positive crossmatch. we called such antigens "permissible mismatches" and show that they often included those hla antigens of the patient's mother that the patient had not inherited (noninherited maternal antigens; nima). in of the patients, the permissible class i mismatches included the nimaa. the noninherited paternal antigens (nipas) were analyzed as a control; only two of the nipas tested were acceptable mismatches, which emphasized the preferential nonresponsiveness to nima. recent experiments indicate that what holds true for antibody formation also holds true for t cell activation. of hla class i and hia class i allospecific cd -positive ctl clones. monoclonal antibodies (mcab) directed against the cd structure were only found to inhibit antigen-specific cytotoxicity of a series of class i allospecific cd -positive ctl clones and not of a class i allospecific cd -positive ctl clone. however cytotoxicity induced by cd mcab (used at suboptimal concentrations) or cd mcabs in both types of ctl clone was blocked by cd mcabs. the absence of cd mcab blocking of antigen-specific cytotoxicity of the class- -specific cd positive ctl clone may be explained by assuming that it results from a triggering signal which is to strong to be overcome by the down-regulatory signal of the cd antigen. these combined findings clearly suggest a functional involvement of cd not only in tcr/cd activation, but also in tcr/cd controlled alternative activation routes, such as the cd activation pathway. moreover it shows that even an hla class i allospecific cd -positive ctl clone expresses a functional active cd antigen. the absence of hia class i expression on the target cells (daudi cells) used in the experiments described indicate that the cd antigens not act solely in an adhesion-like fashion, but exhibit also a more general regulatory function in t-cell activation. this regulatory role of cd may be explained by assuming the induction of a threshold for activation, which is triggered after binding of cd mcab or binding to its natural ligand, hla class i. in our view, cde-mediated regulation of t-cell activation could therefore prevent non-specific triggering of cytotoxicity by interactions of insufficient affinity. *this study was supported by a grant from the dutch kidney foundation. the cd t-cell surface antigen s felt to have the dual functlon of stabilizlng the interaction of the t-lymphocyte with the antigen presenting cell (apc) a s well as transduclng an independent signal that can potentiate the actlvatlon related alteratlons generated through the t-cell receptor. we have found that upon antibody-mediated cross-linklng of the cd molecules of cloned murlne t-lymphocytes there is a time and temperature dependent decrease in the abundance of the lymphocyte-speciflc tyroslne klnase p lok. this co-modulation is speclflc for cd and p lck slnce cross-linking of other t-cell surface antlgens (cd . t , thyl. ) does not result in detectable alteratlons in the abundance of the lck protein and slnce cd cross-llnklng does not induce any alteratlon in the abundance of p *.. another srcrelated tyrosine klnase highly expressed in t-cells. such data suggest that cd and the internal membrane lck protein are in close proximity within the cell. further analysls has revealed that slgnlflcant amounts of lck can be immunopreclpitated by antl-cd antibodies. in addltlon. cd can be speciflcally preclpltated by anti-lck antibodies. our data imply that cd and p ok are physically associated in cd + t-lymphocytes. the flndlngs that cd is msoclated to the lck proteln in either murlne or human t-cells and that cd is also complexed to p lck ln cd * t-cells suggest that the lck tyroslne klnase is involved in the functlon of the cd and cd accessory molecules. these apc do not appear to present processed klsa determinants. in light of these findings, of apparent interest is the issue of which cells types are responsible for hlsa-specific t cell tolerance induction. studies in mice treated from birth with anti-p antibodies suggest an important but perhaps not exclusive role for b cells in this process. we are currently pursuing the identity of other cell types which may be involved. in addition, lmmunogenlclty c university of texas southwestern medical center at dallas, dallas, texas qlo is a soluble class i-like major histocompatibility antigen produced specifically by the liver. previously, it has been shown that mice possessing soluble qlo can generate anti-q cytotoxic t lymphocytes (ctl), suggesting that this soluble molecule does not function as a tolerogen. we have recently constructed c h transgenic animals which express an exon shuffled q (al, p )/ld ( , tm) molecule. this qio/ld molecule is expressed specifically in the liver on hepatocytes but not on nonparenchymal liver cells, spleen, thymus, kidney or brain. the expression of qio/ld in the transgenic hepatocytes is equivalent to la expression on balb/c hepatocytes, suggesting the animals are expressing physiologic levels of the transgene. the presence of membrane bound qio/ld in c h animals has not caused anti- ctl precursors to be deleted, however, because primary in vitro ctl assays show these transgenic animals can specifically lyse qio/ld targets. histopathologic examination of the livers of these animals does not show extensive lymphocytic infiltration or inflammation. in addition, serum levels of alanine aminotransferase. aspartate aminotransferase, and alkaline phosphatase are also normal, confirming that these animals do not show overt signs of liver rejection. results are ampatable with a t least two different pathways of antigen hardling, a pathway for degradaticm of antigen, and a "pmcessiq" pathway for antigen presenbtim. my+ l monocytcq enes appear t o i n t e r f e r e with t h e processing pathway, either by i n h i b i t i n g production of antigenic material t h a t can associate with ia o r by i n h i b i t i n g putative intracellular event@) imr lvb-q the binding of ia to processed antigen and tmnsprt of annplexes to the cell surface the immunogenicity and antigenicity of synthetic peptides (sp) derived from the sequences of a streptococcal antigen were investigated in macaque monkeys. immunization with the free peptides of and residues failed to elicit serum antibodies or t cell responses. however, both serum antibodies and lymphocyte responses were elicited by immunization with the sp linked to tetanus toxoid (t) as a carrier. indeed, spl -lt and sp -tt elicited serum antibodies and proliferative responses of lymphocytes, not only to the sp but also to the native strtptococcal antigen. recall of sp -tt or sp - t immunized monkeys w i t h suboptimal doses of the native srreptococcal antigen resulted in a significant increase in antibodies, both to the sp and native antigen, confirming that the two sp share antigenic epitopes with the native antigen. the b and t cell epitopes were then determined and the b cell epitopes resides in residue - , whereas the t cell epitope overlaps and consists of residue - . the t cell epitope has an amino-terminal leucine and carboxy-terminal glycine and alanine added to residue - of the b cell epitope. in spite of the b and t cell epitopes being expressed in sp (residues - ), the monomer failed to induce serum antibodies without a carrier. however, immunization with dimers of peptide-linked or disulphide-linked residues - , without a canier, elicited both serum antibodies and proliferative responses of lymphocytes. the results suggest that the monomeric sp is not immunogenic, whereas the dimeric peptide elicits both antibodies and t cell responses. the minimal t cell-b cell structure required for immunogenicity is now being determined. lmmunogenlclty section a departments of immunology and rheumatology, mayo clinic, rochester, pin . susceptibility to collagen induced arthritis (cia) in mice maps to the i-a loci in h- mice. however, swr (h- ) mice are cia resistant, suggesting a role of non-mhc genes. we have recently shown gene complementation between h- from swr and tcr v genes from several non-susceptible strains. cia has been induced in b , c h.a and a fackcrosses with swr with similar high incidences; , and % respectively. c l shares a similar background with b and is h- b, but has the same v tcr mutation as swr. backcrosses showed a very low incidence ( %) of cia, and tte arthritis observed was of a much milder and transient nature. the invariant chain associated with hla class i molecules is a - kd glycoprotein implicated in antigen processing and assembly and intracellular transport of class i molecules. class i molecules and invariant chain are expressed primarily by b lymphocytes and antigen-presenting cells such as macrophages and can be induced by interferon-y in a variety of cell types. to define sequences involved in the human invariant chain gene regulation, bp ' to the initiation of transcription were subcloned upstream of the cat gene. transfection into invariant chain-producing cell lines and non-producing cell lines demonstrated that this ' region displayed tissue specificity and responsiveness to interferon-y. deletion mutants were constructed to ascertain the functional properties of specific regions of the invariant chain upstream regulatory regions. these deletion mutants have led to the identification of putative regulatory regions: to , to , and to bp ' to the cap site of the invariant chain gene. deletion of any one of these regions results in decreased cat activity. protein-dna interactions of these sequences have been characterized by mobility gel shift assay and dnase i footprinting. two regions have been identified that exhibit cell type dependent binding of nuclear proteins. two color flow cytometry was used to characterize the surface phenotypes of human bronchoalveolar lymphocytes (n= ). the cd /cd ratio was highly variable ( . - . , mean- . ). a high proportion of the t cells expressed hla-dr ( - %, mean= l%) indicative of t cell activation. however, detectable levels of the i+- receptor were expressed on < % of the cells. cd r was absent from cd cells in most preparations ( - % mean= %) suggesting that the cells are inducers of ig synthesis. uchl , a marker of memory cells was present on - % of lung t cells. uchll+ cd r-lung lymphocytes responded poorly to pha and cona but did respond to il- in the presence of accessory cells. together these data suggest that lung lymphocytes are recently activated memory cells. il- induced lung t cell lines were also characterized for antigen expression and w ( activity. high lak activity was obtained in preparations containing a high proportion of cd cells. these cultures appeared to be suicidal. in contrast, lines with a high proportion of cd + had low or absent lak activity but proliferated in the presence of il- for at least months expressing a cd cd r-phenotype. this abstract is a proposed presentation and does not necessarily reflect epa policy. the polymorphic second exons of the hla-dp, and dpd genes have been specifically amplified in vitro by the polymerase chain reaction (pcr) method, using the thermostable dna polymerase of aauaticus. sequence analysis of mi clones containing the amplified dp sequences from a panel of thirty-four df typed cell lines revealed only the two previously characterized alleles for dp, . fourteen allelic variants were defined for dpw eight of these are associated with the t-celldefined dpwl- types; two subtypes were found for both dpw and dpw . six additional dp alleles which were previously typed in the t cell assay as blanks were also idenlfied. based on this sequence information, non-isotopic sequence specific oligonucleotide probes have been developed and used to type a margarita betz, dominic dordai, brian e. lacy, and barbara s. fox. department of medicine, university of maryland school of medicine, baltimore md . murine type helper t cells (th ) secrete interleukin (il ) in response to antigen. despite the likely importance of these cells, little is known about their priming and expansion in vivo. we have demonstrated il production in response to a cytochrome p peptide following t cell expansion in vitro. this antigen has not previously been shown to induce th cells. bio.a mice were primed with a peptide fragment of pigeon cytochrome c in cfa. lymph node cells were restimulated in vitro with antigen for - days, ficolled and rested for days without antigen. cells were then tested by limiting dilution for the presence of antigen-specific il producing cells. il was detected using the il sensitive cell line ct s (provided by dr. w. e. paul, nih). the specificity of the response was confirmed by blocking with the anti-ll antibody b . following in vitro restimulation of the primed lymphocytes with antigen, il production was detectable from as few as cells per well. il secretion was antigen dependent and required both in vivo priming and restimulation in order to be detected. it is not clear why primed lymph node cells, placed in limiting dilution culture directly after removal from the animal, failed to secrete detectable amounts of il in response to antigen. suppression is an unlikely mechanism as fresh primed lymph node cells were unable to inhibit il production by restimulated cells. we are now investigating the factors that may regulate the development of il producing t cells. mark r. boothby, ellen gravallese, hsiou-chi liou. and laurie h. glimcher, department o f cancer biology, harvard school o f public health, boston, ma . regulated pattern, and normally expression is limited to certain cell types such as cells and macrophages. cells is accompanied by the loss o f class i mhc expression. these genes also respond to external stimuli such as the cytokine il- , which increases b cell ia. a region o f the aa mhc gene activated expression of a cat reporter gene in a b lymphoma cell line but not in a myeloma cell line. a nuclear protein that bound to two sites within this region was found. this binding activity was present in spleeiis that lack t cells and in b cell lines, but it was absent from all three myeloma cell lines tested. il- treatment of normal and athymic mouse spleen cells greatly increased the binding of this nuclear protein to its a a target sites, concomitant with increased aa transcription; "thus, cells contain a sequence-specific binding activity regulated both by il- and by differentiation. the differentiation o f b cells to plasma lmmunogenicity c peter van den elsen . ldepartment of immunology, the netherlands cancer institute, amsterdam, zdepartment of immunology, erasmus university, rotterdam, department of immunohaematology, academic hospital, leiden, the netherlands. human tcr y occurs in disulphide-linked (type ) or non-disulphide-linked (type ) forms, dependent on the use of the cyl or cy gene segment. the cyz gene segment can contain a duplication or triplication of exon , which gives rise to different protein forms (types bc or zabc). it is not known whether functional differences exist between these receptor types. protein chemical analysis of type and type bc receptors on functional human t cell clones derived from peripheral blood (pb) has indicated that not only the y chains, but also the chains have a molecular mass and charge which set apart type and q p e bc receptors. two sets of fifteen clones were randomly generated from pb of two normal donors after selection with the anti-tcr y - mab, which recognizes all receptor types. dna rearrangement and mrna expression analysis of y and genes allowed us to map the specificity of the anti-tcr y mabs tcs- and tiya to the v l and vy gene segments respectively. subsequently it could be concluded from the analysis of random clones that the majority of type receptors use vy , while this preference seems absent in type receptors. the great majority of type receptors do not use v l. while the majority of type receptors do. this was confirmed by fluorescence analysis of pbl of a large panel of normal donors. we conclude that vy and v gene segments in functional tcr y in pb are used in non random combination and that their expression is correlated with rearrangement of the y gene to cyl or cy . we have previously shown that polymorphic residues in the nh -tenninal half of the p domain (amino acids - ; hypervariable regions and [phvl and ) determine with which allelic or isotypic a chain a particular b chain can achieve efficient cell surface beterodimer expression. this result might be understood in terms of the current model for ia smcture which predicts that w v l would lie adjacent to region. therefore, to examine the role of ahvl residues in conmlling hetcroduncr expression. a mutant a d cdna was created in which the codon for amino acid was mutated to code for the auk residue at this position. in addition, recombinant a d and a& cdnas, in which the segments encoding the three a hypervariable regions were exchanged between the two alleles, were used to study the connibutions of other a chain polymorphisms to this process. interestingly, the polymorphic residues in ahv are predicted to lie in a region of the a a chain a-helix which is adjacent to the phv region of the p chain a-helix. allelic substitutions in this latter region of ad have been shown to similarly affect surface ia h e t e r o d i i expression. taken together, these results suggest that there are at least two spatially separate areas in which the a and p chains interact and that these interactions are affected by polymorphic rtsidues in both areas, conmbuting to the efficiency of heteroditner expression and, most likely, ia quartcmary conformation. the aim of this project is to identify contact residues of the t cell receptor (tcr) with antigen and/or mhc class i molecules. as a model system, a vp -containing tcr has been chosen since the majority of vb ' t cell hybrids react with ie molecules of the k,s,d, and b haplotype. t cell hybrids have been made which have a dual reactivity: they are vp + and recognize ie molecules but also show reactivity towards a known antigen, namely chicken ovalbumin (ova). one such hybrid has been mutagenized with ethyl methane sulfonate (ems). mutants were selected on the basis of their survival after stimulation by either antigen or ie. it was expected that mutations in all different kind of genes involved in t cell recognition and t cell activation would be found. mutants obtained fall into two major groups: ) loss variants of tcr a or fl chains,t or l t : ) mutants with point mutations in one of these genes. we are currently analyzing the mutants biochemically and functionally in order to identify the particular gene affected. point mutations in the a and p genes of tcr mutants will be localized using the polymerase chain reaction in combination with dideoxy sequencing. activation of t lymphocytes requires the intracellular fragmentation of foreign antigens and their presentation by class i or class i major histocompatibility complex glycoproteins. the direct binding of peptides to class i molecules has been shown in a number of experimental systems and its specificity compared to that of t cell activation. in contrast, direct binding of peptides to class i molecules has been difficult to detect; although peptide sensitization experiments and the crystallographic structure of hla-a persuasively argue for its occurence and importance. in this study, we demonstrate specific binding to hla-a of an influenza matrix peptide (flu-m residues - ) that has previously been shown to act as a target for certain hla-a restricted influenza-specific cytotoxic t lymphocytes. we estimate that less than . % of the purified hla-a molecules were able to bind the added peptide. we and others have shown that allorecognition by cytolytic t lymphocytes (ctl) is analogous to t cell recognition of foreign antigens in that both can occur via presentation of antigenic peptides by products of the major histocompatibility complex. we have used peptides corresponding to the alphal helix of selected hla molecules to analyze t cell recognition of this polymorphic region. the alpha alpha helix of hla-b and -b are identical, and show a high degree oh homology with those of hla-bw , -b , and -b . peripheral blood lymphocytes from normal donors were stimulated in vitro with targets expressing hla-b to derive allospecific ctl lines and clones. in some individuals, the allospecific response was almost totally directed against the alphal helix. the ability of peptides corresponding to the alphal helix of these hla molecules to inhibit and induce lysis as well as to modify other assays of t cell activation will be discussed. diseases and *national institute of child health and human development, d bethesda, md . classical transplantation antigens are constitutively expressed on cells of all tissues exce t brain. transaiption is regulated by the interaction of nuclear factors with ' flanking regions tiat include the class i re latory element (cre). previously, the cre has been divided mto re om on the basis of nuclear t%or blndin . several studies have implicated the nuclear protein (ri) wfich binds to the inverted repeat geggattcccca) of re 'on i as necessary for gene transai tion although region i is identicarin all se uencedouse $d and l enes, it is not conservefin qa r y genes. a comparison of the c& from h- ld with that of , a qa region gene expressed o y in the liver and fetal olk sac, shows that there are two changes within the inverted r eat se uence (tgaggactcc$a). these differences disru t the dyad symmetry. another nugotide diierence between h- l and qlo falls within re 'on ifof the cre. however, qlo can bind to the nuclear factor (rii) that binds to region ii of the fit ld cre, whereas qlo region i can not bind to the nuclear factor (ri) that binds to the region i inverted re at. to test whether the differences m region i contribute to the restricted tissue e ression of q l r w e have used site-directed in vitro mutagenesis to make the inverted repeat of]cglo region i like that of the classical class i genes. a change at either base enhances transcription as measured in a transient transfection system. either change also allows binding of the nuclear factor that binds to the classical r alterations in the cre regon i contribute to the limited tissue expression of . the presence of disrupted cre region i in other region genes likely contributes to their tissue restricted expression. on i sequence. thus, molecular analysis of t cell receptor structure/function in sperm whale myoglobin specific t cell clones. jayne s.danska, alexandra m. livingstone, toshi isihara and c. garrison fathman, stanford university medical school, stanford, ca. we have undertaken structural characterization of the t cell receptors (tcr) utilized by a well defined panel of murine dba/ t cell clones that recognize epitopes within the - peptide of sperm whale myoglobin (sp wmb) presented by i-ad or i e . only of independent clones show alloreactivity for whc haplotypes. using the polymerase chain reaction (pcr) and dna sequencing of the tcr a and fc chains from matched sets of clones bearing either whc restriction or epitope specificity in common, we are addressing structural relationship between tcr and mhc/antigen for this model system. among i-ed restricted t cell clones reactive with spwmb - , all have highly homologous tcr chains associated with a minimum of three different tcr a chains, some of which are derived from novel v gene families. to further characterized the specificity of these clones we are generating substituted peptides to identify residues within the epitope important for interaction with tcr or restricting mhc molecule. functional verification of the relationship between given tcr primary sequences, and recognition capability will be addressed by transfer of the a and/or chafns cdnas created by pcr amplification into t-cell hybridomas expressing endogenous tcr genes of known sequence and specificity. with mhc fine specificity. the differential impact of substitutions with the n-terminal and c-terminal portions of the apl domain is consistent with models of auap structure in which the n-terminus interacts with peptide while the c-terminus interacts with both peptide and the tcr. or aauapu-resmcted t cell clones. the antigens tested were l-tymsine-p-lmmunogenicity c expression of the q p gene, patricia m. day, katherine e. lapan and jeffrey a. frelinger, department of microbiology and lmmunolog university of north carolina at chapel hill, chapel hill, nc . generally, the transcription of class i genes tom the q a a region is limited to tissues of hematopoietic origin. previous work in our lab demonstrated widespread transcription of the q gene in the .p mouse, with high levels of mrna found in liver, lung, lymph node, spleen, testes and thymus. less rna was present in muscle and brain tissues. however,,it is not known whch individual cell types within these tissues are responsible for the transcription of the q gene. we raised polyclonal antisera against a synthetic peptide, derived from the predicted amino acid sequence of the q p transmembrane region. we selected this region since it is the most locus specific. this antisera immunoprecipitates a class lsized protein. a monoclonal antibody, directed against the same peptide, has also been produced. sv -transformed h- p fibroblasts show an abundance of q message. suprisingly, indirect immunoluorescent staining with the monoclonal antibody reveals a cytoplasmic localization of the protein with a perinuclear concentration. different patterns have been observed in examination of the h- b em onal carcinoma cell lines ax and pcc . qgspecific antibodies allow us to identify the cell ty es which express the gene product. the application of in situ hybridization techniques will correlate the cellular site ofmrna synthesis and protein detected by antibodies. understanding the paltern of expressim of the q gene is the first step in determining the so far elusive function of these mhc genes. and il r mrna a f t e r mitogenic stimulation. antibodies against cd r, but not against c common determinants, synergise with suboptimal doses o f mitogen t o induce il and il r mrna expression, suggesting t h a t cd r molecules are operative i n transmembrane s i g n a l l i n g i n immature thymocytes. there i s also an i n d i c a t i o n from northerns using cd probes t h a t cd p mrna i s not induced i n activated cd -thymocytes as i t i s i n mature t c e l l s . these r e s u l t s support the idea t h a t cd r+ molecules are essential f o r generating signals required f o r c e l l survival w i t h i n the productive intrathymic lineage. we examined a panel of thl and th t cell clones for the ability to induce antibody synthesis in a mishell-dutton culture system under cognate b-t cell conditions: our findings indicate that both thl and th t cells are heterogeneous, i.e., some but not all thl and some but not all th clones have the capacity to induce antibody under these conditions. we examined the effect of -irradiation or rnitomycin-c pretreatment of our thl and th clones on their ability induce antibody synthesis. asano et al. (j. immunol : ) have reported that th clones are exceedingly sensitive to -irradiation, with doses as low as rads abrogating the ability of th clones to induce antibody synthesis. we found that while the helper activity of th clones was very radiation sensitive, helper activity in thl clones was very radiation resistant. thl clones given rads of irradiation were as effective as unirradiated clones in inducing anti-tnp plaque forming cells (pfc). moreover, when used at higher t cell/b cell ratios in culture, irradiated thl clones were more effective than unirradiated clones in inducing antibody synthesis. the effect of irradiation on th clones was not simply due to inhibition of proliferation, since mitomycin-c pretreatment of the clones had little effect on helper activity. conservation, alexander l. dent, pamela j. fink and ste hen m. hedrick, department of biology, university of california, san diego, a . the p chain gene of the murine t cell receptor has been shown previously to have an alternative spliced form of message. this message contains a novel exon, termed c& which is inserted between the vdj and constant region exons. we have studi d expression of the cpo exon at the mrna level by rnase protection. we have found that about % or less of of p messages in normal t cell clones contain the cgo exon, whereas p m e s a es. in the thymus contain the exon at - fold higher levels. to address t i e importance of the c exon in the immune ,system, we have undertaken a phylogenetic approach. \y cloning and sequencin the rat analogue of cpo, we have found that while the rat exon is very simifar to the mouse exon, both donor and acceptor rna splice signals are defective in the rat cpo gene. this implies that rat cpo cannot be spliced into rat p m e s a s. furthermore, we have sequenced the analo ous region to mouse cpo in e human p chain locus, and have found no stretct of sequence remotel homolo ous to cpo. because cpo is not conserved evolutionarily, we beieve that ! he cpo gene element does not sewe an important function to the immune system of most vertebrates. ) . we found that one arrdno acid substitution at a vdjp juntional region position found to be highly conserved in pigcon cytochrome c-specific tcr's results in a change in antigen fine specificity, while an* change abolishes all detectable responses characteristic of the d tcr. we will present the results of mutagenesismansfection analyses of two other pigeon cytochrome c-specific tcr's. the murine ctl response to human class i molecules is - orders of magnitude lower than the response to murine alloantigens, due to structural differences between human and murine homologs. we investigated whether this discrepancy could be overcome by exposure of developing t cells to human class i molecules in transgenic c bl/ mice expressing hla-a . . ! c zhe bdixujiar basis of - . lbve digiustn ard ed p d l u b r , div- of s ch mice expressed hla-a .i in spleen, bone marrow and thymus at levels similar to those of endogenous h- molecules. however, the frequency of ctl specific for other human alloantigens remained similar to that of normal mice. the frequency of hla-a . restricted influenza specific ctl was - orders of magnitude less than the frequency of h- restricted ctl. these results indicate that the poor response of murine ctl to human class i antigens is not determined by selection in the thymus, but by species-specific constraints on the interaction of mhc antigens with t-cell recognition structures. while the mice are tolerant to hla-a .i expressed on murine cells, they still respond to hla-a . expressed on human cells. the epitopes defined by such clones are present on hla-a . positive human cells derived from several different tissues. such epitopej are not dependent upon the species of p m associated with the class i molecule, nor upon the structure of the attached carbohydrate. the results suggest that one or more highly conserved normal human proteins contribute to the formation of such epitopes, and provide an explanation for the failure of ctl raised against class i molecules on human cells to recognize the same molecules expressed on murine transfectants. this suggests that normal endogenously expressed molecules may also be important in the formation of epitopes on class i antigens recognized by allospecific ctl. we previously demonstrated t h a t several subclones derived from a c +, cd -/cd -t-cel i l i n e have undergone secondary rearrangements a t t h e t-cell receptor (tcr) a locus w h i l e maint a i n i n g i t s o r i g i n a l tcrb and igh d-j rearrangements (marolleau e t . al., i n press). these secondary rearrangements r e s u l t i n t h e j o i n i n g of germline va and j a gene segments which replace the p r e -e x i s t i n g va-jacmplexes of t h e parental t-cell line. i n an e f f o r t t o examine t h e molecular mechanism responsible f o r these va-ja gene replacements, t h e s t r u c t u r e s o f tcra cdnas prepared from both t h e parental and subcloned t-cell l i n e s were determined. i n addition, northern b l o t and southern b l o t analyses were performed on both t h e parental and subcloned t-cell l i n e s using a panel o f va and j a probes. our r e s u l t s i n d i c a t e t h a t : ) secondary rearrangements r e s u l t i n both productive and non-productive va-ja j o i n s , ) the mechanism whereby secondary rearrangements occur i s a d e l e t i o n event t h a t involves germline va genes ' t o t h e p r e -e x i s t i n g va-ja complex j o i n i n g t o j a the class ii major histocompatibility complex (mhc) antigens are a family of integral membrane proteins whose expression is tissue-specific and developmentally regulated. a pair of consensus sequences, x and y, separated by an interspace element, is found upstream to all class ii genes. deletion of each of these sequences eliminates expression of class ii genes in vitro or in transgenic mice ( - ). furthermore, the absence of a specific binding protein for the hla dr a x box in patients with severe combined immunodeficiency disease whose cells lack class ii suggests a critical role for these proteins in class ii gene transcription ( ). report the cloning of a agtll cdna encoding a dna binding protein (human x-box binding protein, hxbp- ) which, like the proteins in whole nuclear extract, recognizes both the x box and interspace elements of the human dra and murine aa genes. the hxbp- cdna hybridizes to two rna species, . kb and . kb in human, that are expressed in both class ii positive and class ii negative cells. hxbp- does not cross-hybridize to two murine aa x box binding cdnas recently isolated in our laboratory which also recognize the dra and a ax boxes. these observations provide evidence for the existence of multiple x box binding proteins which recognize a common or overlapping motif. chromosome mapping studies demonstrate that hxbp- arises from a multi-gene family two of whose members map to human chromosomes and . taken together, these data suggest a high degree of complexity in the transcriptional control of the class ii gene family. france . in an attempt to analyze positive or negative in vivo regulation of clonal expansion of cytolytic t lymphocytes (ctl), we immunized blo.br mice with the kb specific ctl clone kbs-cu). and we tested whether t cells obtained from such mice would influence the in vitro development of the ctl clone kbs-c?o. a clone-specific helper effect has k e n observed, which is mediated by cd + splenic cells from immunized mice. control immunizations of b o.br mice with ti negative variants of suggest that this growth regulation involves the recognition of the ti of kbsc . the precise nature of the antigen recognized on the ctl clone, the possible involvement of ti determinants with or without mhc products u e now under investigation. we have shown that thy-]+ dendritic cells present in the epidermis of mice (dec) express cd associated v and v gene products. we have produced a monoclonal antibody directed against v and found that a wave of cells appearing at the earliest stages of fetal thymic development express v . phenotypic and functional analysis of v + cells in the early fetal thymus indicates that they have characteristics in common with the v + dec. both populations express high levels of ly- and are ly- c+. neither express cd or cd . interestingly, the v + fetal cells express elevated levels of il- receptor, indicating that they may have been activated. functional analysis demonstrated that, unlike other fetal thymocytes, the v + cells can be stimulated to produce lymphokines and lyse a panel of target cells which are also lysed by the adult thy-l+ dec. these results raise the intriguing possibility that the first receptor-bearing component of the t cell system to appear in ontogeny might give rise to the thy-]+ dec. ( ritical to an understanding of the function of cells bearing the gamma-delta t cell receptor will be an understanding of when and where such cells function. in order to investigate this, we have used a variety of techniques (in situ hybridisation, cdna cloning, and pcr) to examine m a s of tcr gamma delta gene expression. one conspicuous site of expression is the intestinal epithelium, which is by contrast almost devoid of tcr alpha beta expression. interestingly, the v gene segment usage in this location is quite specific and is different to the specifcity that we have found in the spleen and in the thymus, and that others have found in the skin. this specificity suggests in turn that expression of the resmcting elements recognised by gamma delta may also be spatially nxticted. extensive analysis of junctional diversity can pmvide infomation on the diversity of antigen nxogniscd by gamma delta. the basis for selective expnssion of v gene segments may in part lie in different requkments of the v-gannna gene pmoters. to examine this, the wnscriptional capabilities of the various gamma gene promoters in t cells murine tcr gamma genes: distinct spatial restriction of v-gene segment usage, adrian thyday*, susan kyes*, simon carding#, charles a. janevay, being compared by linkage to the chloramphenicol acetyl transferase gene. biochemistry, university of wisconsin-madison, madison, . murine strain a sublines a/j and a/wysnj have a genetic polymorphism that regulates serum immunoglobulin responses to several protein antigens. strain a/j secondary igg responses to bovinv gamma globulin, oralbumin, hemocyanin and galactosidase l-ere -, l o -, i -and -fold greater, respectively, than a/wysnj responses. subline a/hej is a low responding strain like .l/wysnj. analysis of h- class and class i molecules provided no evidence for a breeding error to account for the genetic polymorphism. instead, an important immune response gene outside h- may hare been heterozygous when the sublines diverged, and the polymorphism resulted from sezregation and differential allele fixation. a mutation subsequent to subline divergence is also a possible source of the polymorphism, but is less likely. the high responder phenotype inheritance pattern in (a/wysnj x a/j)fl, f , and backcross mice was consistent with segregation of a single, recessive gene. we named this locus l a for the strain a sublines that define it; strain a/j represents the k a h allele and strains a/yysnj and a/hej represent the allele. hayes, keith d. hanson, faye nashold, and david j . miller, department of the secondary iggza responses were also affected. several different proteolytic digests of denatured seb have been tested for their ability to stimulate t cell hybrids to produce il- . a tryptic digest that retains activity has been fractionated by hplc and the stimulatory component is being analyzed. examination of the amino acid sequence of seb and the proteolytic cleavage sites has led us to synthesize several peptides for analysis. these peptides, and their analogues, will be tested for their function in vivo and in vitro. to understand the interactions involved in the famation of peptide-mhc complexes, an assay has been developed to detect dr specific binding of peptide analogues of t e l l determinants to cell surfaces. ebv msformcd b cell lines (bcls) w m incubated with biotinylated peptide followed by fltc conjugated streptavidin, and then anaiysed by flow cytomctg. a panel of bcls homoygous for diffmnt dr types bound analogues of peptide - from influenza virus haemagglutinin (previously shown to be a helper t cell determinant restricted through dr ) to varying degrees, w h m no binding was observed to the dr-bcl rj . binding could be specifically inhibited by the natural unbiotinylated t cell determinant or other drl restricted determinants. competition by a range of peptides revealed quantitative diffmnces in their ability to bind drl. the assay is currently being used to generate a detailed model of the complex formed betweenha - anddrl. and trp for leul . and hla-a . differs from hla-a .i by the substitutions of thr for ala glu fo??a and trp for zeu . residues and in the -sheet of the molecule, and residues , , and in the a-helix are thought to interact with bound peptide or the tcr. to evaluate the role of these residues on ctl-defined epitopes, two genes were constructed that encoded novel molecules which differ from hla-a . only at residues , , and , or at residue . the effect of a-helix substitutions on serologic and ctl-defined epitopes that varied between hla-a .i and hla-a . were evaluated by constructing genes that encoded the individual differences at residues , , and , as well as additional non-naturally occuring substitutions at these same positions. hla-a . specific ctl were found that were: ( ) insensitive to substitutions at either residues , , and , or residue , but were lost when all four positions were changed; ( ) dependent upon the residues , , , but not residue ; ( ) dependent upon residue , but not residues . , and ; and ( ) dependent upon residues , , , and residue . further epitope mapping with the a-helix mutants demonstrated that a substitution at residue often destroys an epitope not affected by substitution at residue . even conservative substitutions at position were more disruptive than nonconservative changes at residue . residue , while important in defining an mab epitope, had no effect on any ctl epitopes. these results indicate that spatially separate residues in the a-helix and -sheet of the molecule can contribute to the epitope recognized by a given ctl. furthermore, considerable complexity must exist in the spectrum of t cell receptors utilized to recognize hla-az, as ctl clones exhibited distinct fine specificity patterns. to follow the evolution of these class i types, to discern the chief selective pressures on its members and thus indicate the probable functional properties of the antigens. a cosmid library was screened for class i genes. clones were mapped and could be grouped into clusters of contiguous dna spanning , kb. by hybridisation studies, class i genes/ gene fragments could be distinguished. transfection analysis revealed that genes could be expressed as cell surface antigens: two genes, in a block of duplicated dna encoded serologically defined rt .c products, the other genes gave rise to novel class i antigens detected by the xeno-antibody x . using region specific probes, we could detect clear rat homologues of the mouse qa and h- genes, however there were only two rat genes with limited homology to the mouse tla genes. the analysis showed extensive remodelling of the class i region in the evolutionary gap between rat and mouse. while the immunological role of t cells bearing the ap t cell receptor (tcr) has been well characterized, much less is known about the function of t cells bearing the $ tcr. we investigated the role of tcr $cells in the immune response to complete freund's adjuvant (cfa). after immunizing mice with cfa, we observed a greater than -fold increase in the number of tcr $ cells present in lymph nodes draining the sites of immunization, compared to a - -fold increase in the number of tcr ap cells. there were at least three different species of tcr's expressed on these cells in the draining lymph nodes, including two protein products derived from the rearrangements of cyl and cp, and one product derived from cy . % of tcr ys cells from immunized lymph nodes expressed the il- receptor in vivo. and these cells constituted roughly % of the proliferative response of total lymph node t cells to - . tse, et al. (j.lmmun..vol.l , p. . ) have demonstrated that at least three cell types are involved in the t cell proliferative response to antigen, including an antigen specific-t cell, an antigen-presenting cell, and a t cell that is found in unprimed lymph nodes or spleen, which has been termed the recruitable cell. we have utilized their approach of analyzing the slope of log cell number-log response curves to examine whether tcr @ cells can function as "recruitable" cells. we found that tcr ys cells as well as tcr ap cells can function as recruitable cells in this system. these data suggest that tcr $ cells can participate in the immune response without being specific for the antigen. analysis of the membrane associated phosphoprotein profiles of b cells harvested from cultures of resting cells exposed to il- for - hrs reveals the presence of phosphoprotein with an mr in the range - , . destroy the autoradiographic signal from this phosphoprotein suggesting that it is phosphorylated upon tyrosine residues. appearance of this molecule, and lps also apparently fails to result in the presence of a kd structure in the phosphoprotein profiles. anti-il- antibody. , in the cultures prevents the appearance of the kd phosphoprotein. the genes for t n f -a and tnf-p are tandemly arranged on mouse chromosome , with only . kb separating the ' end of the tnf-p mrna from the ' end of the tnf-a mrna. yet, the two genes are independently regulated. in vitro transcription and nuclear run-on experiments indicate that the two genes are transcribed from independent promoters. in macrophages, which express tnf-a but not tnf-p, only the tnf-a promoter is active. in t lymphocytes, which can synthesize both proteins, both promoters are active. activation of either cell type results in a moderate (up to -fold) increase in the level of transcription, while mrna levels increase more than wfoid under the same conditions. interestingly, the tnf-p gene is aanscribed -fold less than the tnf-a gene in t lymphocytes, although the corresponding mrna is more abundant. these results indicate that the accumulation of both tnf-a and tnf-p mrna after cell activation and their relative steady state levels are controlled mostly at a post-transcriptional step. acanomycin d chase experiments reveal that tnf-a mrna stability in macrophages is not significantly altered after activation by lf's, and therefore that stabilization done cannot account for the observed accumulation of tnf-a mrna. in order to examine more closely which elements are required for the regulation of tnf-a and tnf-p mrna abundance, we constructed hybrid genes combining putative control regions of tnf-a and tnf-p with known constitutive control elements. results obtained from the transfection of these hybrid genes into various cell types indicate that elements located both ' and ' of the coding sequence are required for the proper regulation of tnf-a and tnf-p mrna abundance. celiac disease is characterized by small intestinal mucosal injury and malabsorption. disease is activated when a genetically susceptible host ingests wheat gliadin or similar proteins (i.e., prolamins) in rye and barley. d region specif icities -dr and -dqw . class i d-region haplotype associated with celiac disease is extended and also includes genes in the hla-dp subregion. chain gene with those encoding dr and dqw may indicate that the hla haplotype associated with celiac disease exhibits an unusual degree of linkage disequilibrium or, alternatively, that disease susceptibility involves the gene products of more than one hla locus. to characterize possible hla structural variants unique to celiac disease, the polymorphic second exons of the expressed dr, dq and dp genes were amplified from genomic dna of celiac disease patients, and their nucleotide sequences determined. our studies indicate the presence of a unique constellation of d region genes associated with the celiac haplotype, and exclude the presence of a disease specific dr, eq or dp structural gene variant in this disease. disease susceptibility is strongly associated with the hla class i we recently determined that the hla this same population of t cells contains a high frequency % ) of cells which will respond to a given allogeneic mhc protein, or to differences at two other genetic loci termed mls, in conjunction with mhc. we have transfered the a and b chain genes from a pigeon cytochrome c/el specific, alloreactive. and mis' specific murine t cell clone into an unrelated host t cell. we demonstrate that the genes encoding a single a b receptor chain pair can transfer the recogntion of self mhc molecules c m p l e x e d with fragments of antigen, allogeneic mhc molecules. and an m sc (hls- ) encoded determinant. in this case the transfer of antigen specificity and alloreactivity requires a specific a receptor chain combination, whereas mlsc reactivity can be transfered with the chain alone into a recipient expressing a randomly selected a chain. site directed mutagenesis of the ja region has also been performed in an attempt to identify sites involved in the alloreactivity of this t cell clone. in addition. we demonstrate that a single amino acid change in the v-j junction of the a b receptor can alter mhc restriction a s well a s antigen fine specificity. department of genetics, washington university school of medicine, st. louis, i( . the s tumor sublines are variants isolated from a sing parent balb/c tumor which demonstrate locus-specific shut-off of their kd, dd and l genes. four phenotypically different sublines were characterized at the dna and rna level. southern blot analysis indicated that no major chromosomal deletions have occurred, and treatment of the sublines with -azacytidine had no effect on class i expression. between loci are unlikely. none of the repressed class i antigens could be induced with interferon even though the expressed antigens were fully inducible. northern blot analysis revealed message only for the expressed antigens, showing that the repression mechanism is acting at the transcriptional level. rnase protection analysis confirmed this result and demonstrated that the transcriptional repression is exquisitely specific for the kd, dd and ld genes as other "class i-like'' messages are present in. all the cell lines. expressing class i antigens from both fusion partners, but the negative class i antigens originating from the s partner were not expressed. lymphokine gene expression was examined in a panel of short-term murine t lymphocyte clones derived by single-cell micromanipulation from allogeneic mixed leukocyte cultures. about % of clonable t cells, including both cd +cd -and cd -cdw cells, could be expanded for assay at an average of days after cloning. following stimulation with concanavalin a or anti-cd antibody, all clones secreted detectable granulocyte-macrophage colony stimulating factor (gi(-csf), interleukin- (il- ) and il- , but cd + clones on average secreted higher 'levels of each lymphokine than cd + clones. clones ( %- %) expressed detectable gm-csf, interferon-y and il- mrna and % expressed il- mrna. when the frequencies of co-expression of any pair of lymphokine mrnas vere determined, all were found to correspond to the values predicted for random assortment of the individual frequencies. for example, among il- -positive clones, also transcribed interferon-y, giving the frequency of double-positive clones expected for random association ( . % . %). expression of the four lymphokine genes therefore segregated independently among the clones and did not allow the division of t cells into subsets vith distinct patterns of lymphokine synthesis. greater than -fold in the adult liver cell line, to fold in the macrophage cell line and just slightly in l-cells. we have subcloned the region ' to the li gene which contains sequences that may be important to regulating expression of the li gene. this region includes a -mer (cctagaaacaagtga) which occurs ' to many ifn?i regulated genes. current research has been directed towards identifying and comparing proteins from nuclear extracts prepared from control and ifn- treated cells which bind to this region (- to - ). this data indicates the li molecule may be expressed in cells not known to be directly involved in the immune response. although there has been considerable interest in the recently identified gamma, delta t cell receptor, relatively little is known as to its function. during our studies of the human immune response to autologous b cell lymphomas, we generated cytotoxic t lymphocytes (ctl) specific for tumor idiotype. these ctl lysed only autologous tumor cells and none of a large panel of other autologous and allogeneic cells. inhibitable by anti-idiotypic and anti-immunoglobulin antibodies but not by a panel of classical anti-mhc antibodies. phenotypic analyses showed that these ctl were cd +, cd -, cd -, and express the delta, and presumably gamma! t cell receptor. such ctl can be used to gain new insights into the function of the gamma, delta t cell receptor and t cell recognition of immunoglobulin, and may prove clinically useful in adoptive immunotherapy. tumor lysis was for ebv-induced antigens. furthermore, lcl variant . , which does not express any hla -a, -b. or -c determinants. is killed by cultures primed to lcl-. . antibody blocking experiments suggested that this killing was mediated by t cells, and was not restricted by known class i antigens. depletion of leu positive cells from the effector population did not eliminate cytotoxicity on lcl-. . cold-target blocking studies further suggested that the class -nonexpressing lcl-. and the class i-nonexpressing lcl , share residual deterninant(s) other than hla class i or class i that can restrict cytotoxic t cell responses to ebv-induced antigens. national jewish center for immunology and respiratory medicine, denver, co it is uncertain to what extent lymphokines can be differentially produced by activated primary t cell populations. to determine if il and ifnr were differentially regulated in uncloned human t cells from adults (ad) and neonates (nt), these mrnas vere measured by in situ hybridization after maximal stimulation by ionoaycin and pma. il mrna was detected in . % of total (tl), . % of cd ', % of cd ' cd r-, and . % of cdb' ad t cells, but in none of the tl, cd +, or cd ' nt t cell populations (virtually all nt t cells were cd r'). in contrast, ipnr mrna was found in . % of tl, . % of c d ' , % of cd ' cd r-, and % of cd + ad t cells, but only . % of tl, % of c d ' , and % of cd ' nt t cells. these results agreed with other estimates of il and ifnr production based on ria of cell culture supernatants, rna blotting, and gene transcription assays. in contrast to il and ipnr, il was expressed in similar amounts by ad and nt t cell fractions, as well as the ad cd ' cd r' and cd r' subsets. thus, the capacity for increased il and ifnr production by ad t cells appears attributable, in large part, to the postnatal acquisition of the cd r' subset (putative memory t cell population). aowever, additional mechanisms exist which act transcriptionally to limit il production by both neonatal and adult t cells. such selective expression may be important for restricting the potentially pleiotropic effects of certain lymphokines t o appropriate responder cells. we observed significant inhibition (> % at ng/ml) of the presentation of wova and of ova - by the anti-ap - peptide mab's. exhibited significant inhibition. peptide mab's. after incubation with antigen +/-mab, indicate that the inhibition occurs at the level of antigen presentation. dg , was also observed for the anti-p chain peptide mab's and to a lesser extent by the anti-a chain peptide mab's. peptide sequences are capable of interfering with antigen presentation, in vitro. supported by nih grant, ai- . the ovalbumin (ova) i-ad restricted t cell hybridoma, do l. was used to the anti-i-ad mab, mkd , also much less inhibition was observed with the anti-% -experiments with glutaraldehyde fixation of the b d.p cells before or inhibition of i-ad allorecognition by the t cell hybridoma. these results indicate that mab's generated against class i rijllinghoff, institute for clinical microbiology, university of erlangen-nurnberg, erlangen, f.r.g. and the *institute for clinical immunology and rheumatology, university of erlangen-niirnberg, erlangen. f.r.g. recently we have shown that cloned l . major-specific l / t-helper cells of type (th cells), when stimulated with antigen, are able to induce polyclonal b-cell proliferation ( ). we here present evidence demonstrating that this process is dependent on a direct cellcell interaction between t-and b-cells. which in the effector phase, i.e. during stimulation of the b-cells by activated t-cells, can be mediated by a mechanism other than cognate interaction. this conclusion is derived from experiments, in which highly purified resting b-cells were polyclonally stimulated by l / t-cells triggered by an anti-t monoclonal antibody, in the absence of antigen. the triggering process was independent of the presence of the fc part of the antibody and occurred in cultures devoid of macrophages. thus, the well established cognate recognition does not appear to be the only way of b-cell induction by t-helper cells of type . studies show that a proportion of the peripheral blood cd ' t lymphocytes do not express cd or cd and are called double negative t cells. they normally have a tcr. however, another population of double negative t cells exists that expresses the a@ heterodimer. w e have purified and expanded such a population isolated from the peripheral blood of a healthy individual and studied i t s phenotypical and functional characteristics. the c e l l s are cd ' cd -cd -, positive for wt and negative for the nk markers. they express a and p mrna b u t lack ymrna. from surface iodinated cells were precipitated w i t h monoclonal pf two closely running bands ( & kd) . functional studies demonstrate that they proliferate to anticd and pha, t h i s response was blocked by cyclosporin a. there was no nk lysis b u t anticd induced l y s i s of target cells. the cells responded t o il- and il- as previously shown for other t c e l l s , b u t also t o il- , a lymphokine thought t o affect mainly stem cells and not previously shown t o stiaulate growth of mature cells. long term growth of these c e l l s was also maintained by these cytokines . roberto biassoni , silvano ferrini , rafck p. sekaly , and eric . long , laboratory of immunogenetics, national institute f allergy and infectious diseases, nih, beth-md , and istituto nazionale per la ricerca sul cancro , genova, italy. cd -cells grown in vim in the presence of il- acquire the ability to l~s e a wide variety of tumor cells in an mhc-unrestricted manner. we have previously shown that cd - clones expressed the cd epsilon gene but no functional transcript from cd gamma, cd delta, tcr alpha, tcr beta and tcr gamma genes. this result suggested that these cd - ' cells represented an early stage in t cell differentiation. to test for expression of the tcr delta gene in these cells, rna from a panel of cd - ' clones and from three highly enriched populations was hybridized with several dna fragments of the delta locus. abundant transcripts were detected with a c delta probe and a j delta probe in out of clones and in all three populations. at least four different transcripts were present with sizes similar to those found in cd ' tcr gamma-delta' cells. however, the tcr delta transcripts in cd - ' cells are most likely derived from unrearranged genes because no rearrangement could be detected in dna from an enriched population using a j delta probe, and because these aanscripts hybridized to a dna fragment corresponding to the unrearranged genomic sequence '-upstream of j delta . expression of unrearranged tcr delta genes in cd -cells provides further evidence that these cells belong to the t cell lineage. functional capabilities and by differential release of either il or il upon activation. we have produced a new monoclonal antibody to cd which has allowed us to separate normal murine cd + cells into two populations based on the density of expression of cd epitope. the separated populations seem to be analogous of subsets found in cloned t cell lines. cd + t cells with high density of cell surface cd after polyclonal activation produce il and mrna encoding ifw and il . it does not produce il or il mrna. cd low density population on the other hand transcribes mrna for il and secretes il protein. data will be presented to demonstrate that the two subsets of normal cd + cells also differ in their proliferative response to mitogenic stimuli and to exogenously added growth factors. the substitution of v to l at was the only change that could be discriminated by of allospecific ctl lines. suggesting that those ctl lines recognize a . plus a peptide whose presentation andlor binding is affected by the v to l substitution in the floor of the peptide binding site. in contrast, the l to w substitution at (but not the other substitutions) abolished the ability of the a molecule to present the viral peptide to out of peptide-specific a . -restricted ctl lines, suggesting that this substitution alters the presentation of the influenza matrix peptide but does not inhibit the ability of the peptide to bind to the a molecule. although y tcr.s have a great potential for diversity, it remains to be determined whether this potential is realized in terms of expressed y tcrs. preliminary studies in several laboratories have indicated that y tcrs expressed in earlg t h r c y t e s and adult epithelial tissues are more restricted in diversity com ared to adult tc expressin thymocytes. we have derived a panel of cloned dendritic epigrmal t cells (jetc, lines and ybridomas that express at least three types of y receptors -c , cy and c n . immunoprecipitation, northern and southern blot analyses, and sequence anazses of l gt cloned cdna or olymerase chain reaction ( k r ) amplified cdna segments have been used to anal ze in detail &e extent of diversit in the expressed y and chains and whether restricted airin o?y and chains occurs. our resu& indicate that for this panel of cloned cell lines and ! irkg is nonrandom and that variability in certain types of receptors appears to be restricted. %owever, we have observed significant chain diversity in these cells that is obtained by the use of multiple v-regions, and n-region and junctional diversity. we are investigating whether the observed y and chain pairing, and pattern of chain diversity are present in other $tcr bearing cells or whether they are only characteristic of detc. activation of ctl precursors from murine unprimed spleen cells with ril- or ril- results in distinct lytic spectra, depending on which lymphokine is present. we have used allo-stimulation in limiting dilution analysis with subsequent testing on an allo-specific target (a ) and an mhcdeficient, non-specific target (rle). in the presence of ril- exclusively allo-specific ctl are generated, while ril- supports a proximately equal numbers of precursors that k~ll a and rie targets. dose response analysis of ril- -supported killing activity indicates that the lytic spectrum is independent of the amount of ril- used, and therefore this il- effect is intrinsic in its activity on unprimed spleen cells. mixing experiments indicate that ril- can partially override the effect of il- on the generation of non-specific killer cells. split well analysis and cold target inhibition experiments are in rogress to ascertain the actual proportion of specific killer cells which can be generated with ril- . be. are also testing the ability of cofactors, such as il- and il- , to optimize the response of il- generated ctl. we conclude that il- , not il- , must be used when ctl are generated from unprimed spleen cells in mice. t r a n s c r i p t s i n y/ tcr populations. i n t e r e s t i n g l y , these same v genes, as well as a further+crosshybridizing v gene previously designated va . , are expressed by peripheral a& tcr c e l l s as . kb tcra transcripts. these data suggest t h a t b a -dn th represent a developmentally unique subset i n which both v and vg segments are non-randomly expressed. furthermore they i n d i c a t e t h a t there i s considerable overlap between the v a and v gene repertoires . indianapolis, in in order to detect the small amounts of lymphokines generated in vivo following antigen stimulation, we developed a co-culture system which allows for detection of il- / , il- /csf and tnf from ln cells stimulated in vivo with picryl chloride (pcl). utilizing thb system in combination with facs analysis and receptor binding studies, we examined the production of these lymphokines in primary and secondary immune responses. during a primary immune response, the production of il- was not readily detectable on dl, peaked on d and was gone by d . at no time were we able to demonstrate the presence of il- . alternatively, the presence of il- /csf and tnf was w i l y detected on dl, but olso peaked on d . in comparison to primary responses, secondary immunization lead to at least two alteraticns. (i) peak production of all lymphoki es shifted towards dl. ( ) although most lymphokines did not demonstrate increasea in the amount produced/lo cells, the amount of lymphokine generated/ln was vastly increased due to an increased number of cells. utilizing single and dual color facs analysis we also examined the ln cells for alterations in t cell subpopulations. during the course of the primary response: (i) the percentage of thy i+ and l t + cells decreased until d and then began to recover, ( ) the percentage of thy-i+, t -,t -cells peaked at the time of greatest lymphokine production (i.e.-d ) and ( ) the il- receptor was expressed solely on thy-i+ cells, was detected on both t + and t + subsets and peaked on d . most of these alterations also occurred during the secondary response, but their timecoune was shifted so that maximal effects occurred earlier (e.g., dl). finally. the maximal binding of radiolabeled il- by the ln cells following both primary and secondary sensitization correlated with the expression of the il-zr as detected by facs analysis. in addition, binding of radiolabeled il- demonstrated similar patterns except for the detection of significant binding on dl. these results demonstrate that (i) an ordered timecourse of lymphokine production occurs in vivo following exposure to antigen and ( ) the secondary immune response to pcl is characterrd by an accelerated tempo of lymphokine production, rather than an increased level of lymphokine production/lo cells. activation as direct g-protein activation by a f pi-hydrolysis using phorbol diester stimulation of pkc restores the inhibi$$ble phenotype and the ability to upregulate c-fos. even more interesting, sig-linked ca responses by vs . -c . are equivalent to those observed in the wildtype wehi- . resul$g suggest that contrary to current thought, sig-generated signals may not be coupled to ca fluxes via inositol phospholipid hydrolysis. thus, vs . -c . is a new and powerful tool with which to analyze signalling through sig at the molecular level. unlike the wildtype, crosslinking of sigm on vs . -c . did the signaling defect in vs . -cl. -appears to be proximal to phospholipase c triggers pi-hydrolysis and bypassing these latter lmmunogenicity c analysis of t cell receptor chains from adult cd -,cd -thymocytes mark w. moore, i. nicholas crispe and michael j. bevan, department of immunology, research institute of scripps clinic, la jolla, ca . the role of tcr genes in t cell development has not been determined. to extend out understanding of the repertoire of tcr expression, we prepared a cdna library from cd -,cd -adult balb/c thymocytes and cloned and sequenced tcrq genes from this cdna library. we found that clones were transcripts of the unrearranged c , gene and that clones terminated in the j , region. nine of the remaining clones were v , -j c genes and five of these were in frame. only one clone corresponded to c and was v -jy c jofned'in frame. sds-page analysis of the -chain proteins from the surface of both balb/c anzcdbl) adult cd -,cd -thymocytes did not detect the , mw vy c protein, but did detect the , mw v c protein. these results suggest that despite the abundanc$%f pull-length, functionally joined, v c transchpts in the thymocyte subset, the protein product is not expressed on the cell surface as the prehfcted , mw protein. finally, our analysis of the v-j jointing of the genes reveals both flexibility at the v-j junction and extensive n-region nucleotide addition that lead to diversity of the predicted protein sequence. il in response to the same stimuli. e identification of these two subsets of cd ' helper cells is mostly based on studies performed with long-term cultured t cell lines and it is not clear whether these two subsets exist in vivo and represent distinct lineages of t cells. in particular, the frequency, tissue distribution and ontogeny of cells capable of secreting il in vivo is not known. these studies have been hampered by the fact that freshly isolated t cells from unprimed animals failed to secrete detectable amounts of ila and il when stimulated in vitro by lectins or alloantigens, whereas iu is readily detectable in these same cultures. data presented here indicate that freshly isolated t cells from unprimed animals can be induced to produce il in a receptor-de endent, antigen-independent manner upon stimulation by anti-cd antibodies. our results also stow that only cd ' and not cdst cells can be induced to secrete il and that cross-linking of the receptor is required for o timal activity. we believe that this approach will be useful in identifying in vivo cells recomittefto the th pathway and study their ontogeny, activation requirements and tissue distriiution. hlb, brussels, belgium. we have studied the murine tcr repertoire against the c-terminus of cytochrome c in association with certain alleles of the mhc class i molecule, eakepk (iek) and eakepb (ieb). for mice possessing these alleles, the majority of responsive t cells utilize one member of the variable val gene family in conjunction with a limited set of vp genes. as an extension of these studies, we have examined ie specific, alloreactive hybridomas derived from ie non-expressing (eab) cytochrome c non-responder mice to determine their usage of va and vp genes. tion assay showed that fourteen utilized the same val gene segment used by the majority of cytochrome c specific, ie restricted t cells and eight utilized a closely related val gene that also is associated with this antigen response. element most commonly used by cytochrome c-specific t cells was not found among the alloreactive hybridomas tested, @ genes less frequently used in the cytochrome response were expressed by seven of the alloreactive hybridomas whose va segments were defined by rnase protection. determining recognition of ie molecules both in mhc-restricted, antigen specific immune responses and in alloreactive responses. the t-helper cells of seven mouse strains, representing class i haplotypes (ias, ia , iab, iakiek, iadied) were responsive to immunization and restimulation with parent peptide. the ied determinant was shown to be a presenting element by monoclonal antibody blocking and by use of l-cell-transfectants as af'cs to purified t cells and to t cell hybridomas. a series of overlapping synthetic peptides identified two minimal t-cell sites within the parent peptide: mice expressing ia and ie responded to a fragment at the n-terminus of the parent peptide (site ) while mice expressing only ia responded to a distinct but overlapping fragment at the c-terminus (site ) . these minimal sites identified in vitro could be used to immunize mice in vivo in an mhc-restricted manner. the human tcr locus is strategically located within the atcr complex between the cluster of va/v region and the ja segments. which can be spliced to ca in pre t cells, separates from the ja segments. pulse field gel mapping as we as molecular cloning link diversity (ds), j g , c, and tea within kb. considerable tcr diversity is generated despite the predominant use of one v and j segment. d and d are and bp long, are frequently recombine as d, /d ? and reveal exonucleolytic trimning with extensive "n" segment addition. specialized ' and ' deleting elements, rec and p j a , separate the locus from the a locus. cells with rec/$b ja recombinations comprise most deletion events although rec recombines with other major acceptor sites in fetal and post-neonatal thymic dna. the ' deleting element ( rec) is evolutionarily conserved in the mouse and functional comparisons are underway. delete the locus may prove to be the pivotal event establishing separate y and ae lineages. to study the mechanism of t-cell tolerance, transgenic mice were generated that expressed the mlsa reactive t-cell receptor (tcr) o-chain vb on - % of peripheral t-cells. in transgenic mice bearing mlsd, the numbers of high tcr expressing thymocytes and of thy . + peripheral t-cells were reduced. the cd /cd ratio of peripheral t-cells was decreased fourfold compared to negative littermates. both mlsa and mlsb tcr &transgenic mice were able to mount a t-cell dependent antibody response against viral antigens whereas the capacity to generate alloreactive and virusspecific cytotoxic t-cells was impaired in tcr &transgenic mlsa, but not in transgenic mlsb mice. rna analysis and immunof luorescence with tcr vb-specific mab further revealed, that the expression of endogenous tcr -genes in these mice was suppressed. tolerogen-reactive lymphocytes, as measured in the mlr, in spite of their long-term acceptance of a skin graft bearing the tolerated antigens. lymphokine production by mlr+ tolerant lymphocytes is different from that of syngeneic normal lymphocytes. normal lymphocytes produce only il- in primary response to tolerogen, while tolerant lymphocytes produce il- and il- . using limiting dilution analysis, we have to estimated the frequencies of pil- and pil- (precursor) cells in these cultures. after primary k vitro stimulation, normal responders have a low but measurable frequency of pil- cells, while tolerant responders have a much higher pil- frequency. however, following subsequent & restimulations, the pil- frequency of normal responders rises and begins to approach that of the tolerant responders, such that the two populations are indistinguishable based on pil- frequencies following the third round of in vitro stimulation. these data suggest that the high frequency of il- producers (presumably t,, cells) among the tolerant lymphocytes resembles unexpectedly a "primed" state, rather than "unprimed"as in nontolerant responders (where th , dominate the early response). the existence of "primed" t cells in phenotypically tolerant animals raises the possibility that precocious activation of tr (by neonatal exposure to tolerogen?) suppresses the later emergence of t,,, which would be expected to contain the cells responsible for graft rejection. a large number of cd + t-cell clones, obtained from peripheral blood t lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human cd + t-cell subset. six out of cd + clones were able to lyse daudi or p cells in the presence of anti-cd antibodies. the remaining cd + tcell clones tested did not acquire this cytotoxic capacity during a culture period of weeks. in the absence of anti-cd mab, no lytic activity against daudi. p and k target cells was observed under normal culture conditions. these two types of cd + t cells showed high reactivity with anti-cdw ( b ) mab and no reactivity with anti-cd r ( ) mab. the cd + clones without anti-cd mediated cytotoxic activities (th ) consistently showed a higher expression level of cd antigens. th cd + clones did produce il- , ifngamma and tnf-alpha.beta. whereas the th t-cell clones produced minimal amounts of il- . ifn-gamma and tnp-alpha. beta in response to anti-cd mab and pma. not all cd + clones did release il- , but there was no correlation with cytotoxic activity. moreover, as compared to the th cd + clones, tr cd + clones proliferated moderately in response to anti-cd mab. however, anti-cd mab induced proliferation of only the th cd + t-cell clones was enhanced by anti-cd mab. both cd + subsets provided help for polyclonal b-cell activation with anti-cd mab. our data suggest that the human cd + subset, in analogy to the murine system, comprises two functionally distinct t-cell subpopulations. in the mouse, when la antigens are isolated immunochemically, the predominant species isolated are the isotypic matched pairs, aaap and eaep. however, when la ap dimer expression is studied using an l cell transfection model, it is found that the isotype-mismatched dimer apdea is readily expressed at the cell surface. these results suggest that differences in assembly andl or transport of different la pairs may be most readily visualized in a competitive environment where multiple distinct la chains are available. to investigate this possibility, the relative efficiency of inter-and intra-isotypic dimer formation and expression was evaluated using a sequential l cell transfection system. l cells already expressing an ap dimer on the cell surface (apdea or apdaad) were supertransfected with a third la gene (aad or ea, respectively). synthesis of this second a or p protein led to competition for the unique partner chain. individual clones were scored for cell surface expression of the distinct dimers (e.g., apdea vs epdea or apdaad vs apdea) using facs analysis with chain specific monoclonal antibodies. in addition, each species of mrna was quantitated by northern blot hybridization using bcus specific probes. our results indicate that in the h- d haplotype. isotype-matched dimers are expressed with - x the efficiency of isotype-mismatched dimers. this result suggests that, regardless of the cell type studied, if each of the four murine la genes is expressed at equivalent levels, intraisotypic dimers will be expressed to the virtual exclusion of the interisotypic dimers. however, if chain synthesis asymmetry occurs, the isotype mismatched pairs may be expressed at immunologically relevant levels. differential we have identified a series of discrete stages among the cd -double negatives which seem to form a sequence, with tcr gene rearrangement and rna expression gradually progressing, but with potential for expansion and repopulation of irradiated thymuses diminishing along the series. on this pathway cd must be expressed late or after the acquisition of cd and cd . cell cycle analysis shows the highest rates of cell division to be among the rsa+ il- r-pgp- -population which probably precedes the transition to cd +cd + and tcr expression. thus it seems unlikely that tcr/antigen interactions play a role in cellular events occurring among the double negative cells which lead on to mainstream t-cell development. egr-l is a murine early growth factor inducible gene which encodes a protein with zinc fingers. its expression was investigated in murine b-lymphocytes stimulated through their antigen receptor (sig) with anti-recptor antibodies (anti-ig) . rapid (by minutes) upregulatlon of egr-l mrna expression was observed at doses of anti-ig sufficient to drive the majority of go cells into cell cycle. agonists and inhibitors of protein kinase c (pkc) showed that expression was coupled to the pkc component of receptor immunoglobulin transmembrane signalling. interestingly, signalling through sig on the murine b lymphoma wehi- did not upregulate egr-l expression even though similar signalling pathways are associated with this receptor in these cells. southern analysis showed that egr-l is not deleted or translocated in this cell line. importantly, cell growth and proliferation of wehi- is inhibited by anti-ig stimulation suggesting a relationship for egr-l expression and differential processing of receptor ig signals. this notion is further supported by the finding that murine b lymphomas whose proliferation is not inhibited by anti-ig showed receptor immunoglobulin coupled egr-l expression. reeulation of exdression of a class wc transeene. dinah s . the expression of the transgene product. the patterns of expression of the transgene parallels that observed in situ, indicating that regulatory elements necessary for normal patterns of expression are contained within the injected kb dna segment, and that trans acting factors involved in its regulation function between species. included among these elements are those specifying preferential expression in b cells relative to t cells. in vivo treatment of transgenic mice with a/@-interferon results in increased expression of the transgene in a number of tissues. the response parallels that observed for the endogenous h-zkb, but differs markedly from qa- . analysis of the chromatin structure of the transgene reveals a single constitutive dnase i hypersensitive site present in both spleen and thymus, which is not altered by interferon. both a novel negative and positive regulatory elements have been identified in the 'flanking region of the transgene. the negative regulatory element reduced the activity of both the homologous class i promoter and a heterologous viral promoter. in vivo competition experiments indicated that the functions of the positive and negative elements are mediated by distinct cellular trans-acting factors. the negative regulatory element requires the presence of a positive regulatory element to function. this interaction between elements represents a novel mechanism for regulating gene expression. mcdevitt. department of microbiology and immunology, stanford university school of medicine, stanford, ca . published data show that encephalitogenic h-zu murine t cell clones with specificity for the n-terminal eleven amino acid peptide of myelin basic protein display a restricted fine specificity when tested on substituted analogs of the native peptide. for example, substitution of alanine at certain positions in the peptide totally abolishes the response of each clone (acha-orbea et al.. . cell : ) . recent experiments also have shown that the ability of some peptide analogs to bind to h-zu i-a gene products does not always correlate with their ability to stimulate the t cell clones (see accompanying abstract by david c. wraith and hugh . mcdevitt). this suggests that h- u mice may lack a t cell repertoire capable of recognizing these peptides complexed to h- u i-a gene products. to test this possibility, h- u mice were immunized with a panel of peptide analogs, as well as the native peptide. the in vitro t cell proliferative response to each of the peptides then was measured. the results show that in vivo immunogenicity of the peptide analogs also does not strictly correlate with their capacity to stimulate the t cell clones. in this way, the polyclonal t cell repertotre of h-zu mice for the myelin basic protein peptide analogs was examined, and could be compared with the i-a binding characteristics of the peptides. terms of antigen and mhc recognition. this response involves a limited repertoire of t cells which crossreact on species variants of the antigen. in addition, t cells specific for the antigen in association with syngeneic mhc can recognize antigen on similar allogeneic mhc molecules. the groupin of clones by functional phenotypes defined by these crossreactivities allowed us to corrcfate tcr gene usage with either antigen or mhc recognition. some of the pigeon cytochrome c-specific clones within one functional phenotype use receptors that differ by as few as two amino acid residues. other clones e y s s very different tcrs but exhibit similarities in antigen/mhc reco nition. the efect of these tcr differences on recognition was assessed using a pane? of anti en analogs with single amino acid substitutions presented on different mhc molecufes. each clone exhibited a unique pattern of res onse to the antigen analog panel, even clones with very similar receptors. also, eace residue in the antigenic region of the peptide was critical for interaction with at least one t cell receptor. therefore, the antigen must either be a linear molecule with each residue available to interact with the tcr or be able to assume several conformations to interact with mhc and the tcr. lmmunogenicity c thy- + cd + ly- (b )+ cd -cd -tcrx- ' helper cells. anne i. we have found that these cells can be preferentially stimulated to proliferate when cocultured with the b lymphoma, ch . one to % of nylon wool non-adherent, ia-, jlld-, and cd -lymph node cells from normal unimmunized mice have the phenotype thy-l', cd ', cd -. and cd -. these cells proliferate when co-cultured with a syngeneic surface ig' lymphoma, ch , even in the absence of any added antigen, mitogen, or fetal calf serum. prior to stimulation we find that approximately % of thy . ' cd ' cd -cd -express the marker ly- (b ), however after culture with ch the majority of cells with this phenotype express the marker ly- (b ). after ch dependent proliferation the ly- (b )' t cells are able to provide help for secretion of ig by fresh ch b cells. surface labelling and precipitation of t cell receptor molecules reveals that most of the thy- ' cd ' ly- (b )* cd -cd -cells express tcr(r- ). furthermore, cd precipitation shows that as many as four different - heterodimers are utilized within the entire responding population. this suggests that a heterogeneous population of double negative tcri- cells are involved in the response to ch . college of kedicine at east tennessee state university, johnson city, tn interferon-producing (t ) and interleukin (il ) producing (t ) clones were assayed for their ability to diregtly induce cytostatic activity in macro:hages generated from splenic myeloid precursors (m -c). in the presence, but not in the absence, of antigen, t clones activated the m -c to inhibit the growth of p tumor cells in vitro. th cjlones were not able to activate such effector activity in the i -c. effectively present antigen to the t clones as evidenced by the proliferation of t cells cultured with antigen in the pfesence, but not in the absence, of m -c. thereyore, although both t and t were activated by cognate interaction with antigen presenting (ba) or nippostrongylus brasiliensis (nb). spleen cells from these mice were cloned at limiting dilution with alloantigen stimulation, and every two weeks, lk production in response to con a was measured. clones derived from, and stimulated with, cells from unimmunized mice initially tended to secrete low lk levels, with few clearly defined th or th clones. by days after cloning, some clones had acquired th or th patterns. cfa, ba and nb-imnunized mice gave rise to clones that were mostly th or th even at early times. cfa and ba immunizations induced almost exclusively th clones, whereas nb induced more th clones. these results are consistent with a model in which resting, previously unstimulated t cells produce low amounts of lks, and progress through stage(s) where they secrete both th and th lks before finally differentiating into th and th cells. the results with cfa, ba and nb-primed mice suggest that this process occurs in vivo as well as in vitro. strains as carriers of melioidosis antigens to the immune system, deja tanphaichitra, mahidol university, p.o. box - , bangkok , thailand the attenuated gale mutant, salmonella typhi strain, tyzla, served as the recipient in a conjugal dna transfer experiment. conjugal dna transfer was obtained by the mating procedure on an appropriate blood agar medium. were examined serologically. one selected strain was found to have the serological characteristics of the recipient s . typhi, tyfla strain and also expressed the pseudomonas the donor strain was a pseudomonas pseudomallei mu . the resulting antigen clones were repurified by restreaking on the medium and pseudomallei antigen. the s. typhi transconjugant strain is due to the presence of the pseudomonas pseudomallei plasmid. a group of subjects when received four doses of this bivalent vaccine strain in this study it appears that pseudomonas pseudomallei synthesis in developed antibodies against pseudomonas pseudomallei up to %. pseudomallei, an intracellular pathogen, produces a characteristic antigen probably to be plasmid coded, we considered that the gale salmonella typhi tyzla oral vaccine strain, highly effective against typhoid fever, might be modified so as to be protective also against melioidosis due to pseudomonas pseudomallei. terminal deoxynucleotidyl transferase (tdt) is a lymphoid-specific nuclear enzyme present in early lymphocytes. to investigate the regulation of tdt gene expression, pre-b and pre-t cells were treated with phorbol -myristate -acetate (pma) o r three analogs, and tdt steady-state mrna levels were determined by northern blot analysis. treatment of early lymphocytes with pma results in a rapid and reversible decline in steady-state tdt mrna levels within six hours. this rapid decline can be blocked by pretreatment of the cells with a protein kinase c inhibitor, implicating protein kinase c activation in the decline of tdt mrna. nuclear run-off studies demonstrate that tdt transcription is rapidly down-regulated within minutes after pma treatment, indicating that this regulation occurs mainly at the level of transcription. furthermore, cycloheximide blocks the decline in tdt in rna showing that new protein synthesis is required for transcriptional inactivation. the nucleoprotein gene from the influenza virus a/nt/ / was stably cloned into the attenuated aroa-strain of salmonella typhimurium sl . nucleoprotein purified from pnp - was tested for the ability to generate virus-specific immunity. immunization with recombinant derived nucleoprotein induged immunity to all type a influenza tested but not against type b viruses. cd helper t cells were primed but no evidence was found for priming of class i restricted ctc. mice immunized with recombinant nucleoprotein were protected against a subsequent challenge of influenza virus. the information obtained from the study of the immunity and protection generated by the purified recombinant protein was then used to design experiments to investigate the possibility of using the attenuated salmonella vector to deliver the nucleoprotein molecule to the immune system by the parenteral or enteral routes. we characterized the extrachrom- circular i n i s in -day-fetal and -week-old m u r i n e thpmcytes and -week-old m u r i n e splenocytes. f popllation of circular chias was clone into the kgtll phase vector. we screened ca. tna cl-by plaque hybridizations with all far kirds of tcr gene probes derived from jal , val , db , db , jyl , j and loci. cut of , cna cl-from fetal and -week-ld thymocytes, hybridized with tcr aprobes and hybridized with tcr &probes. positive cl-with tcr yand probes were to in fetal thymocyte erived library, but few in -week-old thymocyte. of fetal tcr clcnes analyzed, cl-had dd or vd reciprocal joints and clcne had vd ar dd d i n g joint. relative frequencies of circular dna clones for four different tcr genes are consistent with the order of the expression of the genes the t cell developnent. of , tna cl- signalling could be studied. llzmambxane signalling was maasured by ability to t?z nslocate fkc frcrm the cytcplasa to the nw leus after surface i-a was banrl by a or p dxdn specific monoclcnal antibody. i(pmwing either or amino rids fmn the a chain cvtoplasnic (cy) damin did not affect the ability of tkse i-a r m l d e s to trarslocate pkc to the nucleus. normal splenic b c e l l s were rendered non-responsive t o subsequent challenge w i t h lps, as measured by a decreased a b i l i t y t o generate antibody forming c e l l s (afc), by incubation overnight ( - hours) w i t h ug/ml a n t i -i g . both i n t a c t and f(ab)', a n t i -i g , as well as monoclonal anti-igm (bet and b- - ) , were able t o induce c e l l non-responsiveness t o subsequent lps challenge, suggesting t h a t sig/fcr i n t e r a c t i o n s are not necessary i n the induction o f lps non-responsiveness. i n contrast, induction o f nonresponsiveness t o subsequent challenge w i t h fitc-prucella abortus required i n t a c t a n t i -i g . the a b i l i t y o f mitogenic a n t i -i g (rab f(ab)', o r - - northern blot analysis and bioassay data were used to analyze separate lymphokines as well as the il- receptor (murine tac). northern blot comparison of fresh and primedt enriched rna revealed that primed t cells produced -fold more lymphokine than the fresh t cells. the only lymphokine that showed equal amounts of mrna for both fresh and primed t cells was il- . a time course of fresh and primed t + cell lymphokine production was also analyzed. the primed cells produced a short burst of lymphokine mrna that peaked between . and hr after con a stimulation and declined after hr. the fresh t cells produced a longer burst of lymphokine mrna that peaked - hr after stimulation. the il- receptor @- r) mrna time course from activated primed cells showed different kinetics than lymphokine mrna. this suggested that molecular regulation of the il- r might be different than lymphokine regulation. to further examine molecular regulation in the primed t cells polysome profiles were evaluated for lymphokines, l r , and other cellular genes. the recently developed method of gene amplification by the polymerase chain reaction (pcr) has proven to be particularly suited for the analysis of t cell receptor (tcr) genes. we adopted existing methods for the preparation of cytoplasmic rna from as little as cells and used this material as template for first strand c-dna synthesis. pcr amplification of this c-dna, using v-and c-specific oligonucleotide primers yielded enough material to produce single-stranded dna in a second pcr which could then be sequenced without cloning. in case of unknown v-usage, the pcr was employed for screening for v-beta elements by sequential reactions with different v-beta specific primers. we have used this method to reinvestigate the h- b restricted cytotoxic t cell response to tnp in c b mice. beta chain sequences of ctl clones obtained by direct cloning of immune spleen cells were compared to sequences of clones obtained by cloning of individual short-term in vitro ctl lines. it was found that a) in vitro bulk-stimulations reduced the heterogeneity of the beta-chain responses to tnp, b) similarities between different tcr-beta-chains concentrated on the usage of certain jb-elements ( jb . , . , . ) rather than v-region or nid-region sequences, and c) the majority of jb . containing beta-chains was associated with alpha-chains expressing v-segments of the val family. these expression of genes which encode the t cell antigen receptor is cenval to the generation of the t cell repemire. our labomtory has been investigating genes for both the alpha and beta chains of this receptor in inbred strains of runus norvqicus (the laboratory rat), a species in which several autoimmune disease models have been developed. and which is used extensively in transplantation studies. using genomic southern blots and mouse probes specific for five different v a subfamilies, we have estimated the size of the v a repertoire in ten inbred strains of rat. results show a significant increase in the size of one subfamily and suggest increases in two others in all ten strains. the rat v a l subfamily has about twice as many members as the mouse, while the va and vu subfamilies, depending upon the enzyme used, show a similiar duplication. the va and va subfamilies have a comparable number of members in both species. these data are most easily explained by a single duplication event in the rat invoking at least one and perhaps three subfamilies, but not encompassing the entire v a locus. this implies that the val subfamily (perhaps together with va and va subfamilies) is regionally clustered and not interspersed with either the va or va subfamily. based on restriction fragment length polymorphisms, we find evidence for six distinct v a haplotypes in the ren strains tested. we have also cloned eight unique germline v a l gene segments. one of these has been sequenced. and has a coding region % identical to the most closely related mouse v a l sequence. this degree of relatedness is similiar to ra#nouse vg homologues. which share % nucleotide sequence similarity. we are using these clones to generate angle copy probes from flankiig regions to further map the v a l locus. current approaches to mhc-peptide binding studies require either large quantities of highly purified mhc protein and/or the use of sophisticated detection apparatus. i n order to simplify detection of peptide-mhc interactions we have investigated the use of photosensitive-crosslinkers. two reagents have been successfully tested. a benzophenone derivative of peptide - from rat myelin basic protein (rmbp) was only effective after the introduction of a glycine spacer residue between peptide and crosslinker. an azido-nitro-benzoyl derivative of peptide . , a heteroclitic analog of rmbp - ( ). had a high affinity and bound specifically to the peptide binding site. the . photoaffinity probe has been used to test the binding properties of other analogues of rmbp - and is currently being used to define (a) the kinetics, (b) ph and (c) temperature dependence of the binding event. this particular photoaffinity conjugate retains both the mhc binding and biological properties of the original peptide and is helping us to define the roles of "determinant" versus "t cell repertoire" selection in the mhc linked autoimmune response to mbp the antigen-specific t cell repertoire is diverse in its ability to recognize a wide universe of foreign antigens. this t cell repertoire is composed of a set of clones each of which is specific for a given foreign antigen. therefore the precursor frequency of t cells specific for any give foreign antigen is extremely low. however, two prominent exceptions to this general rule exist, and these are the t cells present at high precursor frequency which are specific for foreign hhc products or for the products of the minor lymphocyte stimulatory (mls) genes in the mouse. the present studies were undertaken in order to examine factors involved in t cell repertoire formation by assessing the relationship between t cell repertoire for conventional foreign antigens and for mls products. studies indicate a striking degree of overlap between the set of t cells specific for pigeon cytochrome c and the set of t cells specific for mlsc gene products. demonstrate that the basis for this overlap lies in the predominant expression of one tcr vp gene, vbs, by those t cells which recognize mlsc. involvement of specific tcr afl dimers in recognition of mlsc and further suggest that t cell reactivity to these gene products may play an important role in establishing the t cell repertoire for foreign antigens. conclude that, rather than destruction of some essential apc structure, ecdi fixation prevents the apc from actively responding during the encounter with the t cell. this results in a failure to express new structures (probably located on the apc plasma membrane) that appear to be essential for stimulating t cell proliferation. these structures are distinct from ia or il . the induction of these structures during t-apc interaction occurs in six hours, requires protein synthesis, and can be elicited by il , il or lps, but not ifn-gamma. in the absence of these induced structures, the apc stimulates a partial t cell response, il release, but the t cells fail to proliferate. these induced structures on the apc may be either adhesion molecules that stabilize the t-apc interaction, or they may provide additional stimuli to the t cell. were not c m n t o the three s t r a i n s o f mice (balb/c, regions , , , and ; c h/he, regions , , , , ', and '; and c bl/ , regions , , , and ). immunisation with type i collagen (cii) leads to development of arthritis in mice with certain mhc haplotypes and is associated with an immune response against cii. we have been studying the t-cell response in the arthritis susceptible strain dbm (h- q) . analysing the proliferative response in cultures of lymph node cells from immunised mice a s well as t-cell lines and clones established from such cultures it was found t h a t li the t-cell response after immunisation with heterologous cii was preferentially directed against foreign determinants on the cii molecule with little o r no crossreactivity against autologous cii. ' both the primary response and the reactivity of established lines and clones were directed against the cbll fragment of the cii molecule, using c b l l fragments prepared from chick, bovine or rat cii. / pepsin present in cii preparations after using pepsin digestion for solubilisation of the collagen is strongly immunogenic even in very small amounts and it was therefore necessary to use cii prepared from lathyritic cartilage without pepsin digestion for immunisation. in contrast to the pattern in lymph node cultures from immunised mice we found that when culturing spleen cells from unimmunised mice there was a t-cell response against collagen that was preferentially directed against autologus cii. since we earlier have found that autologus cii may induce an immune response and also arthritis in dbn mice we conclude that there exist t-cells capable of reacting with autologus collagen and inducing an immune response as well as arthritis but that these cells are under regulation so that they not readily can be activated into proliferation but may be induced to perform certain effector functions. tested. the characterization of these two cd rdsas will be presented. analysis of hla polymorphism using sequence specific oligonucleotide probe hybridization to amplified dna, lee ann baxter-lowe, jay b . hunter, and jack gorski, the blood center of southeastern wisconsin, milwaukee, wisconsin . hla polymorphism plays a key role in antigen:mhc interaction. the polymorphism of the first domain encoding exon of the hla-dr p chain has been studied by in vitro dna amplification and use of sequence specific oligonucleotide probe hybridization (ssoph) to detect polymorphic sequences. a bp segment of genomic dna was amplified and hybridized with synthetic oligonucleotide probes ( - bases) under conditions that detect single base pair mismatches. identification of these mismatches can be used to predict micropolymorphism in the protein products, including single amino acid changes. haplotype specific patterns of oligonucleotide probe hybridization were defined for a panel of homozygous typing cells. analysis of family data demonstrated the expected inheritance patterns. most known serological specificities are encoded by multiple allelic forms of dr p chains and ssoph can identify these differences. this was exemplified by detection of unique ssoph profiles for subtypes of dr , drw and drw alleles. this procedure was also used for analysis of hla-dr polymorphism in large numbers of heterozygous individuals, including an hla-deficient scid patient. the ssoph data were correlated with serological specificities and will be useful for delineation of hla restriction in alloand autoimmunity. different cell membrane receptors have been shown to be involved in human t lymphocyte activation induced by either monoclonal antibodies or mitogenic lectins. these t cell surface molecules can be divided into two categories : a) the t cell antigen receptor (tcr) associated with the non-polymorphic cd antigen b) t cell differentiation molecules not linked to cd /ti such as cd (t ) and tp ( . ). monoclonal antibodies directed against these t cell surface structures triggered different t lymphocytes functions : mitogenesis, il- receptor expression, il- secretion. our knowledge about early events involved in t cell membrane activation is not complete, especially involving the transduction mechanism mediated by gtp-binding proteins ; nevertheless, numerous authors have demonstrated that cd /ti complex triggering induces the activation of phospholipase c, leading to the phosphoinositide cascade associated with an increase of free cytoplasmic calcium ions. in the present report, we show that different activating cell molecules (con a , pha and pma) can trigger oxygen free radical liberation when incubated with the human jurkat tumor t cell line. since membrane oxidative metabolism has been shown to be related to the stimulation of the phospholipase a , and to be the final consequence of a membrane nadphoxidase : this could represent a previously undescribed pathway of t lymphocyte activation. high affinity monoclonal antibodies (mab), specific for staphylococcal nuclease (nase), were produced and characterized. competitive inhibition assays were conducted resulting in a series of complementation groups that define eight overlapping epitopes. it is estimated that these epitopes account for % or more of the accessible surface of nase. mutagenesis of the coding sequences for nase was carried out to produce a series of variant molecules (each differing from wild-type. nase and from each other by a single amino acid) that will enable mapping of nase epitopes, determination of residues involved in antibody binding, and the contribution of various physical and chemical factors to affinity and fine specificity. screening some of these mutants with the panel of mab enabled us to map several nonoverlapping epitopes and further subdivided some of the mab complementation groups. oligonucleotide-directed mismatch mutagenesis has been done on codons encoding the original amino acid residue and other surface residues in its immediate vicinity. determination of enzyme activity and structural analysis by cd spectropolarimetry of several of the mutant proteins suggests that any structural changes that may occur are local and not global. supported by grants ai , l ca and s rr from the national institutes of health. activation of t cell proliferation is believed to occur via the hydrolysis of inositol phos holipids, which, through the second messengers inositol- , , -tris hosphate and diacylglycerof(dag), promotes the elevation of intracellular calcium levels anjactivation of protein kinase c (pkc), respectively. the role of pkc in t cell activation was investigated by comparing the effects of stimulation by - -tetradecanoyl phorbol acetate (tpa), and the dag, oleoylacetyl glycerol (oag), on a > % pure population of t cells cultured in rpmi medium containing % autologous serum. treatment with either tpa or oag caused down-regulation of the t cell rece tor, a consequence of its hosphorylation, but only tpa, in syner leuiin receptor (il -r), expression and, sgsequently, proliferation. immunohistochemical staining with antisera specific for the pkc subspecies a, pi, pii and shows that restin t cells express a, pi and pii pkc subspecies which are diffusely distributed throughout the celt. after minutes treatment with either oag or tpa all three subspecies are redistributed to a focal area within the cell. the redistribution is transient in oag stimulated cells, where the pkc distribution is similar to that in untreated cells after hour of treatment. in tpa stimulated cells, however, the pkc redistribution is prolonged, becoming more marked until mitosis occurs after - hours of treatment. these results suggest that transient intracellular redistribution ofpkc causes phosphorylation and down-regulation of the t cell receptor, but that prolonged redistribution is required or t cell proliferation. sm is a nucleoprotein complex associated with small rna molecules in eukaryotic cells. the spontaneous generation of anti-sm antibodies is specific for patients with systemic lupus erythematosus (sle) and develops in % of mrl mice. the response has been shown to be t-cell dependent in mrl/lpr mice. t-cells specific for sm are found only in mrl (h- k) mice and mice bearing h- s and h- f haplotypes (which do not develop anti-sm antibodies). we are currently working to define the variable regions of the t-cell receptor genes used in the sm response. a series of t cell hybridomas from mrl mice has been generated and are being screened for sm positivity. a technique has been designed to amplify specific alpha and beta chain tcr genes using the polymerase chain reaction allowing for a more rapid sequence analysis. it is also our intention to locate the sm specific epitopes of the t-cell hybridomas. d. bloom, p.l. cohen, and s.h. clarke, department medical institute and experimental immunology branch, nci. nih, bethesda. md . to determine whether prior activation history affects t cell receptor mediated activation of t cell clones, the murine type i helper clone ae was maintained in tissue culture by stimulation every ten days with either ( ) antigen (cytochrome c), irradiated h-zk spleen cells. and il- or ( ) il- alone. ae cells grown with antigen and antigen presenting cells (ae -ag) proliferated and produced t cell growth factor activity (tcgf) in its culture supernatants following stimulation with immobilized anti-t antibody. the tcgf activity was shown by bioassay using indicator cell lines and specific blocking antibodies to be almost entirely due to gm-csf with little or no il- activity detectable. detectable il- mrna levels. (ae -ilz) displayed substantially greater anti-t induced proliferation than did ae -ag cells. in contrast to ae -ag cells, ae -il cells produced large quantities of il- in response to anti-t stimulation. furthermore. one cycle of stimulation of clone ae -ag with il- in the absence of antigen and irradiated spleen cells was sufficient to cause this clone to produce substantial amounts of il- upon subsequent anti-t stimulation. these data suggest that t cell receptor mediated stimulation of t cell clones by specific antigen and antigen presenting cells inhibits subsequent anti-t induced il- production. t cell proliferative responses and sera antibody levels of myasthenic patients to several synthetic peptides representing different epitopes of the human achr were examined. we detected significant differences in the humoral and cellular responses of mg patients compared to healthy controls to peptides of the human achr alpha-subunit with sequences p - , p - and ~ - . proliferative responses of lymphocytes from myasthenic patients to p - and to p - correlated significantly with hla-dr and with hla-dr , respectively. in order t o investigate further the immune responsiveness to selected sequences of the human achr, t cell lines and clones specific for peptides p - and p - were established from lymph nodes of c h.sw mice. the recognition specificities of these lines were tested by examining crossreactivity to a series of shortened and/or extended peptides of the above sequences. deletions of amino acids in positions and ( =p, =l) resulted in a decrease of the peptides' stimulatory activity on the ~ - specific t cell line, whereas deletion up to position on the n-terminal end had no effect on the triggering potential of the peptides. similar results were obtained when deleting residues and ( =v, =p) in stimulation assays of the p - specific t cell line. help in determining important t cell epitopes on the human achr. the role of guanine nucleotide binding regulatory proteins (g proteins) in the regulation of phosphorylation of the y subunit of the cd antigen has been examined. cd y chain phosphorylation in isolated t cell microsomes or permeablised t cells was stimulated by the g protein activator, guanosine '- thiotriphosphate (gtpys), but other nucleotides such as camp or gdpbs were ineffective. dependent. these data are consistent with the involvement of a g protein in the signalling mechanisms that regulate the phosphorylation of the cd y chain. the regulatory effects of calcium and gtpys were compared in normal peripheral blood derived t cells and jurkat cells. there were differences regarding g protein regulation of cd y chain phosphorylation in normal t cell and jurkat cells and current models explaining these differences will be described. expression of the gamma-delta t cell receptor has been thought to first occur in a population of thymocytes shortly after their precursors populate the thymus between and days of gestation. in the course of our studies investigating the ontogeny of t cell receptor expression in the mouse embryo we have identified an extrathymic site of gammadelta expression in a population of cells present at distinct times of gestation. evidence will be presented demonstrating two periods of activity of the murine gamma locus in the developing embryo. are colonizing the thymus from the liver and the gene segment useage detected is different to that first expressed in cells of the developing thymus. around the time of birth) involves the functional rearrangement and expression of a gamma gene segment corresponding to the initial functional rearrangements detected earlier in gestation in the thymus, which can occur independently of thymic influence. demonstrate a new site of gamma-delta receptor expression in the liver of newborn mice that can occur in the absence of any thymic influence. the primary (in vivo) response of cs bl/ animals to the class i antigen qa- is a helper (th) dependent event as indicated by the requirement for copriming with a distinct antigen capable of activating helper cells. in contrast, the secondary (in vitro) response to qa- demonstrates no need for costimulation with the helper antigen. in attempts to more closely examine the helper requirements for activation of primed ctlp, we have observed that depletion of l t cells from spleens of qa- primed mice abrogates the in vitro generation of anti-qa- effectors. the response is restored by the addition of concanavalin a induced supernatant (cas) or by the addition of syngeneic but not qa- allogeneic l t cells. indeed, even in the presence of cas, l t cells expressing the qa- alloantigen specifically suppress the activation of anti-qa- ctl in a manner reminiscent of that seen with lyt- veto cells. although the mechanism whereby l t cells exert suppression is unclear, we have determined that ctlp are susceptible to veto only within approximately the first hours of culture, after which they resist suppression. results from further studies of the nature of suppression and the l t veto cell will be presented. group i proteins induce ige ab responses in - % of mite allergic patients. murine mab and human igg and ige ab. unrelated. crossreactive epitopes on gpi and gpii allergens from different mite species. in contrast, igg ab in balb/c mice immunized with loug specific" epitopes and < % was a n t i -u i (a gpi homologue, with - % amino acid sequence homology to i). four non-over lapping epitopes were defined by mab, with one species specific immunodominant site on each gpi allergen. cross-reactive gpi epitope and this mab could inhibit human ige ab binding by - %. specificity of the murine anti-gpi response was not h- restricted, but could be altered by immunizing balb/c mice with lower ag doses (lug) in alum or . dertussis. using these regimes, up to % of the murine igg ab responses was gpi cross-reactive. responses to gpii allergens appear to be strain dependant. unresponsive to gpii. however, balb/b, a/j, cba, c h c b all produce gpii cross reactive igg ab. anitgenic sites on gpi allergens are conformational, whereas those on gpii may be sequential. known to affect ige expression in mice may also affect the epitope specificity of igg ab. we have compared the b cell epitopes on these allergens using panels of however, ag binding ria on sera showed that human igg and ige ab recognize the gpi and gpii allergens are antigenically i in cfa was directed against "species murine ab balb/c are completely thermal denaturation and reduction and alkylation expts suggest that the results with the gpi allergens suggest that immunization regimes which are c this report demonstrates for the the exclusive recovery of - -specific t cell clones c further molecular analysis should identify and characterize achr reactive autoimmune clones and/or suppressor cells. cohplex, mogens h. claesson, p e t e r bkams a n d s t e e n d i s s i n g , l a b . e x p . h e m a t . immunol., d e p t . med. anatomy a, a n d d e p t . g e n e r a l p h y s the ly- alloantigens represent a family of phosphatidylinositol anchored proteins that function as accessory molecules in the process of t lymphocyte activation. the expression of these alloantigens is often induced on t and b lymphocytes after activation by mitogens or antigens. previous studies have shown that the induction of ly- alloantigens in t cells is at least in part due to the action of ifn-a/b or ifn- . in the present study, we have demonstrated that ifn- also induced ly- molecules on b lymphocytes and bone marrow cells. furthermore, we now show that tnf also participates in the induction of at least one of the ly- proteins, ly- a/e. tnf was found to synergize with ifn- to induce ly- a/e expression in thymocytes, t lymphocytes, and bone marrow cells, but not b cells. for t lymphocytes, the synergistic induction of ly- a/e by tnf was restricted to cells from the ly- . haplotype whereas ifn- was sufficient to fully induce ly- a/e expression in cells from the ly- . haplotype. this result is consistent with the notion that there is more complex regulation of the ly- afe molecules in t cells obtained from the ly- . haplotype. for t cells from balb/c (ly- . ) mice, ly- a/e, but not ly-cc, molecules were induced by ifn- and tnf. furthermore, when compared to ly- a/e, the regulation of mhc class molecules in these t cells by tnf was minimal. the induction of ly- afe molecules on balbfc t cells resulted in an enhanced capacity to activate these cells through the ly- t cell activation pathway. one transformed t cell line, . . . was also identified whose ly- a /e molecules were synergistically induced by ifn- and tnf. optimal expression of ly- a/e molecules on . . cells required continuous culture of this cell line with these two cytokines and resulted in the detection of optimal levels of cytoplasmic ly- afe mrna by northern blot analysis. this latter result suggests that ifn- and tnf regulate ly- a /e at the level of transcription and/or mrna stabilization. ut southwestern medical center, dallas, texas . an igm antlcd mab ( . ) was found to modulate cell surface cd on highly purified human t cells within hours in the absence of a secondary antibody or accessory cells. inhibition could be overcome with accessory cells or il . the inhibitory effects of . could be mimicked by briefly pulsing cells with the calcium ionophore, ionomycin. . or ionomycin pulsed cells were inhibited in their subsequent capacity to resp nd to pha even when exposures were carried out in the presence of egta to prevent increases in [cap*]. from extracellular sources. inhibition was not the result of an inability to respond to pha 'by increasing [ca +] .. moreover the newly expressed cd molecules were capable of generating increases in [ca *].' after reacting the cells with anti-cd + a cross-linking secondary antibody. these studies dem'onstrate that a state of nonresponsiveness in resting t cells can be induced by modylating cd with an anti-cd mab in the absence of co-stimulatory signals. a brief increase in [ca ' . resulting from the mobilization of intracellular calcium stores appears to be sufficient to induce'this state of t cell nonresponsiveness. cd . laurie s. davis, mary c. wacholtz, and peter e. lipsky, dept. of internal medicine, lmmunogenicity c a central lab.blood transf.service, lab. of exp. and clin.immunology of the univ. of amsterdam, amsterdam. the netherlands monoclonal antibodies (mab) directed against the human cd molecular complex induce a strong proliferation of t cells, when immobilized on microtiter wells. this activation system, that was shown to be independent of accessory cells, accessory-cell derived factors or lfa- mediated intercellular adhesion ( ). allows one to study the requirements for t-cell proliferation and differentiation in a well defined manner. il- and ifn-gamma but no il- could be detected i n culture supernatants of coated anti-cd stimulated t cells. the addition of ril- or ril- had only a moderate effect on t-cell proliferation, vhereas helper activity for ig production was strongly enhanced in the presence of these factors. in this system differentiation of precursors to cytotoxic t lymphocytes (ctl), as measured in anti-cd mediated cytotoxicity, could be demonstrated within days after initiation of the activation. allospecificity of the induced effector ctl was demonstrated using a panel of hla class-i p -transfectants. in this system the regulatory role of the cd molecule in tcell activation and differentiation was studied. addition of anti-cdz mab to t cells stimulated with coated anti-cd mab enhanced il- production, proliferation as well as ig production. interestingly, pctl differentiation was also enhanced by anti-cd mab. this system seems valuable for the analysis of requirements for differentiation of human t cells subsets. . van noesel et al., nature , - , analysis of the requirements for human t-cell differentiation, rolien de jong. vivienne rebel, g i j s van seventer. miranda brouwer, frank miedema, rene' van lier, the newly described t cell receptor (tcr) locus is located inside the tcr a locus between va and ja . despite this unique situation, a highly efficient regulatory mechanism results in the complete independence of these two loci. we have recently described, in humans, a site specific recombination which joins a ' deleting element (srec) to the send of the ja's (yja) resulting in the deletion of the tcr- locus in t lymphocytes expressing the a/b tcr. rearrangements of the tcr as well as immunoglobulin genes are mediated by a unique recombination machinery and therefore, the specificity of these rearrangements is thought to be the result of a differential accessibility of the dna involved in the recombination process. as a consequence (and/or cause) of the opening of a segment of dna, the region involved is fxst transcribed as a sterile transcript prior to the rearrangement. in that regard, we have found that the kb of dna u p s a u~n of yja are actively transcribed ('t early a" transcript, tea) early during fetal development. the presence of the tea transcript presumably reflects the opening of the tea sequence prior to the tcr- deletional rearrangement. in order to better understand the mechanisms involved in the dna accessibility model, we started to look for dna-binding proteins which might play a putative role in the opening or blocking of the tea sequence. by the technique of "gel shift assay" we found such a negative regulatory protein in the nuclear extract from a non-lymphoid cell. the binding activity appeared to be specific as it was competed out by an excess of unlabeled autologous dna and not by an excess of irrelevant dna. further studies are now in progress to determine first wether the presence of this binding activity can be correlated with a "closed" configuration of the tea region and second to determine the precise location of the dna binding region. cohen, laboratory of chemical biology, niddk, national institutes of health, bethesda, md . mabs retard lymphoproliferation as well as autoimmunity. interesting, so does the adminisuation of a mab to l t , thus suggesting that the t helper subset, which is not part of the unusual expanding population, is required for initiating the pathology in these animals. as a means of characterizing the expanding population of abnormal cells as well as the phenotypically mature (l t +) cells that may be associated with them, i have generated a series of t cell hybridomas from the enlarged lymph nodes and spleen of m p r mice. in parallel, i have derived a series of control (non-lprflpr) hybridomas from mrulpr x balb/c f animals (which show no sign of pathology), and a series from mrl, mice (which have a delayed onset of autoimmunity without lymphadenopathy). very few hybridomas ( - ) were obtained in the non-lpr derived fusions. when i con a stimulated the lymphocytes from non-lpr mice prior to fusion however, many more hybridomas were obtained(l - ). this is in contrast to the fusion efficiency obtained from lprnpr mice which did not require in viuo lymphocyte stimulation to obtain a comparable number of hybrids. this result suggests that the m u p r lymphocytes are activated in situ. in addition, while less than % of the lymphoid mass is comprised of t helper (l t +) cells , over % of the hybrids are l t +. the fact that a dispropomonate number of t helper cells are rescued by fusion suggests that the cells activated in situ may be autoreactive t helper cells. currently i am characterizing these t helper cells for their lymphokine production, t cell receptor gene usage and auto-specificity and will compare them to the hybridomas obtained from non-diseased animals. goodnow, s. gilfillan, h-j. garchon, j. erikson and m. davis. stanford university, stanford, ca . we have made transgenic mice bearing gene constructs encoding the t cell receptor a and p chains from a cytochrome c-reactive t cell hybridoma. despite a lack of tissue-specificity in mrna expression, cell surface expression of uansgene-encoded protein was limited to t cells, presumably because both chains require cd proteins in order to assemble on the cell surface. in mice carrying only the a chain consuuct, the transgene was expressed in the thymus as early as day of fetal life, - days before endogenous a chain mrna. the first detectable cell surface expression of a transgene was on % of day fetal thymocytes. this vast increase in up-bearing cells in fetal thymus was due to pairing of transgenic a chains with endogenous p chains, of which a substantial number are. normally rearranged by day of fetal liie. the balance between ap-expressing t cell supopulations was grossly disturbed in these mice, the most marked abnormality being an increase in the number of l tnyt- -cells both in thymus and in peripheral lymphoid organs. it therefore appears that premature expression of surface ap t cell receptor may disturb t cell differentiation pathways by allowing t cells to leave the thymus without expressing l t or lyt- . mice carrying the p construct showed no increase in surface expression of t cell receptor in fetal life, since endogenous a chain rearrangement was limiting. in mice carrying either the a or p chain mansgenes, the number and surface phenotype of t cells expressing y& t cell receptors was unaffected in early fetal liie. suggesting that the a p and y t cell lineages diverge before thc rearrangement and expression of the appropriate subset of t cell receptor genes. department of microbiology and immunology, institute and the university of pennsylvania, philadelphia, pa. .the thymic stroma plays a major role in initiating the colonization, organization and differentiation of precursor stem cells into functionally mature t cells. a variety of cell types including thymic nurse cells, cortical and medullary epithelial cells, nonepithelial dendritic cells, and macrophages, combine to form the thymic stroma. the differential role of such cells in thymic development is unclear. we have isolated a number of morphologically distinct stromal cell lines from the thymuses of sv transgenic mice. several of the cell lines are of epithelial origin, while others have features consistent with non-epithelial "dendritic" cells. we have focused on one of these cell lines, bearing the phenotype of a cortical epithelial cell, for its ability to support the growth and differentiation of stem cells from the fetal liver and fetal thymus, and cloned pre-t cells obtained from adult mice. the cortical epithelial cell line produces factors that induce the dramatic proliferation of fetal liver and thymic stem cells . in addition, fetal liver cells cocultured with this cell line are induced to rearrange and express their t cell receptor (tcr) genes. a cloned pre-t cell line is also induced to rearrange its tcr oenes in resoonse to sianals mediated bv this cell line. gugrin, marie c. b n , corinne amiel, nadia coniglio, jacques leclsre, laboratoire d'immunologie and clinique endocrinologique, chu de nancy and facult de mgdecine, vandoeuvre les nancy, france. the lfal molecule, an adhesin of the lfa family involved in cell-cell interactions, is physiologically expressed on all white blood cells. it is absent in some congenital immune deficiencies ((id), and is expressed on a decreased number of peripheral blood lymphocytes (pbl) in aids. we investigated its presence on pbl from patients with auto-immune disorders of the thyroid. a monoclonal antibody (iot , immunotech) directed to a conformational epitope involving both chains of lfal was used in indirect immmunofluorescence on pbl from blood drawn at a similar time in all patients. a calibrated flow cytometer (epics profile, coultronics) was used to measure the percentage and numbers of positive cells, as well as the mean fluorescence (mf) and shape of the fluorescent peak. data were correlated with clinical information,therapeutic, and other pbl features such as the cd icdb ratio. the percentage of lfa + cells was significantly decreased in patients with graves' disease, hypothyroidism and hyperthyroidism. the mf was lower and the shape of the fluorescent peak seldom displayed the bimodal characteristic noted in controls. these data suggest the participation of the altered expression of lfal in the pathogenesis or evolution of auto-immune diseases. pt=ciilat.pd that excess hla r l a s s tt expression, ciinmonly found i n a i t i v e human nutoinimiine tlisrases maintdin:? the *rctivatinn of dutnreactivr t c e l l s which in turn prnducr mediators which maintain r.la:;s i t expression. this hypothesis has been tested i n many ways i n t h y r o i d i t i s . crj t i c a l l y autoreactive t cell:? are €nilrid i n thyroid autoinnline tissues which a r e rrstimnlatrd hy thyroid f o l l j c u l a r r r l l s . more rrcently we have been exploring the s p e c i f i c i t y of the autoantigen reactive t c e l l s in hashinoto's t h y r o i d i t i s where thyroy-lobulin s p e c i f i c clones have been found, i n contrast t o graves' disease, where thyrocyte recognizing clones do not react wi.th tliyroglnhulin. tn rheumatoid a r t h r i t i s , collagen type i clones have been found, persistently in the activated (il- r') t c r l l pmil over several years i n t.he same p a t i e n t . to verify t h a t antigen present,ation is involved i n rhrumatoid art.hritis ( r a ) a disease i n which, unlike thyroi.dj t i s the nature of the major antigen presenting c e l l (apc) is unknown, thc e f f e c t of ,~nticl.ass i dntibodies a t -oncentration which block *ari;j vation of t r-el is mi the synthesis n € rla-dr mrna wa:j waluat.rd. the inhibitory effect supports d i.rifira role nf an d s yet iiriknown apc i n maintaining the i:hronj.ci t y o f r a conversely, all the clones were unable to respond to a substitution at (tyr to asp). nase mutant proteins were constructed with the same single amino acid substitution and t cell responses to peptides and mutants were compared. preliminary evidence suggests that the mutant proteins like the peptides, substituted at residue and , will not induce t cell clone responsiveness. these data suggest that the overall structure of the protein will not compensate for the lose of a particular amino acid which is necessary for t cell recognition. medicine, baltimore, md . to explore the variables important in t cell priming, an adjuvantfree immunization regimen was developed. bio.a mice were primed subcutaneously with syngeneic spleen cells that had been pulsed with high concentrations ( pm) of the peptide - , a cnbr cleavage fragment of pigeon cytochrome e. the t cell response was assessed using a sensitive limiting dilution assay that measures lymphokine production with the ctl-l cell line. the precursor frequency of antigen-specific cells in the draining lymph nodes of mice primed with antigen-pulsed spleen (aps) was in , indistinguishable from the frequency of in found in mice primed in the footpads with nmol of - in complete freund's adjuvant (cfa) (data are given as geometric means, n= , s.e.m = x/t . and . , respectively). despite the apparent similarity in the t cell compartment of mice primed using these different regimens, antibody induction was strikingly different. mice primed with - in cfa developed serum igm and igg responses against the peptide, with antibody detectable in an ellsa assay at a : dilution. mice primed with - owaps, however, produced no detectable anti-peptide antibodies. maximal t cell clonal expansion therefore appears to be possible in the absence of antigenspecific b cells. these data argue against the hypothesis that antigen-specific b cells play an obligate role in t cell proliferation in vivo. the reasons for the lack of antibody induction are currently under investigation. cell receptor (tcr) complex of jurkat cells. the coprecipitation of these peptides with tcr requires treatment with monoclonal antibodies (mabs) directed against tcr (c or r ) prior to cell lysis and immunoprecipitation. treatment of jurkat cells with mabs directed against cd ( - or . ) or hla (w ) does not not induce the association of these peptides with tcr. the signal-transduction mutant cell lines, j.cam and j.cam , have previously been described ( ,z). these cell lines, derived from jurkat, fail to activate the inositol-phopholipid second-messenger pathway in response to anti-tcr mabs. treatment with mab c induces the association of the and kd peptides with tcr in j.cam cells but not in j.cam . j.cam modulates tcr normally in response to anti-tcr mab treatment ( ). hence, these observations suggest that the two peptides are involved in the signal-transduction pathway of the t cell receptor complex rather than receptor internalization. sle is an autoimmune disorder associated with several different hla class i antigens. we studied a large sle patient population by sequencing of the pcr amplified first domain of the dqb and dqa chains and by sequence specific oligonucleotide probes to further define these associations. shared dqb sequences at amino acid positions = eu, =tyr, and fasp may predispose some individuals with hla dr , , , or to develop sle. a novel dqb sequence found in two drw dqwl sle patients shares these amino acids in the dqb hypervariable regions. the association of the drz dqwl.azh gene was greatly increased in the sle patients with lupus renal disease. the hla association may be directly due to structural aspects of the hla genes. when parent -+ f chimeras are prepared with supralethal irradiation ( rad + rad), the donor-derived cd + cells differentiating in the chimeras show partial tolerance to host-type h- determinants, despite the apparent absence of host-type apc. donor-derived cd + cells give only low proliferative responses to host-type apc in primary mixedlymphocyte reactions (mlr); furthermore, in i-e-+ i-e+ combinations, the donor cd + cells show molecules. this finding implies that tolerance is induced intrathymically, presumably through contact with a non-marrow-derived component of the thymus, e.g. epithelial cells. in support of this possibility, thymectomized & + ( a x b)f chimeras given strain j? marrow cells and a strain thymus graft (irradiated) show no detectable tolerance to host-type strain b determinants: the strain & cd + cells differentiating in these chimeras give strong mlr to strain b and do not show deletion of v + cells. = % deletion of cd + cells expressing i-e-reactive v t cell receptor b measured by these two parameters applies not only to lymph node (ln) cd + also to cd + cells recovered from the thymus. interphotoreceptor retinoid-binding protein) is a glycoprotein of residues (bovine) which localizes in the retina and pineal gland and induces inflammatory changes in these organs (eau and eap, respectively) in immunized animals. the experimental disease is considered a model for certain uveitic condiiions in man. we have recently shown that irbpderived synthetic peptides can also induce eau/eap in lewis rats. the present study compared two such peptides, "r " (residues - ) and "r " ( - ). peptide r was found to be immunodominant, shown by its being recognized by lymph node cells (lnc) or line cells sensitized against whole irbp. in contrast, peptide r was not recognized by the whole irbp-specific lymphocytes and is considered nondominant. in addition, lnc sensitized to r , but not to r , responded to intact irbp. r was superior to r in producing eau/eap and cellular immunity (minimal doses: . vs pg/rat). on the other hand, the two peptides were comparable in their capacity to stimulate presensitized lymphocytes. moreover, lnc sensitized against r were similar to those sensitized against r in their capacity to adoptively transfer eau/eap to naive recipients. this study thus provides a unique system in which both immunodominant and nondominant peptides produce autoimmune disease and can be compared for their immunological features. the age-related diminution in immune responsiveness has been shown to result from increased regulatory mechanisms and not from a paucity of immunobgical recruitment (aging: immunology and infectious disease , , ). we present evidence based on "libraries" of monoclonal antibodies (mabs) omained from young and aged donors that there occurs with aging an increase in autoimmunity which is possibly the result of the accumulation of liielong "original antigenic sins". the resultant increased connectivity of the immune system is represented by mabs obtained from aged donors which are multiply anti-self cross-reactive. furthermore increased connectivii is supported by the evidence that anti- . , -trinitrophenyl mabs are ad positive, d positive as determined by i n h b i i n studies using mabs anti-idiitypic reagents. analysis of the vh and vk region genes utilized by these mabs indicate a nonrandom gene usage. life bng stochastic immunological events lead to a pattern of cross-reactiities and non-random usage of vh genes. these immnological events lead to the emergence of the patterns which are partially elucidated by the data presented. these patterns mimick those seen early in ontogeny, but indicate a possible convergence to an ever-increasing connectance of the idiitypic repertoire expression. in other words, life-long immunological experiences contribute to a down-regulation resulting in both paucity of drimaw immune reswnses and an increase in autoimrmnity which are both the earmarks of immunity in aging. (supported by usphs grants ag- to eag and al to cab) lmmunogenicity c stimulation of these cell lines in suspension with saturating levels of mab okt produces total and fractional inositol phosphate accumulation linearly related to receptor number, (r > . ). this technique also allows an approximation of the minimal number of reccptors which must be engaged for second messenger generation in this system, which we estimate as . ~ receptors per cell. or terminate t cell activation. since this molecule plays an important role in human t cell development we sought to identify the murine homologue of cd in order to determine its expression on murine t cell and its role in activation. we have used a human cd cdna clone to isolate a full length cdna encoding the murine equivalent of cd from an el t cell lymphoma library. this clone shows similar domain organization and a high degree of homology to the human cd molecule. the murine cdna clone has been used to examine mrna expression of cd in normal and activated murine t cells, and in various t cell tumors. peptides generated from the translated sequence will be used to produce antisera to correlate the surface expression of cd with mrna expression, and to biochemically and functionally characterize this molecule. pat happ and ed palmer, basic sciences division, dept. of pediatrics, national jewish center for immunology and respiratory medicine, denver, co . we have attempted to determine the frequency of rearrangement and expression of the individual a and g chain v gene segments that make up an unselected, untolerized t cell repertoire. in order to do this we generated over t cell hybridomas from freshly-isolated thymocytes of newborn c blllo mice and subjected rna from these hybrids to northern dot blot analysis using va, vp. cy and c probes. comparison of the expressed repertoire of vp gene segments in this newborn thymocyle population with similar data previously generated from an adult peripheral t cell population reveals two vg genes, vp and vpl , whose expression is decreased in the periphery, possibly due to the effects of tolerance. two additional vp gene segments were expressed more frequently in the peripheral population than in the newborn thymus, vp ( . times higher in the periphery) and val ( times higher). it is possible that these represent ewo instances of positive selection of t cells which is determined primarily by the receptor's vg gene segment. va gene segments were expressed in only % of newborn thymocyte hybridomas (compared to % expressing vp) and determination of va rearrangement frequencies was complicated by the unexpectedly large number ( %) of hybrids expressing cs mrna. further examination revealed that several va gene probes were actually detecting rearrangements to cs. the most notable of these was va , which accounted for approximately % of the expressed va repertoire but was rearranged exclusively to cs. barbara bergman, brenda bradley, kevin lafferty and mary portas. barbara davis center for childhood diabetes, u. colo. health sci. ctr., denver, co . we have produced a panel of islet-specific t cell clones by culturing lymphoid cells obtained from non-obese diabetic (nod) mice in the presence of nod islet cell antigen and antigen-presenting cells (apc). these clones were selected to the panel on the basis of (a) their antigen-specific reactivity to islet cells and apc in an in vitro proliferation assay and (b) their ability to mediate islet graft rejection in vivo in a tissue-specific manner. we have further characterized these lines for cell surface phenotype, - production, and proliferative response to non-nod islet antigen. all of the clones tested to date are of the cd phenotype and make il- in response to islet antigen and nod apc. nearly all of the clones we have tested also make good proliferative responses to islet cell antigen obtained from mouse strains other than the nod or to a mouse beta cell tumor line. preliminary results indicate that at least one of these clones can lead to islet cell damage in a disease transfer experiment in which the cloned t cells are injected into a non-diabetic nod f recipient. we are currently carrying out tests to further characterize lymphokine production by these cloned cell lines, to analyze differences in antigen recognition and mhc restriction requirements among the clones, and to determine their effectiveness in mediating the disease process in nondiabetic animals. in an attempt to identify the epitopes on class i mo ecules recognized by alloreactive cytotoxic t lymphocytes (ctl) we have examined k -specific ctl for tpir recognition of synthetic peptides with sequences derived from the native k molecule. co secutive overlapping peptides molecule were tested for their capacity to inh&bit k -specific ctl clones in their recognition of cells expressing the native k molecule. in these studies inhib ion by peptide was found to be an extremely rare event, although one peptide (k - ) did inhibit recognition by a particular ctl clone (clone ). in a separate set of experiment it was observed that clone could recopize kblll- when presented by h- class i molecules. as clone was of h- origin, this finding led to conclude that inhibition may be due to class i-restricted recognition of the k pegtide on the surface of the ctl clone, peptide and native kb for the t cell receptor. we present evidence in favor of this conclusion. the pepscan method is used for the systematic identification of sequential b cell epitopes i n protein molecules (geysen, meloen, barteling. pnas : ; ) . it was designed for the synthesis and subsequent testing for antibody binding of large numbers of overlapping peptides directly on their solid supports. the mhc dependent presentation required for t cell recognition seemed prohibitive for the use of pepscan to identify t cell epitopes. however we now have shown that by a novel modification the peptides can be recovered from their solid supports and used in t cell assays. holmdahl, department of medical and physiological chemistry, box uppsala university. s- uppsala, sweden. both autoreactive t cells and autoantibodies play an important role in the pathogenesis of type i collagen (cii) induced arthritis in mice. we have earlier reported that only strains with h- q , h- w , h- w and h -r were responders to autologous mouse cii and only these strains developed arthritis after immunization with autologous or heterologous cii. however, heterologous cii induced a more acute and severe disease and a more pronounced autoantibody response. this findings indicate that ) the ability to mount an immune response against autologous cii is a prerequisite for the susceptibility to collagen arthritis and ) that a crossreactive autoantibody response after immunization with heterologous cii may further enhance development of arthritis. we have now studied activation of autoreactive b cells after primary immunization of den mice with rat cii. in hybridoma collections, obtained - days after immunization, . % of the hybridomas produced igg reactive with autologous cii, - % produced multispecific igm and a significant number produced igg rheumatoid factors. the anti-cii antibodies recognized at least different epitopes on the cii molecule and originated from many different vh and v kappa gene families. furthermore, none out of investigated anti-cii hybridoma expressed cd rna message. we therefore suggest that the primary anti-cii autoantibody response involves activation of memory b-cells. these memory b cells have most likely been earlier activated by cii autoreactive t cells. in these aspects the origin of the anti-cii autoantibody response is principally different from the origin of "natural" autoantibodies. t cell receptors (tcr) recognize antigen in association with self mhc molecules, usually following processing to smaller peptides. the t cell repertoire to an antigen, therefore, reflects not only the ability of a given mhc molecule to interact with antigen, but also the affects of initial repertoire selection by self mc. we have t#tn analyzing the tcr repertoire specific for beef insulin ( ) in balb/c mice (h- ), which are high responders to the antigy. these studies revealed that vp. is dominantly used in the tcr's specific for bi/a and our preliminary data suggests that the vgb. chain may be involved in mhc restriction. we have now obtained several t cell hybridmas specific for bi from (balb/cxa/j) f , animals. a/j mice (h- a) are low responders to bi while the f m ce a e high responders. most of the balb/cxa/j hybridmas were restricted to the hin the balb/c hybridmas. interestingly, the analysis of v gene usage demonstrated that vg . was not used in the balb/cxa/j hydridonas. the relevance of these results to the development of the tcr repertoire in different mouse strains will be discussed. this work was supported by the mrc of canada. ctl specific for the q k molecule were generated from normal splenocytes by & vitro culture with a k bearing stimulators. these ctl have been shown to lyse transfected targets expressing hla-a regardless of their murine haplotype, and they specifically kill a bearing human target cells. furthermore. the effector function of these ctl can be inhibited with an hla-a specific monoclonal antibody. thus, the transgene product functions correctly as a tolerogen and is recognized directly as a class i antigen. although transgenic mice have been shown to be tolerant to a k expressed by murine cells, transgenic ctl specific for hla-a on the surface of human cells have been generated. these the major virulence factor of m.pneumoniae was shown t o be a kda protein which is located in the tip structure membranes of these cells. beside the adhesin function this protein is also involved in first massive humoral and cellular responses of the human host during the acute phase of upper respiratory tract infections and interstitiel pneumonia. intranasal inoculation of guinea pigs with the isolated kda protein led to lympho-histiocyte infiltrations around bronchi and small vessels of the lungs which are characteristic infiltrations after an infection with live m.pneumoniae cells. furthermore one peptide ( amino acids long) which was synthesized according to the amino acid sequence of the adhesin, showed a proliferative activity to in vitro cultivated t-cells of bronchial washings, whereas synthetic peptides with th e sequences of the direct neighbourhood showed no in vitro activity. most interestingly this t-cell proliferative activity is located on a surface loop of this protein which is also responsible for the adhesin f uction. christopher a. smith, gwyn t. williams, rosetta kingston and john j.t. owen. department of anatomy, university of birmingham, medical school, vincent drive, birmingham tj, uk. rearrangement of t-cell receptor a and b chain gene segments during t-cell development results in a diverse array of receptor specificities. to avoid auto-immune responses, cells that have generated self reactive receptors are thought to be eliminated or inactivated, to produce self tolerance. recent studies have provided compelling evidence that clonal deletion of immature receptor bearing cells within the thymus makes an important contribution to this process, although the mechanisms involved are not uderstood. of immature mouse thymocytes with anti-cd antibodies added to thymus organ cultures, induces dna degradation and cell death through the endogenous pathway of apoptosis. is in marked contrast to the activation of mature t-cells by the same anti cd preparation and is specific to the extent that apoptosis is not induced by either anti-cd- or anti-thy- . to organ cultures suggesting a role for changes in intra-cellular c a w levels in the signalling pathway leading to the induction of apoptosis in immature cells binding. thus activation of the process of apoptosis in immature cells binding self antigens may be the mechanism responsible for the selective deletion of cells that could generate an auto-reactive response if allowed to mature. we have now obtained evidence that engaging the cd /t-cell receptor complex this in addition, calcium ionophore (ionomycin) also causes apoptosis when added anderson cancer center. houston, tx immunization of patients with bcg and irradiated tumor cells induces specific delayed-type hypersensitivity (dth) to tumor cells and not to normal colon cells. since igg antibodies may require t-cell help, we wished to characterize the igg-defined tumor-associated autoantigens (taaa) of human crc so as to define a subset of the t-cell repertoire for crc. western blots of detergent extracts of primary and metastatic human colorectal carcinomas and paired normal tissues were probed with autologous igg. nine taaa were recognized by % or more of the sera: , , , , , , , . and kda. these taaa may be normal colon differentiation antigens, since they were present in extracts of normal colon. autoantibodies are more frequently present to the kda antigen in patients with metastases ( %) than in primary tumors ( %. p<o.o ) while there is more reactivity to the kda antigen in primary tumors ( %) than in metastases ( %, pso. ). only of patients sensitized with bcg and irradiated autologous tumor cells, developed antibody to new taaa; whereas of control patients developed antibody to new taaa without sensitization. the taaa in the western blots that stimulate autologous lymphocyte proliferation are greater than kda. finally, immunization with a human crc failed to induce dth in mice to a syngeneic murine colon carcinoma that expresses at least of the igg-defined human taaa. thus, taaa that presumably require t-cell help in patients do not appear to be part of the t-cell repertoire for dth to human crc. in several rodent species and strains, the emergence of specificity to a discrete immunodominant epitope of guinea pig basic protein (gpbp) results from selection of cell lines with whole gpbp. these epitope specificities have been found to be strain-specific and define the specificity of the t helper cells which transfer experimental autoimmune encephalomyelitis (eae) in adoptive transfer experiments. line from aci-strain rats which was encephalitogenic and specific for a new t cell epitope of gpbp. the cell line responded in vitro to the peptide cokkesponding to the region - of gpbp but not to peptides corresponding to other regions of gpbp. this cell line is restricted by i-a but not i-e and transfers a dth response to gpbp as well as eae into naive recipients. these results provide support for the hypothesis that the in vitro immunodominant epitope specificity defines the specificity of gpbp-specific encephalitogenic t cells. the definition of a new encephalitogenic epitope in the aci strain also suggests that the mechanism of eae in rats depends on strain-specific extrisic properties of antigen recognition such as the mhc class i genotype or the composition of the t cell antigen-receptor repertoire. we have produced a gpbp-specific t lymphocyte medical center, stanford, ca . a wide variety of t lymphocyte surface antigens have been isolated and shown to play important roles in the t cell response. we outline here a strategy to isolate and characterize surface proteins unique to activated t cells. a rabbit anti-serum was generated by immunizing with allo-activated human t cells and subsequently absorbing with the t cell tumor hpb-all, a tumor that expresses all functionally relevant t cell surface antigens previously described. this absorbed antiserum was able to recognize an uncharacterized group of surface proteins on t cells that was absent from hpb-all. furthermore, the absorbed anti-serum was able to inhibit i n virro cytotoxic and mlr responses; an effect that was abolished by carrying out an additional absorption with the t cells used for the initial immunization. monoclonal antibodies to activation antigens were generated by immunizing with complexes made by adding the absorbed anti-serum to solubilized ctl. the absorbed anti-serum was also used to screen a lcgtll cdna library. one cdna clone so isolated is expressed on ctl as evidenced by northern analysis. dna sequence data indicates no similarity to other sequences in available databanks. childrens hospital of orange county, orange, ca , and university of california, irvine, ca . we examined the ability of cytokines to stimulate the proliferation of pbm from new-onset iddm, long-term iddm, non-diabetic pediatric control, type i diabetic, and non-diabetic adult control subjects. initial results demonstrated the ability of human pbm culture supernatants containing peak natural il- activity to stimulate a significantly higher proliferative response from pbm of new-onset iddm than from pbm of long-term iddm, type i diabetics, or non-diabetic controls. more recent results have shown that human recombinant il-la, -lb, - , - and - , interferon gamma, lymphotoxin, and tumor necrosis factor, as well as con p.-stimulated rat spleen supernatants, also stimulated a significantly higher proliferative respcnse from pbm of new-onset iddm than from pbm of controls. however, human recombinant il- stimulated equivalent levels of proliferation from pbm of both new-onset iddm and controls. these data indicate that, in new-onset iddm, there .e present pbm which are in an early stage of activation not revealed by increased responsiveness to recombinant il- . in the case of t cells, this may represent a stage prior to an il- -dependent stage of activation. this would be consistent with the active induction of newly responsive cells during a stage of iddm when islet cells are still present. this response is not the result of the metabolic aspects of iddm but a reflection of the immunologically activated state of t cells, b cells andlor monocytelmacrophages in iddm. houghten, c. david pendleton, james l. comette, charles delisi and jay a. benofsky, metabolism branch, national cancer institute, national institutes of health, bethesda, md . analysis of known helper t cell m) cpitopes suggests that th epitopes tend to be amphipathic alpha helices, that is, helices with hydrophobic residues on one side and hydrophilic residues on the other side. based on this hypothesis, a computer program "amphi" was generated which may aid in prediction of th epitopes. within residues to of spermwhale myoglobin (swmb), a th epitope is known to exist but has not yet been found. as an approach to further test the hypothesis, this undetermined th epitope was sought both by an unbiased test of ten evenly overlapping -residue peptides that span the region, and by computer analysis. of the ten peptides, only one was able to stimulate two clones that are specific for the - region. this peptide gave remarkably high stimulation index. analysis of this region by the computer program also predicted the site covered by this peptide (residues ) to be the most likely site for th epitope.. therefore, unbiased in v i m testing and the computer prediction correlated well. the peptide was able to prime t cells of b o.br mice in vivo for in v i m response to the native swmb as well as to the peptide fragment of residues - . immunization of mice with swmb showed that, of the ten overlapping peptides, the major site of response is to the identified peptide. finally, a peptide of residues - was made to increase the amphipathic score. this extended peptide induced greater proliferation of the clones. thus, this study has identified a new dominant epitope of swmb and confirmed the utility of the amphipathicity hypothesis in a prospective test. amino acids a t a selected site; ) t-cell antigenicity, that is analysis of prirrary structures with -and -amino acid templates obtained from database of knawn y-epitopes; gly, hhydropkbic, pplar or charged amino acid ( ); ) prediction of the secondary structure according to gamier ( ) to exclude sites having high turn or coil forming ptential. ccanparison of t h i s set with published program ( , ) shows that it has high predictive f r . so we used it fo search for probable t-epitopes i n hepatitis a virus capsid pro- and for mediators of delayed hypersensitivity (tdh ~ assayed by ear swelling induced by co-injection of responder cells with irradiated tumor cells). lymph node cells recovered days post-inoculation contained tdh cells and ptc (freq / , ) , but tc effector cells. by contrast, graft-infdtrating cells not only contained tdh and ptc (l/l,oao), but direct tc effector cells ( lytic units). all three sets of t cells were antigen-spedfic. the finding of significant frequencies of ptc within the lymph nodes and the graft site, but tc q& at the latter, suggests that differentiation of tc from unprimed ptc may be a multistep process, the first of which occuis in the draining lymph nodes. the data further suggest that the terminal step@) in this process take place after the donally expanded ptc have disseminated to the graft site. we suspect that the final step requires help provided uniquely by tdh cells within the graft. the fmal step may not occur (as might have been expected) in the lymph node because priming of ptc and tdh take place at noncontiguous anatomic sites. interference & seed, pnas a, , ) . the n-terminal amino acid sequence of normal t cell cd was found to match the hpb-all sequence except in residue and (...ts...) . in principle this might reflect somatic mutation in hpb-all or cd -gene polymorphism: erroneous residue assignment in the gas phase sequencing cannot be excluded entirely. to investigate the significance of this sequence variation a genomic cd dna clone of a normal human t cell clone was analysed around residues . and found to be identical with the hpb-all cdna sequence. sumtic mutation in hpb-all cd therefore can be ruled out. the molecular characterization of t cell receptor (tcr) structure in a panel of mouse t cell clones specific for sperm whale myoglobin (spw mb) has shown that -ed-restricted clones specific for the spw mb region - use highly homologous v bchains. four of these clones are indistinguishable in their response pattern to a panel of overlapping synthetic peptides spanning the spw mb sequence - , yet use different v a gene segments. we are currently trying to find fine specificity differences among these clones, in order to correlate them with tcr expression. t cell hybridomas have been made from these clones, and are being screened on panels of substituted peptides. we hope that conservative substitutions at positions important for interaction with the tcr will reveal fine specificity differences among these clones. sloan-kettering institute, new york, ny we previously showed that clonal deletion, non-suppressor mediated immunological tolerance neonatally induced to mls allo-determinants was specific in that the frequencies of t cells responding to a variety of other stimuli were unaffected while the frequency of t cells responding to mls were drastically reduced. here we show that mls tolerance is detectable in the thymus prior to a detectable positive response in the periphery (spleen and lymph node). tolerance induced by spleen cells from mlsa congenic mice in balb/c mice is as "potent" as that induced by whole background incompatible dba/ spleen cells, indicating that incompatibility at the ml s locus ( the ly- alloantigens have been shown to participate in the process of t cell activation based on the ability of anti-ly- monoclonal antibodies to induce il- production and proliferation of t lymphocytes. in the present investigation, we have demonstrated that peripheral t lymphocytes from a strain mice exhibited abnormally low proliferative responses after stimulation through ly- a/e and ly- c molecules when compared to responses of t cells from numerous other mouse strains. the abnormal activation of the ly- pathway of a strain t cells was not due to ineffective fc receptor cross-linking of the anti-ly- monoclonal antibodies, to inappropriate cellular expression of the ly- a/e alloantigen in a strain t cells, or to an active suppressive phenomenon. identification of this defect provided an opportunity to compare t cell activation by the ly- and tcr pathways. interestingly, t lymphocytes from a strain mice proliferated normally when the cells were activated by monoclonal antibodies to thy-i or the cd /tcr complex suggesting that this defective activation was selective for the ly- t cell activation pathway. cell separation studies of t cells and accessory cells demonstrated that this defect was quantitative rather than qualitative and that it was complex, residing at both the t cell and accessory cell levels. these results suggest that activation of t lymphocytes via the ly- molecule may involve unique signalling pathways and that activation via cd /tcr can proceed normally in the absence of a fully functional ly- pathway. the h-u)d transfected l cells were recognized by the cil when they were infected with either influenza a virus or a vaccinia recombinant virus encoding the pb . thirty-five synthetic peptides derived from pb , which had either an amphipathic suucture or contained a rothbard' epitope, were tested for their ability to render uninfected targets susceptible to lysis by influenza specific effectors. three epitopes were identified, each of which contained a rothbard epitope. department of microbiology and inununolag, albany medical college, albany, ny our laboratozy has developed a new, powerful technique for hvestigating the inmplne ~n s = to peptide epitopes, involving the covalentcoupling of peptide to phosphatidylethanol-, follmed by amplexing with additional lipids and phospholipids to form a peptide-phoqholipid complex. these cmplexes can be used to inununize mice in the absence of protein carriers or adjuvants, thus facilitating the study of the hmne response to a mall chemically defined antigen. use of this technology has allmed us to identify two t helper cell epitopes in conserved regions of kiv gp not previously identified by computer algorithims, defined by amino acids - and - . inununization with these peptides in peptide-phoqholipid mmplexes results in the prduction i g and igg antibodies, which truss react with cloned fragments of the whole protein. us& this tecfinolosy we have begun to characterize the irmnune reqonse to individual peptide antigens. ?he response of h -k mice to amino acids - of gp of hiv, has been analyzed. lhe optimal dose of a peptide containing both b and t cell epitopes was found to be - ug, depending on the mute of administtation. im mhization required less antigen for optinarm antibody response than did ip. anchorage in the phospholipid cmplex is a strict requirement for an antibody response. additional variables, such as phospholipid m i t i o n and method of cross-linking have been studied and will be discussed. we believe that the use of this peptide-phospholipid ccnnplex technology will be significant both for studying the i n m u m response to single epitops and for vaccine developrent. virology, national bacteriologica; laboratory, stockholm, departments of neurosurgery, virology and immunology, karolinska institute, stockholm, johnson & johnson biomedical research, la jolla, california, pentadecapeptides, sequentially overlapping by a.a., were syntesized based on the htlv-i sequences of gag and env proteins and used as antigens in igg-subclass elisas andt-cell stimulation assays. sera and cells were obtained from asymptomatic, hiv-infected homosexuals. reactivity in all subclasses could be found to parts of the gag-protein. extensive areas of iggl and reactivity were indentified mainly in the p and n-terminal half of p with an additional region in the n-terminal end of p . the highest iggl and reactivities were detected in a.a. - , rich in glycine, arginine and lysine and thereby hydrophilic with a high segmental flexibility; iggz and epitopes were found in the hydrophilic cooh-terminal of p j; the second highest iggl and reactivities were found in the hydro-?hilic n-terminal of p . for gp , the iggl epitopes identified were in the putativeloopregion (a.a. - ) and in the hydrophilic cooh-terminal end of gp (a.a. - ). the immunodominant region of gp (a.a. - ) showed some igg , and responses inaddition to igg . t-cell proliferative responses were detected against many peptides usually in areas not binding igg. still, simultaneously t-and b-cell reactive peptides could be detected, and the showed a distinctly preference for igg . the variation between different patients was considerable regarding both t-cell reactivity to peptides and the subclasses of reactive igg. a mapping of subclass restricted epitopes allows an elucidation of anti-viral imunological mechanisms. kristin komschlies, laboratory of experimental immunology, brmp, dct, and bcdp, program resources, inc., national cancer institute-frederick cancer research facility, frederick, md and jackson laboratories, bar harbor, me . "double positive" (dp) thymocytes are precursors for cd + and cd mature subsets have been based on indirect evidence with in vivo anti-cd or anti-cd treatments. we have approached this question directly by isolating subsets of dp thymocytes from fetal or adult thymus and have shown that only a subset of dp cells ( % of adult thymocytes) can generate donorderived thymocytes after i.t. injection into irradiated hosts. low-density dp cells are injected i.t., up to - % donor-derived cells can be seen at to days after transfer. cd ' cells can constitute up to % of these donor-derived cells at day . in contrast, an equivalent i.t. transfer of dull ly-l/cd (dly- ). early precursor thymocytes would not generate cd ' cells until days - but would produce some cd ' cells by days. thus we have detected differentiation of cd + cells and not cd ' cells from adult dp cells. depleted host and recovered donor-derived thymocytes to days after i.t. transfer of dly- cells, when most of the donor-derived cells are dp. when retransferred, these dp cells displayed a more rapid progression of thymic localization patterns than dly- cells, but were unable to produce enough donor thymocytes to analyze subsets. thus cd ' cells appear to differentiate via a very minor (low density) subset of dp thymocytes. may not be generated from these intermediate dp precursors but directly from dly- cells. the intrathymic differentiation process by which bone marrow-derived progenitor cells develop into mature, immunocompetent t lynyhocytes remains poorly understood. the majority of all intrathymic lymphocytes are cd "double positive" cells that have no well-documented function in thymic differentiation, but have been proposed to be either a critical intermediate between cd - precursor cells and mature t cells, or, alternatively, a "dead-end" cell type. furthermore, although signals mediated through the tcr complex are critical for t cell tolerance induction and repertoire selection, the functions of the cd and co differentiation antigens expressed by the vast majority of thymocytes are unknown. in the present study we addressed the possibility that cd functions as a signalling molecule during t cell development. we engaged cell surface cd on murine thymocytes by in vivo injection of anti-cd mab, and monitored the effects of that engagement on distinct thymocyte subsets. c d ' + cells respond to cd cross-linking by dramatically increasing their cell surface expression of cd and tcr, and decreasing their cell surface expression of cd . in contrast, mature cd ' -cells respond to cd engagement by decreasing their cell surface expression of both cd and cd . these results provide clear evidence that cd engagement regulates cd /tcr expression on developing thymocytes, and, most interestingly, results in increased tcr expression by cd + + double positive thymocytes. thus, cd engagement is a membrane event to which c d ' ' thymocytes respond significantly, indicating that cd may be a signalling molecule in t cell development and may promote t cell differentiation by inducing increased cd /tcr cell surface expression on cd + + thymocytes in vivo. f.g. terpstra. p.th.a. schellekens and r.a.w. van lier, central lab.blood transf.service, lab.exp.clin. immunol. of the univ.of amsterdam, the netherlands t cells can be activated through several membrane molecules that may use distinct intracellular signalling pathways. t-cell activatiep threygh both the cd /tcr complex and cd is accompanied by riaea in free intracellular ca (ca ) and pkc activation. in contrast, evidence from our laboratory suggests that triggering h a cdz occurs through a pkc-independent route. in this study we used mab to various t-cell membrane antigens to localize the activation defect in t cells from asymptomatic hiv-infected men that we previously showed to have an intrinsic defect in their proliferative response to soluble anti-cd nab. compared to hiv-homosexual controls, the monocyte-dependent response of t cells from hiv+ men to soluble anti-cd and a mitogenic combination of anti-cdz mab was decreased as was the monocyte-independent resgpnse to immobilized low-dose anti-cd . in t cells from both groups a normal rise in (ca ) . was induced by anti-cd and anti-cdz mab. however, t cells from hiv-infected men respondad poorly to direct pkc activation by pma in the presence of healthy donor monocytes, whereas control t cells responded vigorously. in contrast, induction of proliferation by anti-cd mab was comparable in t cells from hiv+ and hiv-men. our results indicate in hiv+ men a selective qualitative t-cell defect in activation routes that are dependent on pkc activation, whereas pkc-dependent alternative activation appears to be unaffected. these findings will be discussed with repect to hiv immunopathology and normal t-cell physiology. we have studied the role of the protein kinase c (pkc) in the activation of a prototype th- cell, d .g . . blocking of this enzyme with the drug h- [ -( -isoquinolinyl-sulfonyl)- -methylpiperazinet leads to an increase in cellular proliferation of d cells activated with specific antigen or mitogen. n addition, an increase in the amount of il- and il- mrna is seen under these conditions compared to the level observed in mitogen activated cells in the absence of h- . this superinduction of il- and il- lymphokine mrna is also detected in mitogen activated d cells that are depleted of pkc protein by pretreatment of the cells with phorbol esters. in contrast, mitogen activation of s lenic t cells measured by proliferation is blocked in the presence of h- . these data indicate that jkc has a regulatory role in th- cells by exerting a negative feedback on the events that occur after cell activation avoiding an uncontrolled response. this effect is opposite to that seen in a bulk t cell population that represents mainly th- type cells. the mechanism of signal transmission is, therefore, distinct in the two types of cd + t cells. aberrant expression of class i mhc proteins on non-immune somatic tissue has been implicated in several autoimmune diseases, but it was unknown whether this expression was involved in the initiation of disease or resulted from induction by the immune reaction. we ha e engineered several transgenic mouse lines expressing tissues using eith r the pancreatic elastase promoter or the class i kb gene promoter. the i-as on the pancreas is fully capable of presenting peptide antigens to i-ad restricted hybridomas. h-zb mic expressing i-ad on pancreatic acinar cells but not in thymus are not tolerant to i-as in mixed lymphocyte reactions in vitro, but do not spontaneously produce an immune respon e to the pancreas in vivo. peripheral antigen presentation, i-a restricted immune response, i-a expression by t-cells, and widespread somatic i-ad expression. expression, whereas another has only medullary expression. the effect that these different patterns have on mhc restriction is being examined. ctr., stanford, ca . a transient increase in the intracellular cat+ ion concentration has been shown to occur rapidly following activation of t lymphocytes. this response i s a more immediate and direct indication of t cell activation than either production of interleukins or proliferation. we examined ca++ flux analysis using indo-l and the facs to measure ag-speclfic t cell activation and also to sort for an ag-specific t lymphocyte subpopulation. human t cell clones were exposed to elther allostlmulatory lymphoblastoid cells (which cause proliferation of the t cells) or nonstimulatory lymphoblastoid cells. in both cd + and cd + clones, we observed an ag-specific cat+ flux response similar t o an lonomycin positive control. we then attempted to enrich for antigen specific t cells from a mixed population. two phenotypically different human t cell clones were mixed in equal amounts. after coculture of the cell mixture w i t h lymphoblastoid cells shown to stimulate only one of the t cell clones, the cell mixture was sorted on the facs based on specific cat+ flux. a ten fold enrichment was observed for the ag-specific clone. this approach allows rapld observation of t cell activation and provides a means of sorting antlgen reactive t cells from a heterogeneous cell population. previously attemps to characterize the function of beta- -microglobulin (beta- -m) have used anti-beta- -m antibodies, to block the functional activity of the protein. we now demonstrate that human beta- -m and the proteolytic derivates beta- -m lys and beta- -m deslys added in nanomolar concentrations to allogeneic, pha and con-a stimulated mouse lymphocyte cultures modulate the response induced. thus beta- -m and especially beta- m deslys when added day and augment the generation of specific cytotoxic t lymphocytes in mlc, problably due to stimulation of endogeneous lymphokine production in the culture. both the con-a and pha induced response were modulated. in the presence of beta- -m and its proteolytic derivates the proliferation was optimal at hours, as opposed to the hours optimum in the abscence of beta- -m. these results strongly suggest an active role of beta- -m or proteolytic derivates hereof in the t cell activation and generation of specific cytotoxic t lymphocytes. a ( . fold) . the increase peaked at min and returned almost to baseline by mi?. igm b cells showed random x inactivation, while more mature igg and iga b cells had only the normal x active. we conclude that the xla gene product is required for b cell development, while the xscid gene product is required for both t and b cell maturation. gene mapping has placed xscid in xq - . , linked to phosphoglycerate kinase (pgk), lod . , = . mouse xid, which affects only b cells, is also linked to mouse pgk, suggesting the possibility that xscid and xid belong to related gene families. c institute at denver, national jewish center, denver, co . mapping data for the bxd and akxd recombinant inbred strains shows that the mhc and the products of at least two other genes control the expression of vp . one of these is linked to ly- , and the second for the akxds is linked t mtv- on chromos me . a backcross of b o.br x (c h x b o.br)fl (mls- x ( m l~- ~ x mls- ) ) has been used to confirm the linkage of one of these controlling genes to ly . using the vp -specific monoclonal antibody, exceptions have been found to the general rule, that strains which have eliminated t cells expressing a particular vp specific for a defined self antigen in the thymus, also express this self antigen on their spleen cells, and so these cells are capable of stimulating t cell hybridomas bearing the particular vp in quest ion. the t cell receptor (tcr) of the alloreactive t cell clone c recognizes the h- ld specificity. the cloned a (va . ) and p (vg . ) tcr cdnas have been inserted into plj retroviral vectors containing ltr and regions of the momulv and neomycin resistance genes. three different constructs have been used to transfect v cells. g -resistant clones showing the highest titer of viral particle psoduction have been selected, and injected subcutan ously lacking the h- l vt cells producing retroviruses carrying the a gene have been examined for the expression of the new specificity on the infected t cells. northern blot analysis using the va and the neo-probes showed, in the lymphoid tissues, a transcript with an expected size of . kbs hybridizing with both probes. in the spleen of treated balbfc mice, but not in dm mice, the expression of the exogenous va resulted in proliferation of infected t cells, as assessed by an auto-mlr proliferative assays. into newborn balb/c mice (h- l +) o r into the congenic strain dm loose most of their lytic activity as well as the ability to form conjugates with specific tc. preliminary experiments show that spdp-anti-cd -derivatization of specific tc overcomes the trypsin effect, so that both conjugation and lysis are observed between trypsin-treated pel and sdpd-anti-cd -derivatized specific tc. many investigators, the ti/cd complex on pel surface is central to specific lysis. the observed inhibition of trypsin may be due to its action upon it. since trypsin-treated pel could be triggered toward conjugation with and lysis of tc by anti-cd , the cd at least must have remained intact and is the important molecule for both the critical conjugation step and lysis. trypsin treatment possibly disturbs the interaction of tc with ti/cd of pel so as to prevent proper engagement of the cd . concanavalin a (con a) treatment of tc appears not able to bring back the lytic activity or the ability to form conjugates of trypsin-treated cells, indicating a dichotomy in the mechanism of killing by derivatization with anti-cd versus con a mediation. comparing pel and their blasts ipeb). unlike pel, peb do not show ldcc or lytic activity against spdp-anti-cd -derivatized nonspecific tc. ldcc by con a has been proposed to function via ti/cd interaction. unlike pel, peb also do not show fluorescence. it may be that the ti/cd complex on peb differs from the one on pel. at the same time, total lack of cd is contraindicated for peb kill specifically. the region important in ti interaction for ldcc and that recognized by anti-cd may be within the aberrant area of cd , so neither the con a nor the anti-cd trigger could be transmitted on peb. the importance of the cd is indicated further by immunolabeling both cells with anti-cd revealed that c combination of cd + and cd + islet specific t cell clones are pathogenic in the nod mouse, eva-pia reich and charles a. janeway. jr., department of pathology, section of imunobiology, yale university school of medicine, cedar st., new haven, ct . although diabetes in the nod muse is thought to be initiated by t lymphocytes, thus far no t cell clone has been derived from nod islets. we therefore isolated islets from acutely diabetic nod mice and islet derived lymphocytes were propagated in il- and stimulated every weeks with fresh mitomycin-c treated nod islets to maintain islet specificity (i.e. a proliferative response to nod islets). t cell clones were generated by limiting dilution followed by recloning by single-cell manipulation. facs analysis showed that all clones were positive for thy-l+ and cd +, confirming that receptor bearing t cells had been cloned. cd +cd -and cd -cd + clones were isolated that proliferated in the presence of nod islets. respond to nod but not balblc or b o.br islets, while the two c d ' clones respond to nod and balb/c islets but not blo.br. islet-specific, i-anod restricted, while the c d e clones are islet-specific and dd restricted. injection of individual cloned lines into young, irradiated nod mice leads to no overt or microscopic disease. a syndrome of dehydration, rapid deterioration and death about - days after transfer. mice sacrificed at this time show lymphocytic infiltration around the islets. this suggests that the cd + clones are however, mixture of c d ' and cd + cloned lines causes these cloned lines may be useful in the search for islet cell autoantigens. dusseldorf (frg). autoantibodies to nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. while animal models which spontaneously develop abundant anti-nucleolar antibodies have not yet been described, high titers of such antibodies may be induced by treating susceptible strains of mice with mercuric chloride. we have identified the nucleolar autoantigen against which the hgci -induced igg autoantibodies from mice of strain b o.s are directed. it is a protein of apparent molecular weight of kda and pi value of about . which is associated with the nucleolar snrna u , and by these criteria, is identical with a polypeptide called fibrillarin. it is striking that scleroderma patients spontaneously form autoantibodies against the same u rnp protein. the hgc -induced murine and the scleroderma-specific human anti-u rnp autoantibodies were indistinguishable in their reactivities towards fibrillarin. they further resemble each other in so far as both recognize epitopes on the -kda protein which have been highly conserved throughout evolution. our results provide a basis to investigate at the molecular level whether similar immunoregulatory dysfunctions may lead to the preferential a n t i rnp autoantibody formation in the animal model and in scleroderma patients. forty bp-specific cd + t-cell clones were isolated from the peripheral blood o f a patient with multiple sclerosis. studies with a panel of xenogeneic bps of known amino acid sequence and with laree fragments of the bp molecule demonstrated the existence of at least epitopes recognized by human t-cells. at least of these epitopes were potentially clustered in the middle or at the c terminus of the molecule. in studies with synthetic peptides corresponding to residues - and - of human bp (hbp), clones proliferated in response to - and recognized - . ctl assays were carried out with targets consisting of an autologous b-cell line coated with hbp. nineteen of the clones demonstrated bp-specific ctl activity; these were the same that had recognized peptide - i n proliferation assays. these clones also killed targets coated with peptide - but not targets coated with - . clones that proliferated in response to peptide - or to epitopes located outside of these two regions failed to kill hbpcoated tarpets. to a lack of binding of - to the target cell, as the same b-cell line successfully presented peptide - , in proliferation assays, to clones that had previously recognized this peptide when presented by e-cells. activity against bp is more restricted than is the specificity demonstrated for proliferative activity. the lack of killinp of targets coated with - could not be ascribed prystowsky, department of pathology and laboratory medicine, university of pennsylvania, philadelphia, pa as an approach to studying regulatory factors important during t cell proliferation, the expression of genes induced during il -stimulated t cell growth was examined. to identify genes induced by il , a cdna library was made from il -stimulated cloned t cells in late g of the cell cycle and was screened by differential hybridization, selecting those clones which had higher steady-state mrna levels after il stimulation. of , clones originally screened, positive clones were identified, which represented different genes. when partially sequenced and compared with sequences in the nih and embl databases, clones were identified as cdnas encoding glycolytic enzymes, were cdnas of cytoskeletal genes, were cdnas coding for proteins important in protein synthesis, and clones were not identified. the maximal steady state mrna levels generally occurred during late gl/early s phase, and the induction of most m a s was not inhibited by cycloheximide at a dose that inhibited cell proliferation. nuclear run-on transcription demonstrated an increase in the rate of transcription for gapdh. in addition, there is post-transcriptional regulation of expression, as some mrnas were stabilized upon il stimulation. having cloned these mrnas, we can now begin to study the regulation of expression of selected genes. supported by nih grants. cyciosporin a (csa). a potent immunosuppressive drug, caused organ-spedfic autoimmune disease, such as gastritis with anti-parietal cell autoantibodies or oophoritis with anti-oocyte autoanliies, in balwc mice when the drug was administered daily for one week to newborns. administration to adult mice did not. csa abrogated the production of l t ' t cells and lyl- ' t cells in the thymus. consequently, these t cells were substantially depleted from the peripheral lymphoid organs, especially when the drug was administered from the day of birth. autoimmune disease was prevented when csa-treated newborn mice were inoculated with splenic t cells from normal syngeneic mice. on the other hand, removal of the thymus immediately after neonatal csa treatment produced autoimmune disease with a higher incidence and in a wider spectrum of organs, i.e.. thyroiditis, sialoadenitis of the salivary gland, gastritis. insulitis of the endocrine pancreas, adrenalitis, oophoritis, or orchitis. each autoimmune disease was accompanied by the development of autoantibodies specific for the corresponding organ antigens. it is likely that csa caused organ-specifc autoimmune disease, when administered to newborns, by depleting certain regulatory t cells (suppressor t cells) controlling self-reactive clones. (this work was supported by a grant from the lucille p. markey charitable trust.) artur scherf, philippe dubois, marika plat and luis perelra da sllva, instltut pasteur, - , rue du dr. roux. paris, 'u inserm, hopltal st. louis. , place du dr. alfred fournier. paris, france. we isolated from a genomlc expression library of the malarla parasite p. falciparum a clone coding for an antigen associated with the membrane of infected erythrocytes. intensive sequence analysis of this gene, called - , showed that the major part of this molecule consists of at least repeats of nine amino acids. the consensus sequence of these degenerated repeats is e-e-v-v-e-e-v-v-p and secondary structure analysis predicts t h a t they have a high tendency t o form amphipathic alpha helices. the -and t-cell response t o the two most frequent types of repeat were analysed in five different h- congenlc mice strains using two synthetic peptides (pqa: y-p-(e-e-v-v-e-e-v-v-p)n-k: pqb: the -cell response t o both peptides is restricted to the h- d and h- k xaplotype. pca and pqb specific antibodies do not dlscriminate between the two peptides (measured by elisa). the mhc restriction was confirmed by anaiyslng the tcell response to both peptides. t-cell lines speclfic for each of the peptides pqa/pqb were derived from h- d responding mice. although the peptides differed in four positions of the hydrophoblc part of the helices (valine replaced by isoleucine and leucine) no signiflcant dlfferences were observed in the antigen presentation between peptide pqa and pqb. in contrast, both peptides were unable to induce a proliferation of the heterologous t-cell ilne. suggesting t h a t the variable hydrophobic amino acids of the repeats are involved in tcell recognition. we are currently investigating if other variant repeats of the - antigen are restricted t o a different mhc haplotype and that the totality of repeats may be subjected to no restriction. glven the type of amino acid replacements within the two repeats tested, the t-cell receptor appears t o discriminate between very similar amino acids. auphan, claude boyer, annick guimezanes, claire langlet, cenae dimmunologie inserm-cnrs de marseille-luminy, case , marseille, cedex , france. the yinterferon gene is activated upon cell surface triggering of the aansmembrane t cell receptor (tcr)/cd complex or via phosphatidylinositol anchored thy- molecules cell clones. both these activation events are inhibited by antibodies directed at the lyt- jlyt- molecules and are lost on tcr a-chain deletion variants ( , ). biochemical analyses failed to reveal any association between the tcwcd complex and either lyt-wyt- or thy- molecules. however, such studies consistently revealed a non-covalent, cis-type association between lyt-ulyt- and h- class i products as well as between thy- and h- class i products. the structural features of the molecules required for such interactions are being determined using hybrid engineered molecules and possible implications of such interactions for t cell activation are being evaluated. ( ) schmitt-verhulst, a.-m. et al. nature , - ( ) . ( ) guimezanes, a. et al. cellular immunol. , - ( ) . i t i s increasingly apparent that the association of both chains is critical to reconstitute t,he g-mhc binding site. combinational mechanisms for generating diversity play a major role in increasing the total size of the repertoire. however, evidence for preferential associations have been described between other heterodiaeric proteins of the immunoglobulin gene superfamilg. i t is therefore important to determine if association between different sets of vas and ybs occur at random or if, alternatively, preferential associations exist. we have devised a system which should enable us to determine, by dna mediated gene transfer of cloned cdnas encoding different vas o r vbs, if such preferential associations do exist. we hare also used a panel of monoclonal antibodies specific for a particular va or vb to derive t cell clones expressing these vas or vbs. in a polyclonally activated t cell population expressing a specific va or vb chain, we will present data indicating that the association between these chains is preferential or random. !nteractions between t cells and mhc molecules are thought to select a t-cell repertoire skewed towards recognition of antigens in the context of self mhc molecules. in addition, t cells which react strongly to self mhc molecules are eliminated by a process called self-tolerance. we have recently described generation o f transgenic mice expressing the a t-cell receptor (tcr) from the cytotoxic t lymphocyte c (sha et al., nature : ) . the clone c was derived from a ealf .e (h-zb) anti-eale/c (h- d) mlc and is specific for the ld class i mhc antigen. in transgenic h- b mice, a large fraction of t cells in the periphery expressed the c tcr. these t cells were predominantly cd -cd + and were able to specifically lyse target cells bearing ld. in the periphery of transgenic mice expressing ld, functional t cells bearing the c tcr were deleted. this elimination of autoreactive t cells appears to take place at or before the c +cd + stage in thymocyte development. in addition, we report that in h- s mice, a non-autoreactive target haplotype, large numbers of cd ' t cells bearing the c tcr were not found providing strong evidence for the positive selection o f the c tcr specificity by h- molecules. the requirements for cd expression during t cell differentiation in fetal thymus organ culture woc) were investigated by analysis of the effects of addition of anti-cd mab. control etoc cult& for days contained predominantly cd +cd + cells as well as cd or cd single positive cells expressing high levels of cd . t cells from ftw cultured for days in the presence of anti-cd m a b expressed markedly diminished cd expression. when anti-cd m a b was removed hours prior to phenotypic analysis, it was found that cd was recxpressed on cd + cd + t cells. after days of culture, control roc contained high frequencies ( - ) of cd +cd and cd -cd + thymocytes expressing high levels of cd . in contrast, anti-cd mated cultures, while containing cd -cd +, cd + t cells, were well depleted of cells which reacted with anti-cd antimy. when anti-cd mab was removed hrs prior to analysis, cd was reexpressed on cd +cd + cells, and low frequencies ( %) of cd + cd -t cells were detected, but these cd single-positive cells did not express high levels of cd or detectable ap heterodimrs. while normal frequencies of cd -cd +,cd + cells expressed vg determinants, the a n t i -w mated cultures were relatively enriched in cd -cd +cd and cd -cd -cd -t cells compaml to control day eoc. these data demonstrate that while cd +cd + t cells can differentiate in the presence of anti-cd mab, differentiation of cd +cd + cells is inhibited and cell surface cd is down-modulated. cd +cd + cells can develop in the presence of anti-cd m a b and do express t cell receptors. these results suggest that cd expression, while not required for development of cd +cd +cd + t cells, is necessary for differentiation of mature cd +cd -cd + t cells, and that the interaction of with its ligand is critical for thymus selection of class-ii specific t cells. uppenkamp, and alfred singer, experimantal immunology branch, nih. bethesda, md . in order to investigate the role of the t cell receptor (tcr) in thymocyte maturation, we have studied thymocytes from the immunodeficient c.b-l /scid mouse, which due to a genetic defect, is unable to express tcr genes. despite this mutation, scid thymocytes were functional as they produced lymphokines and proliferated in response to stimuli (pma, ionomycin, il- , il- ). phenotypic analysis revealed that, like normal immature thymocytes, sci+dd'&mocytes were large, thy . +, cd -, cd -, tcr-and were enriched in cd , il-zr+, pgp-lf, and hsa+ cells. however other tcrpopulations present in normal mice, (i .e. cd - +tcr-and c d ' ' t c r -cells) were absent from scid mice. in order to determine if tcr expression was required for cd and cd expression, we analyzed thymi from the occasional scid mouse which possessed small numbers of tcr-bearing thymocytes. interestingly, expression of cd or cd was detected only in those scid thymi that possessed tcr' thymocytes, although cd and cdb expression was not confined to those tcr-bearing cells. further, the introduction of tcr' cells from thy congenic normal mice into tcr-scid mice consistently promoted the expression of cd and cdb (but not tcr) by scid thymocytes. moreover, scid thymocytes from these chimeras possessed cd + + cells, however no mature, single positive thymocytes of scid origin were detected. together, these results suggest that tcr-bearing cells within the thymic milieu are required for the expression of cd and cd and for the differentiation into cd + + cells. however, final maturation into mature single positive thymocytes requires that the thymocyte itself express tcr. tcr in thymocyte differentiation: evidence from the immunodeficient the cd t-cell surface receptor has been implicated in the stabilization of lntercellular interactlons. in addition, it is felt to mediate the transduction of an independent lntracellular signal. we have recently shown (veillette et al.. in press) that the cd molecule expressed in t-lymphocytes is complexed to the internal membrane tyrosine kinase p 'ck. suggestlng that alterations in the properties of thls enzyme mediate the cd related signal. to gain further insight into the structure and function of the cd -lck complex, we have generated nih t flbroblasts that co-express the cd and lck proteins. nih t flbroblasts expressing a murine lck cdna were transfected by calclum-phosphate preclpitatlon with a construct containing a murine cd cdna and the neomycin resistance gene. stably transfected clones were selected for resistance to the amlnoglycoslde c . we have now demonstrated that the cd and ~ '~~ expressed in these non-lymphoid cells can form a stable non-covalent complex. the structure and function of the cd -lck complex expressed in murine fibroblasts is currently being evaluated. h- b t c e l l s from h- b+hh- bxd responder animals fail to generate one (type a) of these two major clonotypes. environment i s permissive for the t cell specificities of both poly- clonotypes (type a and b) and also rovides the determinants for t h e i r selection. the absence of type a clones in the h- b+h- gxd chimeras can therefore only be attributed to the failure of h- b t c e l l s to generate the appropriate t cell receptors. these results suggest a genetic hole in the t cell recognition repertoire of h- b mice for the lysine-containing epitope of the poly- antigen. we have used oligonucleotide-primed gene amplification to study the association between hla class i alleles and autoimmune disease. pemphigus vulgaris (pv) is a severe autoimmune skin disease highly associated with dr and drw . we have shown that the nucleotide sequence of the first (and most polymorphic) domain of a new dq allele is found in / israeli drw pv patients but only / or& healthy israeli controls. this allele differs at only position from two other dq alleles not associated with pv. further, we show that a shared setuence in the third hypervariable region of the first domain of two common subtypes o f dr and drw cannot be the sole determinantdrgf susceptibility to pv. none of dr /drw + pv patients had this epitope. we also report a new dr first domain sequences from a drw pv patient. the frequencly of t h ! $ allele in patients and controls is being assessed. similar studies to identify disease associated class i sequences are underway in patients with multiple sclerosis and rheumatoid arthritis. the frequency of these autoimmune responses is determined genetlcally. and is essentially % in the projeny derived in crosses d transgenic mice to some inbred strains of mlce [e.g. those of h- d haplotype). and yet only in others (e. g. h- b) . recently. we initiated an analysis of the i m m~~l~g i d nonresponsiveness in two lineages of insulin/t antigen transgenic mice with early onset of expression of the transgene (rip- tag # and rir tag # ). while the presence of the transgenic protein durlng the prenatai/neonatal period is the primary requirement for the nonresponsive phenotype, it further appears that the levels of t antigen expression durlng embrydnic development quantjtatively influence the subsequent nonresponstvmess in adult me. an additional modification is exerted by a genetic component whlch seems to map to the mouse major histocompatibility complex [h- ). interesiingly, it appears that the h- d haplotype. whlch is associated with the most complete impairment of immune responses when t antigen is expressed prenatally. is also assodated with the highest frequency of autoimmune responses when the developmental expression of the transgene is delayed. this correlation is considerably strengthened by the observation that outcrosses to c b / j m-zb) produce mlce with either a weaker tolerance in the case of the early expressers. or a less hquent and weaker autoimmune response in lines with a delayed onset of expression of the transgene. thus. the two distinct biolcgical consequences of the presentation of this antigen (tolerance or autolmmunity) are &ly to result from differences of the developmental maturity of the immune system, and both effects appear to be similarly mhc restricted. lmmunogenicity c mapping the pzb geneti contribution t o lupus-like autoimmune disease. i-division o f basic science, national jewish center for immunology and respiratory medicine, jackson street, denver co ; -the jackson laboratory, bar harbor, me. nzb mice carry a geneb) that contributes to the lupus-like autoimmune disease seen in (nzw x nzb) fi hybrids. however, the precise locus of this gene has yet t o be determined. to map i t s location, we are breeding a panel of nzbxsm/j recombinant inbred (ri) with nzw mice. by determining which of the ri strains produce fi animals that develop autoimmunity, the location of this nzb gene can be deduced based on the strain distribution pattern of the known genetic markers among the nzbxsmlj ri strains. cells were then fractionated on a percoll gradient to obtain small dense 'resting" b celb and large 'activated' b cells. the small dense b celb demonstrated increased inhibition at every dose of ic examined, when compared to large b cells. for instance, pulsing with ug of ic resulted in a % reduction in anti-fl, but not anti-trinitrophenyl, plaque forming cell (pfc) response for small dense b cells. in contrast, the pfc response of large "activated' b cells was reduced only %. in addition, when subtolemgenic doses of ic were used ( ng) prostaglandin € ( x - y) could function as a potent secondary negative signal rendering these b cells susceptible to negative signalling. we concluded that small dense b cells are more sensitive to ic induced unresponsiveness and that prostaglandin € may further sensitize b cells along this pathway. . were developed in the mouse against antigens present on humans, hamster and rat nk and t lymphocytes. pre-incubation of lymphocytes with moab did not block the ability of the nk cells to bind to targets but did block their ability to lyse nk sensitive targets. the pre-incubation media contained lytic factors that lysed nk sensitive targets in a h chromium release assay. interestingly pre-incubation of thymus cells or resting peripheral t lymphocytes with the moab induced the t lymphocytes to release lytic factors and acquire nk activity against nk-sensitive targets. lytic factors and acquired lytic activity were unable to lyse lak targets. signalling events did not involve cross linking or fc receptor participation since factors were immunoprecipitation and western blot analysis using the moabs defined a molecule of kd on the surface of thymus or lung mononuclear cells. isolation and amino acid sequence analysis is being done to assist in the characterization of the g . we have been studying in vitro activation of cd -, cd -thymocytes. in the present studies, highly purified double negative thymocytes were cultured with il with or without il . following a hour culture with il + il (in the absence of mitogens) significant proliferation was detected, although all cells remained cd -, cd -. the frequency of cells staining for cd cell surface protein increased from % to % and tcr vbeta from % to %. facs sorted cd -cells from the cd -, cd population showed similar increases in cell surface tcr suggesting differentiation. northern blot analyses showed an increase in c -m rna at hours, no change in cd delta or epsilon expression at , , and hours, and a marked increase in the ratio of . to . tcr beta transcript between and hours. these data suggest that il + il can induce cd cell surface protein expression on progenitor thymocytes. furthermore, this induction is not associated with increased cd transcription, but rather is associated with an increased proportion of functional tcr beta transcripts. the p antigen of schistosoma mansoni has been shown to be able to induce protective immunity against schistosomiasis in rodents and primates. synthetic peptides were used to localize the major b and t-cell epitopes of this protein. the primary structure of the p molecule was analyzed using various predictive algorithms : surface localization, hydrophilicity, flexibility, secondary structure, amphipathicity of predicted a-helical regions, rothbard pattern. according to these criteria, several peptides were synthesized and tested in immunological assays in vitro and in vivo. correlations between these results and the physico-chemical predictions will be shown. simian virus (sv ) t antigen is recognized by cytotoxic t lymphocytes (ctl) in association with class i major histocompatibility antigens.we have identified four distinct antigenic sites on sv t antigen defined by h- db restricted ctl clones. we have therefore attempted to map these antigenic sites precisely by using overlapping synthetic peptides corresponding to sv t antigen amino acids. the synthetic peptides of - amino acid length were incubated for five hours with lcr labelled-non-sv transformed h- b cells in the presence of ctl clones specific for each of the four antigenic sites; and the released radioactivity was used as a measure of specific recognition of synthetic peptides by the ctl clones. the results demonstrated that the antigenic site i resides between amino acids - ; sites ii and between amino acids - . site can be separated from site ii as it was found to reside between amino acids - . the antigenic site v was localized between amino acids - . these results show that sv t antigen is the target protein for ctl and the regions of t antigen recognized by ctl are tightly clustered in the amino terminal half. attempts to identify amino acids that selectively interact with the class i antigen and the t cell receptor are in progress. peripheral blood lymphocytes of cancer patients demonstrate an early humoral and specific response to application of tumoral extracts. the response is measured as a change in the spectrum of fluorescence polarization of fluorescein molecules. this is probably a measure of sol-gel transformations induced in the cytoplasm upon cellular activation of resting lymphocytes. this measurement is carried by epifluorescent microscopy on single lymphocytes, sitted on a regular holes matrix that is scanned under computer control. it is possible to follow changes within each cell in a population of upto ten thousand cells. since the cells are sitted safely in their holes, it is possible to rinse them with various reagents and follow the kinetics of individual responses by repeated scanning. further studying of the stimulative process might be useful in identifying the responding lymphocytes and the tumoral reagents. this test of the immune system is intended for early and specific detection of cancer and for follow-up of immunotherapeutic manipulations as well. c katharine s. ullman, j.p. shaw, elizabeth emmel, and gerald crabtree. stanford university, stanford, ca . following antigenic stimulation, t cells undergo an orderly series of changes that result in cell division and immunologic function. in hopes of working backward in the cascade which links the membrane events to the genes essential for these differentiated functions, we have begun an analysis of the nuclear proteins responsible for gene activation in stimulated t cells. in previous studies, using a panel of internal deletion mutations of the l- enhancer, we found that when the region from - to - was mutated, the level of induction dropped -fold. this region is protected in a dnaase footprinting assay by a protein, nf-ilz-a. although the protection is seen with nuclear extracts from both stimulated and resting jurkat cells, in vivo this is a site of dnaase hypersensitivity found only in activated jurkats and peripheral blood t cells. a tetramer of the protected site directs transcription from an unrelated promoter in jurkat cells only when the cells are stimulated by lectin or antibodies to the antigen receptor. furthermore this activation can be blocked by ng/ml of cyclosporin a. as a means of detecting more subtle changes in protein binding following stimulation. the methylation-interference patterns of stimulated and non-stimulated jurkat cell nuclear extracts were compared and found to be identical. we propose that though the dna-binding protein, nf-il -a, is present constitutively, it becomes functional only following specific signalling through the antigen receptor. further characterization of this protein will help to clarify its particular role in the activation of il- expression following t-cell stimulation. the negative e f f e c t of f l a n k i n g sequences on t c e l l lmmunogenicity c central lab. blood transf.service, lab. exp. and clin. immunology of the univ. of amsterdam, the netherlands the hydrolysis of phosphatidylinositol-diphosphate and the formation of inositol-triphosphate (ip ) and diacylglycerol (dg) re among the first events that can be monitored after t-cell triggering. ip mobilizes c a ' from intracellular stores, whereas dg is the physiological activator of protein kinase c (pkc). although it is suggested that activation of pkc is essential in the induction of mitogenic t-cell responses, no direct evidence for this hypothesis has been presented yet. in this study we used two novel pkc inhibitors, .e. -alkyl methyl-glycerol (amg) and staurosporine to assess the role of pkc activation in human t-cell stimulation. we observed that the inhibl$ors did not interfere with the anti-cd or anti-cdz monoclonal antibody (mab) induced ca rises in peripheral blood t cells, whereas cytoplasmatic alkalinization initiated by the same stimuli or the phorbol ester pma was strongly reduced. amg as well as staurosporine gave a dose dependent inhibition of both interleukin (il- ) production in jurkat cells and t-cell proliferation. in contrast, the induction of il- receptor expression was highly resistent to the action of both compounds. it was shown that anti-cd mab were able to partly overcome the action of both inhibitors. these studies support the important role of pkc-activation in human t-cell triggering. moreover, since anti-cd mab were able to counteract the action of the pkc-inhibitors, it is suggested that mitogenic stimuli generated via different t-cell membrane molecules may use distinct intracellular signal transduction pathways. a f t e r incubation w i t h ndlal- o r al- , t a r g e t s o f @ phenotype were lysed by a n t i -a k i l l e r c e l l s . the a n t i t h e s i s was a l s o observed: a phenotype t a r g e t s were lysed by a n t i - t c e l l s a f t e r incubation w i t h ndlgl- . ndla we have developed a monoclonal antibody that recognizes an apparently t cell-specific antigen, ltac, expressed only in the late stages of t cell activation. when peripheral blood lymphocytes are activated with allogeneic stimulators or pha and examined by facs, ltac is expressed only after day or . ltac is expressed by all t cell clones tested, both cd ' and cd '. a wide range of cell types were tested and found negative for ltac expression, including b cells, fibroblasts, endothelium, and neural cells; thus ltac expression seems to be restricted to the t cell lineage. immunoprecipitation of cell-surface labelled proteins and sds-page revealed a kd band under reducing conditions and three bands of , , and kd under non-reducing conditions. ltac is distinct from all other reported t cell activation antigens on the basis of its pattern of expression and molecular weight. we have purified ltac protein using wheat germ agglutinin-agarose chromatography followed by monoclonal antibody-agarose chromatography, and have used it to produce an antiserum suitable for screening bacterial cdna expression libraries. the regulation and function of molecules expressed in the late stages of t cell activation is an important frontier in understanding the immune system, and reagents such as monoclonal antibodies may have important diagnostic and therapeutic uses. balb.h.p mice were capable of responding to all antigens tested but the magnitude of the response to some of these antigens ( of antigens) was reduced - fold when compared with balb/c mice. responses to the remaining antigens were either comparable ( of antigens) or occasionally even enhanced ( of antigens) compared to balwc mice. these results suggest that jp and dg elements are required to maintain the strength of the t cell repertoire and that, in the wild, mice carrying this deletion a u l d be at a significant selective disadvantage. the identification of - was carried out in radiolabeled murine macrophages by immunoprecipitation and sds-page, using antibodies to were predominately of mr . and . , respectively. a s assessed by estimating radioactivity in each band, -larwas about fold more abundant than - . most of the cell associated - activity, as measured by the d .g . proliferation assay, could be inhibited by anti -iswhereas no inhibitory effect was observed with anti - b. after removal of -ltc from cell lysates by immunoadsorbtion the residual activity accounted for only about % of the total - activity, and was completely neutralized by anti - b. however, in culture supernatants anti i - r reduced the - activity by approx. % whereas a combination of both, anti -leand anti - completely neutralized the activity to background levels. interestingly, only one radiolabeld polypeptide of m, . was precipated from the extracellular medium by the anti - antibody. these data therefore suggest, that externalization is required in order to generate bioactive - molecules, but apparently a proteolytic processing step of the precursor is not involved. moreover, in view of previous reports, demonstrating that cellular interactions during antigen presentation can be mediated via a membrane form of - , o u r experiments substantiate the hypothesis that membrane-associated, biologically active - is predominantely i - q. david l. brandon and joseph w. corse. usda agricultural research service, western regional research center, albany, ca in order to prepare antibodies which would recognize -glucosides of cytokinins (purine phytohormones), we investigated an alternative to carbodiimide reagents. carbodiimides induce the formation of amide bonds, and thus can be used to couple haptens containing carboxyl or amino groups to proteins. the use of water-soluble carbodiimides is limited by the sparing solubility of some haptens in water and by the formation of the intermediate -acylisourea, which can be less soluble than the hapten and which can itself act as an unwanted hapten. to overcome these problems, we have used -morpholinoethyl isocyanide and a suitable catalyst to effect amide bond formation. these reagents are the basis of recently developed chemical procedures for peptide synthesis (aigner et al.. ) . the reaction conditions permit coupling compounds which are soluble or insoluble in water to proteins in a simple, homogeneous system. we have demonstrated that this procedure is widely applicable for preparing enzyme-labeled and immunogenic conjugates of cytokinins and other biologically relevant compounds with an available carboxyl group. we hypothesize that conjugates made with these reagents will not induce the allergic responses and unwanted crossreactivities associated with the use of carbodiimides. supported, in part, by bard grant us- - . mlman r i g , not uwuse rig , uxluces a furctional y-btut-e subset of mdlse thymqtes to. secrete - , shichin turns iniuces cells w i t h i n this subpapllation to becane cytotcouc. nolmal mxls~ thymocvtes beaw ctl in to con a * il- , and that the cplresponses induced by-agentsm blocked canpletely by the addition of the anti-- nyib lb . in addition, normal muse thymxytes produced il- in response to con a f i t . the fijmtionally-ture lobster agglutinin (mgl)-positive t h y r e p subset responded to con a f i g in a similar mmer. ylhen the functionally-nunature subset of mgl-negative thymzyk subsets was incubated with con a f muse r i g , they did not becane ctl and &d not prcduce il- . haever, when wl-negative thymcytes were cultured w i t h con a f human r ~ , cells w i t h i n this subpsxllatim developea gccd ctl activity and also secreted 'ihese data qmnstrate that cpl generatim and i g pmauction occur mnccanitantly i n dl thymqte m a t i o r r s studied. momever, stirmli which f a i l to induoe cn generation also f a i l tostinulate erd qencu.s iir praduction. immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of t lymphocytes including those which produce gamma interferon. since the surface protein antigen (spa) derived from typhus group rickettsiae has been shown to be an effective immunogen in animal models, human t cell clones specific for the spa of rickettsia tvoh were isolated and tested for their antigenic specificity as well as for their ability to produce gamma interferon. eighteen cm-positive clones specific for the spa of r. t v p b exhibited considerable diversity in their response to the spas derived from two strains of p. orowa zekii and from r. c a n a b . the vast majority of clones also recognized the spa's from & or w w but not from r. c w . two heteroclitic clones demonstrated significantly higher proliferative responses to the spa's derived from one or both of the of r. orowazek i i strains than to the spa of r., and one clone demonstrated a significantly higher response to the spa of r. t v o k as compared to the other spas. all clones produced gamma interferon in response to spa stimulation. one of the clones demonstrated significant proliferative responses to a fragment of the spa produced in e. coli infected with a recombinant lamda gt phage containing a kb fragment of the spa gene from r.. we conclude that the spa's from typhus group rickettsiae can elicit both a diverse t cell response in humans as well as the efficient stimulation of gamma interferon-mediated immunity. a monoclonal anti-mouse rbc antibody (g- ) has been prepared which appears to represent a pathogenic autoantibody related to those that arise spontaneously in aging nzb mice and which cause autoimmune hemolytic disease (aihd). this autoantibody recognizes native erythrocytes from mice but not from other species, and northern blot analysis revealed strong hybridization with the j probe but not with the other vh gene family probes tested. thus, g- is clearly distinct from autoantibodies that recognize bromelain-treated mrbcs and which belong to a unique vh gene family. when g- -producing hybridoma cells were grown as tumors in balb/c mice, the mice developed aihd characterized by a decrease in the number of erythrocytes (hematwrit) and the development of coombs-positivity. an anti-idiotypic antibody (e ) was prepared against g- and was found to recognize an idiotypic determinant present on most autoantibody forming cells derived from old (coombs-positive) nzb mice. previously, we demonstrated that treatment of spleen cells from young nzb mice with g- + c or with anti-ly antibody + c resulted in the elimination of suppressor cells and the generation of afc specific for mrbc. treatment with a control nzb igm mab (g- ) + c or with a rabbit anti-mrbc antiserum + c had no effect on the afc response to mrbc. similar deletion of suppressor cells was obtained following treatment of spleen cells from balb/c mice with g- + c. greater than % of the resulting afc expressed the g- idiotype. the results indicate that the response to self-erythrocytes is comprised of autoantibodies that express a recurrent idiotype. in an attempt to determine the optimal molecular parameters for the synthesis of anti-pathogen vaccines, we have studied the murine antibody responses to two types of model vaccines. the vaccines were: ( ) purified polysaccharide type derived from pneumococcal bacteria, and ( ) synthetic conjugates made from a peptide region of the circumsporozoite coat protein of the murine malaria pathogen, p. berghei, coupled with dextran of high molecular weight. in both cases the molecular size was systematically vaned, and an optimal size was found to be important in determining the level of immune responsiveness. in the case of the malaria peptide-dextran conjugate, epitope multiplicity (epitope density) was also found to be an extremely important parameter, immunogenicity increasing with increasing epitope density. the james p. allison. cancer research laboratory, university of california, berkeley, ca. . we have produced an antiserum to a synthetic peptide corresponding to a portion of the murine cd gene product. this antiserum reacted specifically with cells expressing cd . flow cytometric analysis of normal lymphoid tissues revealed that thymocytes were heterogenous in the level of cd expression while splenic t cells expressed cd at uniformly high levels. cd expression with that of other t cell differentiation antigens (il- r, j l l d , cd and cd ) in the murine young thymus and during fetal ontogeny was analyzed using three-color flow microfluorometry. our results indicate that cd expression is correlated with the stage of thymocytes maturation and demonstrate that il- r expression in the thymus is not induced via stimulation of the cd pathway. klinische argeitsgruppen fur rheumatologie/immunologie,schwabachanlage , erlangen. accessory cell depleted resting (la) t cells are not able to produce autocrine growth factors when stimulated via the t cell receptor complex (tcr), although il- receptor expression takes place. the addition of accessory cells restores the proliferative capacity. we have tested a series of recombinant lymphokines and growth factors for their ability to substitute this accessory cell function for human cdb and cd t cells. triggering via the tcr was achieved by a combinatorial stimulation with anti-cd and either anti-cd o r anti-cd that gives rise to il- receptor expression only on the corresponding t cell subpopulation. ll- , tnfa, if+, il- , il- , ll- and gm-csf were either negative or induced only a minor proliferative response. however, the combination of il- with il- was nearly as efficient as il- in cd t cells. cd t cells were much less stimulated by il- + il- . effect, which is in contrast to some reports on accessory-cell depleted mouse t cells whose function require costimulation with il- . this observation might be important for the understanding of t cell proliferation in chronic inflammatory tissues in which il- as well as il- is produced in large amounts. structural and serological studies of these secreted effector molecules have suggested that they are disulfide-linked dimers bearing t cell receptor u and chain d terminants. a t cell hybridoma, mts . , constitutively antibody staining indicated mts . expresses a t cell receptor containing a vg element. northern and southern analyses indicated mts . used v and va genes to encode the t cell receptor. the secreted mts . suppressor molecule was bound by monoclonal anti-v antibodies and by antibodies specific for constant region determinants of the u chain. to assess the role of a and chain genes in encoding the secreted suppressor molecule, we have generated v -and vu -negative variants from the mts . parent. experiments performed to date indicate that the loss of the v gene results in the inability to produce the suppressor molecule. the results are consistent with the idea that the t cell receptor u and chain genes are used to encode these hapten-specif ic/mhc-restricted secreted suppressor molecules. producing a dnp-specific/h-zk -restricted suppressor molecule was generated and studied. sensitivity to clonal deletion and selection for mhc restriction a m to be a feature of a particular stage of thymqte &el*, specifically l a t e mrtical cw+ + cells. in mcent studies we have slwm that early events i n signal trarrwluction i n respnse to antigen r e c p x crosslinkiq differ in the bmdture cw+ + p p l a t i o n and the mature cw' -or a>rl- p p l a t i o n . m t s indicate that antigen receptolg on hath inmature and mature -itive t cells tmnsdwe signals via calcium mobilization, h-er the maqnitu e of fnflux of e&acellular q'+ wfiich follckfi birding of antireceptor a n t q d i f f e r s tetmen these pqulaticns. specifically, imnature cells shcw a m& reduced q influx espcnse carpared to mature cells. we dmw here that dligation of ~~n p has different amepexe w i t h regard to q'+ nnbilization in mature and inmnture cells, no sucfi difference is seen f o l l a d r q ligaticm of the receptnr's transducer, a. ihe zpsults suggest that the signall* cascade leading to the influx of extracellular is intact hen c d~ is ligated, but is inccnplete w i e i i m p , the physiological liw, is ligated. in addition, ligation of cw or cd on bmdture t cells i n a~~ influx of extracellular a ' + canparable to that sem i n mature t cells. a clonal population has been isolated frcm inmnture thymocytes whir has the characteristic signal tramdmtion pxqerties of the tulk of inmnture thymdcytes. 'ihese f i r d h p suggest that "signal" transfer frcm xpnp to may be inefficient in cd + + cells. struchual analysis of the xpnp/cd canplex in hnature and mtum t cells is in progress. flood and alan friedman, dept. of pathology, yale university school of medicine, new haven, ct, . protective immunity to the ultraviolet (w) light-induced sarcoma -re is directed toward a single tumor-specific transplantation antigen expressed by the -re tumor cells. termed the a antigen. a progressive variant line of -re, termed -pro , lacks only the expression of this a antigen. immunization of mice with -re tumor cells haptenated with trinitrophenyl (tnp- -re) leads to the subsequent rejection of tnp-haptenated progressive tumors and to increased delayed-type hypersensitivity and ctl responses to tnp in normal, immunocompetent syngeneic mice. however, little or no humoral immunity to tnp is seen in animals injected with tnp- -re tumor cells. pro did not exhibit tnp-specific tumor protective or cell-mediated immunity, but rather exhibited tolerance to subsequent immunizations with more immunogenic forms of tnp. biochemical and molecular genetic studies have revealed the a antigen to exist on the cell surface as a complex of class i mhc-like molecules. transfection studies with dna encoding each of these molecules into -pro reveals that the expression of one, and only one, of these three molecules mediates this increased immunity. these experiments suggest that an mhc-like antigen expressed on the -re sarcoma acts as a natural adjuvant to increase cellmediated but not humoral immunity to linked antigens. the mechanism of this increased immunity is discussed. efforts to immunize cattle against economically important gastrointestinal nematodes showed that inrmunity is manifested by: ) a response that reduces the fecundity of established and subsequently acquired worms. and/or ) a reduction in the number of worms developing upon challenge infection. however. the ability of individuals to mount such immunity is highly variable. extending these studies to naturally infected populations indicate that there is a great difference in the number of eggs excreted by individual young calves on pasture. to delineate whether these differences were the result of host genetics and to begin to elucidate the mechanisms of resistance to parasite infection, a genetically defined cattle herd was assessed for parasite levels by determining fecal eggs per gram. three years of sampling of the calves during their first grazing season indicates that: ) certain individuals in the herd will consistently excrete high or l o w numbers of parasite eggs, ) the high or low phenotype is significantly controlled by the genetic make-up of the calf, and ) the high or low phenotype is highly heritable (heritability - ). susceptibility is currently under investigation. calves have been determined and mhc class i typing is currently in progress. information is being used to assess the role of the bovine mhc in controlling immunoresponsiveness to parasite antigens. we have been studying the differential effects of il + il versus il on the growth and differentiation of cd -, cd -thymocytes. culture of highly purified cd -, cd -thymocytes with il ( u/ml) + il ( u/ml) resulted in marked proliferation and increased cell size without change from the cd -, cd -phenotype. culture with il or il alone did not cause proliferation. a substantial contribution to the proliferation was secondary il release: addition of anti-il ( b ) blocking antibody inhibited proliferation induced by culture with il + il . il mrna was demonstrated by northern blot analyses after and hours of culture with il + il whereas none was detected at culture initiation and very little was present at hours. effects of il + il on il transcription rate will also be reported. despite a marked inhibition of proliferation with anti-il , there was no affect on expansion of cd + cells following culture with il + il (increase from % to % in hours). thus, in this system, il enhances proliferation of progenitor thymocytes but does not contribute to induction of t cell receptor. carplex. lhese cwplexes can be used to inmmize mice in the absence of protein carriers or adjuvants, thus facilitating the study of the inmum to a sml ckmically defined antigen. use of this tecfinology has allawed us to identify two t helper cell epitopes in cclllserved regions of hn gp not previcusly identified by computer algorithims, defined by amino acids - and - . inummization with these peptides in pptide-@nqhlipid cwplexes results in the prpauction i* and i* antibodies, which cioss react with cloned fragmnts of the w l e protein. us& this &logy we have begun to characterize the innume response to individual peptide antigens. ?he reqonse of h -k mice to amino acids - of p of hiv, has been analyzed. lhe optimal dose of a peptide containing both b and t cell epitopes was found to be - ug, depending on the route of administration. im d z a t i o n requir& less antigen for opthum antibody resporrsf, than did ip. ment for an ant-response. additional variables, as phosfholipid carpasition and method of cross-linking have been studied anl will be . webelievethattheuseof this peptide-phospholipid canplex tehmlogy will be significant both for studying the innwe response to single epitcpes and for vaccine develolment. based on assays in which t cell proliferation was induced via oxidative mitogenesis and exposure to mhc alloantigens, it has been reported that langerhans cells (lc) isolated from normal mouse skin aquire maximum capacity to activate t cells only after hours of culture. we have studied lc from balb/c mouse skins for their capacity to present ovalbumin (ova) and ia doantigens to unprimed t cells and to antigen-specific t cell hybridomas. the data reveal that both fresh and cultured lc presented ova and alloantigens with equal efficiency to previously primed responders (and with fold greater efficiency than spleen cells or the b cell lymphoma a . - ). by contrast g& cultured lc displayed the capacity to present antigen to ynorimed t cells. we propose that the antigen presenting potential of freshly prepared and cultured langerhans cells, respectively, reflect the in vivo functional properties of intraepidermal lc and of lc that have picked-up antigen in the epidermis and migrated via dermis to the regional lymph node. if so, these data suggest that resident epidermal lc are fully prepared to present cutaneous antigens to memory/effector t cells (efferent limb), whereas resident lc must leave the influence of the epidermis in order to develop the capacity to meet the more stringent conditions required for antigen presentation to porimed t cells (afferent limb). we have investigated the structural restrictions placed on residues contained within a minimal t cell determinant, using the balb/c class i restricted t cell response to the site determinant of the influenza hemagglutinin molecule as a model system. to delineate which of the residues comprising the site determinant are involved in interaction with the t cell receptor, we have determinaed the response of a large panel of site specific t cell hybridomas to a collection of peptide analogs differing by single conservative or non-conservative substitutions at positions. the fine specificity patterns of the t cell panel is extremely diverse; t cells varied in both the location and number of residues within the antigenic peptide that effected recognition. our results implicate at least out of residues within the antigenic pepetide as being involved in interaction with the t cell receptor. this result suggest that peptides comprising the site determinant do not form alpha helical structures when in association with mhc molecules. rubella-specific isotype and igg subclass responses were evaluated using elisa techniques in rubella ha seronegative adult females undergoing rubella immunization (ra / strain). responses were evaluated prior to immunization and at , , , , , , and wks post-immunization. pre-immunization sera showed detectable levels of rubella-specific antibody in the igg class ( / ); iga class ( ) and in one or more of igg subclasses ( / ). post-immunization, i subjects failed to develop igm class responses by the ha (sdg) technique while / developed igm antibody by elisa techniques. iga responses were detected at low levels in all vaccinees beginning at - wks and declining by wks post-immunization. antibody in iggl and igg subclasses by - wks post-vaccine with sustained iggl levels but significant decline in igg levels noted between and wks post-immunization. no seroconversion was noted in the igg subclass although individuals had detectable pre-immunization iggz rubella antibody present. igg levels were detected in all vaccinees post-vaccine with a delayed and progressive rise over the study period. subsequent correlation was then performed between rubella-specific antibody responses and the presence or absence of adverse joint reactions occurring in association with rubella vaccine administration. all individuals produced detectable c t lymphocyte responses to varicella zoster virus. anthony hayward, abbas vafai, roger giller & eileen villanueba. departments of pediatrics and microbiology, university of colorado school of medicine, denver co university of iowa school of medicine, iowa city i the proliferative response of blood lymphocytes from varicella zoster virus (v v)-immune donors to live vzv, extracted vzv antigens or purified glycoproteins is predominantly by cd +, hla-d restricted t cells but little is known of the specificies of the responder cells. we restimulated t cells cloned by limiting dilution from vzv-stimulated cultures with purified vzv glycoproteins gpi, gpii and gpiii and found that t cell clones with specificity for each of these mediated both help for antibody responses and hla-dr restricted vzv-specific cytotoxicity. polypeptides of to amino acids length corresponding to predicted amphipathic sequences in the primary structures of g p i, gp i and gp iv were synthesised. proliferative responses were observed to of these peptips (one from each glycoprotein) with responder cell frequencies in the :lo blood t cells range. the gp i peptide additionally defined an epitope recognised by serum antibody. an immunomodulatory approach to treating hsv- corneal disease, hendricks rl, departments of ophthalmology, and microbiology/immunology, university of illinois school of medicine, chicago, il herpes simplex virus type i (hsv- ) corneal infections are a leading cause of blindness worldwide. we and others have demonstrated that the cellular immune response to hsv- contributes to the elimination of virus from the cornea, but in doing so causes the tissue destruction that is responsible for the blinding complications of the disease. we have demonstrated that specifically suppressing the cytotoxic t lymphocyte (ctl) response to hsv- renders mice resistant to corneal disease following topical corneal hsv- infection. in agreement with this observation was our recent finding that in vivo depletion of wt ' (t helper/inducer, and most dth effector cells) neither reduced susceptibility to corneal disease, nor increased susceptibility to disseminated disease. the corneal lesions in wt depleted mice contained numerous lyt- (t suppressor/cytotoxic) cells, and no l t cells. the wt depleted mice exhibited normal hsv-specific ctl precursor frequencies. experiments designed to determine the effect of in vivo lyt- depletion on susceptibility to corneal disease are in progress. our goal is to identify cellular immune responses to hsv- that maximize protection, while minimizing immunopathology in the cornea, and identify hsv- epitopes that preferentially activate those responses. supported we found that affinity purified antibodies to bsa, klh and diptheria toxoid all contain a substantial amount of specific anti-idiotypic activity. against bsa react with mouse anti-bsa antibodies, which suggests that we are dealing with internal image antibodies. mrl-lpr/lpr mice develop spontaneous autoimmunity. we found that these mice make anti-anti-(self h- ) antibodies prior to making appreciable amounts of pathological autoantibodies such as anti-dna, anti-rnp.sm, and rheumatoid factor. the anti-anti-self antibodies are detected using an inhibition of antibody mediated cytotoxicity assay, that also detects anti-anti-(self h- ) in ordinary allogeneic anti-sera. the antibodies are not rheumatoid factors, although the animals do make rheumatoid factors later in the development of the disease. anti-self activity is fully developed at months, when the other autoantibodies are typically barely detectable. important role in the etiology of the disease. the anti-we conclude that anti-anti-self antibodies could play an c feedback regulation of - synthesis in monccytes by t cell products: dual effect of - . mikko hurme, tessa palkama and marja sihvola, department of bacteriology and immunology, university of helsinki, sf- , helsinki, finland. il-i production of human monocytehnacrophages is regulated by several cytokines some of which are themselves able to activate the il- production (e.g. tnf and il- ) while others (e.g. ifn-y) modulate the production activated by other signals. we have now examined the effect of - on the - synthesis. - alone did not induce any - bioactivity or il-la or - mrna expression in freshly isolated peripheral blood adherent cells. in contrast, il- effectively suppressed the lps induced - production. this suppression took place without any decrease in the steady-state levels of il-la and il-i mrna, suggesting that this downregulative effects is posttranscriptional. monocytehnacrophages are known to rapidly loose their ability to produce il-i when cultivated in vitro. if ifn-yis present in the culture fluid, the cells remain capable of producing - . as ifn-yand have been reported to have similar "priming" effects on macrophages (e.g. increasing the tumoricidal capacity and mhc class ii antigen expression) we cultivated monocytes for h in the presence of either ifn-y or - , and after washing the cells they were stimulated with lps. il- activity could be detected both in the ifnyand il- preincubated cultures (but not in the cultures preincubated with medium alone). these data suggest that il- can also display a similar upregulatory function in il-i production as ifn-y. gahreston, tx development of immunity to members of the spotted fever group of rickettsiae is a t-cell dependent response. we have used t-cell hybridomas and cloned t-cell lines from immune animals and convalescent humans to identify the rickettsial antigens that induce antigen-responsive t-cells. in these studies we found that the kda antigen of rickettsia tickettsii. the causative agent of rocky mountain spotted fever, is one of the immunodominant tcell antigens. t-cells from immune animals and humans were responded in culture to a recombinant kda antigen. both sources of t-cells were of the t-helper type (l t * and ' respectively) and produced l- and interferon. it was found that soluble antigenic material of b. rlckettsii obtained by extraction with hypotonic buffer maximally stimulated the t-cell lines. this material was enriched far the high molecular weight polypeptides of s kda and kda. also. ethirwwi ' will induce a long-lived immunity against infection with r. rickettsii. infected guinea pigs develop a minimally cross-reactive antibody response to b. nckettsii. in contrast. a strong cross-reactive t-cell proliferative response is produced. studies are in progress to determine the nature of the common protective antigen of r. humans infected with the parasitic nematode ascaris lumbricoides vary considerably in antibody responsiveness to a kda component of the parasite. this molecule is secreted by the parasite, and is also abundant internally. this heterogeneous reactivity has been modelled in laboratory rodents, and the antibody response to it is h- -and rt -restricted in mice and rats, respectively. using inbred and congenic animals, only mice of h- ' and rats of rtiu were, so far, found to be responders, and this restriction only operated in the context of infection. the specificity of the ige response in these animals was assayed by passive cutaneous anaphylaxis, and in an ige-specific elisa assay. the data show that the above mhc restriction also applied to the specificity of the reaginic antibody response, although animals of all mhc haplotypes responded to other ascaris allergens. amino acid analysis of the kda equates it to a previously identified "allergen a" of the parasite, and we now have its sequence available. these findings have implications for the genetic control of allergic responses in general, and, in particular, to the hypersensitivity responses which are such a feature of infections with parasitic nematodes. there are also implications for the generation of hypersensitivity responses by recombinant vaccines involving certain parasite antigens. the cns. immunohistochemial analysis of both frozen sections prepared from the brains of animals immunized in this manner and of highly enriched glial cell subpopulation cultures for viral gp expression indicated that oligodndrocytes and possibly a subset of astrocytes were the targets of this infection. further, microscopic analysis of frozen sections failed to reveal any overt signs of gross pathologic changes associated with the viral infection. we have been able to demonstrate the presence of virus specific antibody in the serum of these mice as well as virus specific cytolytic t cells in the peripheral lymphoid organs. ments are currently underway to determine whether the lack of pathology associated with wb infection in light of the previously shown virus specific immune responses in these mice is due to a failure of antigen presentation within the cns or some other form of immunoregulatory phenomena. the t lymphocyte proliferative response to pigeon cytochrome in bio.a mice is restricted to the egk:e,k ia molecule and specific for the c-terminal determinant comprised of residues - . blo.a( r) and blo.a(sr) mice are nonresponders to pigeon cytochrome nonetheless, the t cell repertoire of blo.a( r) or ( r) contains some t cell clones capable of recognizing and proliferating to pigeon cytochrome c w h e n presented by bio.a antigen-presenting cells (aft). therefore, one would expect to stimulate such clones in allogeneic bone marrow chimeras of the type bio.a +blo.a( r) or ( r) b o.a apcs and a blo.a( r) or ( r) t cell reperto ! ~.~espectively. ,en(isea c rzave were primed with pigeon cytochrome cytochrome , they showed a good antigen specific proliferative response in vitro. surprisingly, however, if pigeon cytochrome was used for priming, no response was detected, even at priming doses as high as nmol per mouse. - could only be achieved by treating the allochimeras with an anti-cdb monoclonal antibody in vivo during the priming step. clones specific for purified protein derivative (ppd) in the same chimera. thus the regulation which involves cd positive cells is antigen specific. transfer of pigeon cytochrome - primed lymph node cells from the chimera into naive bio.a mice prevented priming of the recipient for a t cell proliferative response to pigeon - , but not priming to the moth synthetic fragment. chimeras of an antigen-specific suppression mechanism involving cd positive cells. faculty of medicine, kyoto university, kyoto , and department of oncology, nagasaki university school of medicine, nagasaki , japan. sera from b mice immunized with a syngeneic ctl specific for fbl- tumor of b origin blocked the cytotoxic activity of only the immunizing ctl clone. therefore, a monoclonal antibody (mab) n - was produced by fusion of the b spleen cells immune to a syngeneic fbl- -specific ctl clone (no. ). the specificity of the mab n - was confirmed by immunoprecipitation, blocking of cytolytic activity, stimulation of proliferation, and induction of tcr-mediated nonspecific cytolysis of the ctl clone no. . in some b mice, - % of the anti-fbl- mltc cells were positive for this n - -defined idiotype, and formed a well demarcated population upon examination by flow cytomehy. even in mice in which no such population was observed some ctl clones established by limiting dilution culture were also positive for this idiotype ( out of clones from mice). the cytotoxic activities of these ctl clones were blocked by n - , which in turn induced the nonspecific cytolysis in redirected assay. however, no positive cells were detected in non-cultured normal or fbg -immune spleen and lymph node cells. this indicates the presence of cross-reactive (dominant) idiotype in the b anti-fbl- cytotoxic t cell responses and may provide a potent tool for analyzing the idiotype-mediated regulation of the anti-tumor immune responses. slade andsylvie gillard, max-planck-institut fur immunbiologie, d- freiburg, federal republic of germany t cells play a n essential role in t h e protective immune response to malaria and a r e associated with s o m e of t h e pathological consequences of t h e disease. however, t h e n a t u r e of their responses and t h e antigens t o which they respond a r e not well defined. w e have developed a limiting dilution assay system in which specific t cell responses to malaria antigens c a n be monitored a t t h e clonal level. i t is possible to determine t h e nature of t h e responding t cell by t h e growth f a c t o r s they s e c r e t e and by their ability to a c t as helper cells for t h e antibody response to malaria antigens. our d a t a suggest t h a t t h e t cell response changes during t h e primary infection and in hyperimmupe animals. o n e to t w o weeks a f t e r initiation of a blood s t a g e infection t h e major cd + t cell which proliferates in response t o parasite antigens s e c r e t e s il- and ifn-y but is not a n efficient helper cell for antibody responses. in c o n t r a s t l a t e r in infection and in immune animals t h e r e is an e f f e c t i v e helper cell response and many of t h e s e cells a r e distinct from those secreting ifn-y and il- . we a r e currently investigating whether these cells retain these phenotypes when grown in long-term in vitro culture and whether defined antigens of t h e erythrocytic parasite elicit different t cell responses. we have localized linear neutralization epitopes on the coronaviruses ibv, mhv, fipv and tgev. the results can be summarized as follows: . linear epitopes of the spike proteins ( - residues) could be mapped to a resolution of a single residue by expression of gene fragments in the prokaryotic pex plasmids and/or pepscan peptide synthesis. . the length the epitopes varied from to at least amino acid residues. we present evidence that the larger epitopes, although conformation-independent according to operational criteria, are nevertheless discontinuous. . in ibv, we localized several overlapping but different epitopes within an immunodominant region of residues. this region is recognized by all polyclonal antisera tested. we propose that its immunodominancy is a consequence of its structure and function and does not depend on antigen presentation or idiotypic networks. an immune response against the mouse testis-specific antigen ldh-c reduces fertility by percent in female baboons. an immune reaction to human ldh-c would be expected to be more effective in primates. since the human testis enzyme is not readily available in large quantities, recombinant dna technologies were uscd to create a source of human ldh-c . antibodies to mouse ldh-c were used to screen a xgtll human testis cdna expression library. a full length human ldh-c clone was identified, sequenced, and the ldh-c cdna was engineered for expression in e.coli. the ' and ' untranslated sequences were removed by restriction enzyme digestion, and synthetic linkers were added adjacent to the start and stop codons of translation. the modified cdna was subcloned into the prokaryotic expression vector pkk - and introduced into w l a c iq cells. cells were grown to mid-log phase, and induced with iptg for positive regulation of the strong hybrid tac promoter. induced cells overexpressed the kd subunit which spontaneously formed the enzymatically active kd tetramer. human ldh-c was purified -fold from liter cultures of cells by two step affinity chromatography to a specific activity of i.u./mg. the n-terminal amino acids sequenced were identical to those predicted from the nucleic acid sequence. antibodies to synthetic peptide epitopes of human ldh-c cross-reacted with the enzyme produced in e.coli. two mg human ldh-c were expressed per liter of bacterial cells. the purified protein is now available for innunogenicity and fertility studies. it is now generally accepted that the principal effector mechanism in the host's defence against leishrnaniasis is gamma-interferon (ifn-y) which activates infected-macrophage to eliminate intracellular parasites. mice by prior sublethal whole body irradiation or treatment with anti-igm or anti-cd antibody. protection can also be induced by repeated intravenous or intraperitoneal immunisation with killed parasites or purified antigens. ly immunised mice produce little or no il- or il- but substantially elevated levels of ifn-y when stimulated with leishmania antigens in vitro. lymphoid cells from balb/c mice with progressive disease can inhibit the maf (macrophage activating factor) and leishmanicidal activities of the culture supernatant of lymphoid cells from mice recovered from l. major infection. maf appears to be ifn-y, whereas the maf inhibiting factors are il- and il- . system can be reproduced with recombinant ifn-y, il- and il- and the maf inhibiting activity of the suppressive supernatant can be reversed by specific anti-il- and anti-il- antibodies. the disease by influencing the ability of macrophage to kill the intracellular parasite. the development of efficacious vaccines against malaria requires an understanding of the mechanisms involved in protective immunity. f'revious studies with plasmodium berghei demonstrated that sporozoite immunity is dependent upon antibody responses specific for the repeat region of the circumsporozoite (cs) protein and cell mediated mechanisms involving cd + t cells. in this study we analyzed the splenic t cell repertoire directed against epitopes on the cs protein of p. berghei and determined whether sporozoite-immune cd + and cd + t cells respond to shared or distinct epitopes. sporozoite-immune spleen cells, cd + and cd + enriched t cell populations of balb/c (h-m), c h @i-%), and c bv (h- b) mouse strains were cultured in the presence of irradiated sporozoites or synthetic peptides representing % of the complete cs protein. surprisingly, none of the cultures proliferated to any of the peptides tested, although proliferative responses to sporozoites were observed in unfractionated spleens and cd + t cell populations. cd + t cells did not respond to any of the antigens tested, even in the presence of exogenously added - . titration of cd + cells into proliferating cd + cell cultures did not suppress the anti-sporozoite response. the lack of anti-peptide reactivity contrasts with uniform responses to sporozoites and may be the result of the context in which cs antigens are presented to t cells. functional analysis of accessory splenic b cells and macrophages revealed that while the anti-sporozoite proliferative responses were not affected by the removal of macrophages, sporozoite-primed b cells were essential for the responses. these data suggest that the cs protein on sporozoites is not processed extensively by macrophages to yield many potential t cell epitopes, but instead is presented by immuncdominant b cells that resmct responses to a limited number of t cell clones. of the primary infection and is also required for optimum protection against reinfection. current studies have demonstrated that relatively few of the viral antigens tested to date ( viral envelope glycoproteins or nonsmctural nuclear proteins) are recognized by hsv- immune ctl populations generated in several different strains of mice (h haplotypes h b, h d. or h k). this failure of hsv specific ciz to recognize the cloned gene products in in v i m assays was demonstrable at the clonal level and could not be attributed to a peculiarity of the recombinant vaccinia conshucts used because studies with adenovims vectors or tranfected l cell constucts yielded the same results. surprisingly, despite their inability to be recognized by hsv specific ciz in vim, when used to immunize mice several of the vaccinia virus constructs would induce memory ciz populations capable of lysing hsv- infected autologous cells. for example, hsv- glycoprotein c (gc) was recognized by h b restricted but not h k restricted hsv specific ctl. however, immunization of either haplotype of mice with a vaccinia gc recombinant induced ctl populations which upon in v i m restimulation with hsv- would lyse histocompatible cells infected with hsv- . this demonstrates that despite the presence of suitable epitopes (intrinsic factors) the context of the immunogen (extrinsic factors) will also influence it's ability to induce ctl. the results of further studies into the nature of these extrinsic factors will be presented and discussed with relevance to future sub-unit vaccine design. w d ~upponrd by public ~~l t h service g~mu, ai md ai fran the ti-^ ~n,litu= md lnrcniour d ,~~~~~~. infection of mice with hsv- induces a brisk ciz response which is necessary for the subsequent resolution we have investigated the structural basis for antigen mimicry by anti-idiotypic internal image antibodies. two mouse monoclonal antibodies (mabs) that bear internal images of a well-defined protein epitope, i.e., the rabbit immunoglobulin (ig) a allotype, were produced and the variable region sequences were determined by rna primer extension sequencing. the results showed that the mab light chains did not contain any allotype-related residues; however, both heavy chain v regions contained a unique sequence homologous to the nominal antigen but in opposite orientation. this reversed sequence was expressed within cdr of both mabs. synthetic peptides corresponding to the putative antigenic regions of rabbit ig and the mab internal images, respectively, were tested for the ability to mimic the al-like determinant. although the homologous residues were presented in opposite orientations, both peptides completely inhibited at similar concentrations the binding of rabbit ig to anti-a antibody. a paired thr and clu was necessary for expression of the a epitope as revealed by conservative substitutions in the peptide sequence. computer-generated, energy-minimized models of rabbit ig and the mabs revealed that the critical a residue side chain placements could be almost superimposable in either context. thus, it appears that an antigenic epitope can be determined solely by md . proliferation of murine type i cd + t cell clones quires simultaneous occupancy of the t cell antigen receptor and delivery of an accessory cell-duived costitnulamy signal in contrast, isolated t cell receptor occupancy induces the cell into a state of reduced proliferative responsiveness to antigen. based on the observation that pkc-activating phorbl esters can at times substitute for the p s e n c e of accessory cells in t cell proliferative response. to mitogens or anti-cd mnodonal antibodies, we investigated the requkment for accessory cells in the antigen-and con a-induced hydrolysis of p m and activation of pkc. the presence of normal accessory cells was found to be unnecessary for the development of pkc-dependent phosphorylations and the addition of normal accessory cells had no effect on the activity of pkc. cell il- synthesis and proliferation presents a paradox. we have studied the effects of ueatment with a calcium ionophore and p h h l ester on t cells and find that increased [ca +]i and pkc activation are in fact insufficient biochemid second messengers in the induction of proliferation. while pliferation was induced at high t cell density in response. to these stimuli, incubation of t cells at decrrased cell density drmonsuated markedly reduced proliferation, and single t cells failed to divide. this suggested that cellular interactions were. q u i r e d in the response. additions of either il or normal accessory cells allowed p l i f d o n at low density, consistent with a requirement for an accessory cell-derived costimulatoq signal in the induction of i l synthesis, even in the plifcrative response to ionomycin and pma. this result underscons the importance of an accessory cell-duived costimulatory signal, acting independently of t cell receptor-mediated increases in [caz+] i and pkc activation, in the induction of t cell proliferation. we describe experiments designed to determine the molecular requirements for recognition by fluorescein-specific ctlps and ctls derived both from n a i v e and from immunized mice. we the production of prostaglandin e, a major immunesuppressor secreted by the macrophages was inhibited by the addition of . m indomethacin to the cultures of monocytes harvested from patients suffering from pulmonary tuberculosis and those from equal number of normal controls. the il-i activity was estimated i n the supernatants of these cultures by their ability to proliferate mice thymocytes. it was found that the supernatants from cultures with indomethacin showed a greater il-i activity than the ones without it( % p . ). this indicates the possibility of pge offering a negative feedback control over il-i production. the defective cell mediated immunity i n patients with pulmonary tuberculosis may be explained through the inhibition on il-i production by pge whose enhanced production is reported i n our earlier studies. the results and our hypothesis on the autoregulation of il-i production w i l l be presented and discussed. in variant viruses which differ from the parental virus (gv) at specific epitopes recognized by monoclonal antibodies directed against the env gene product, gp . biological clones isolated from gv express the gv phenotype suggesting that the loss of specific epitopes is the result of selective de novo processes in the immunocompetent host. additionally, inoculation of adult mice with a biological clone expressing the gv phenotype also results in similar variant viruses. however, inoculation of gv into neonatal or nonlethally irradiated mice results in a population of viruses expressing only the gv phenotype suggesting that the emergence of antigenic variants may be influenced by neutralizing antibodies and/or cellular host res sds-page analysis of immunoprecipitates of sg~s~~,elled lysates of fibroblasts infected with clones expressing gv or variant pehnotypes shows a size difference of the gp precursor. additionally, the recognition of a neutralizing epitope (e- ) associated with gp by mab is dependent on the appropriate native conformation of the epitope which appears to require glycosylation for expression. experiments are in progress to further examine the immunogenetic basis for the generation of these variants and to determine the molecular changes in the virus genome responsible for changes in epitope expression. investigated the capacity of murine splenict cells depleted of acceso cells ( ac ) t o proliferate in response t o stimulation by con a, @ cd ab and activated t cells. the zepletion procedures consisted of carbonyl iron treatment, x "panning " on anti-ig coated flasks, x anti-la cytotoxic treatments and percoll gradient purification of small resting t la-cells. the appropiate concentration of con a ( ng/ml ) and plastic-bound (pb) @cd lg or its f (ab)' fra ments induce proliferation, r expression and (but not secretion in t la-cellscultured for % at x cells/well . responsiveness of tlacells t o con a and dcd in low density cultures ( x cells/well) is restored by the addition of irradiated th cloned cells but not thl ,splenic cellsor r l l l + rll + r . likewise, responsiveness t o non activating doses of con a (lnglml) or soluble @cd is restored by the addition of irradiatedth costimulatory cells . these experiments demonstrate that the ability of t cells t o proliferate in the absence of ac is critically dependent on t-t interactions. t cell subsets prepared by either negative (l t -and ly 'cells) or positive selection proliferate in response t o pb @cd lg . although the proliferative responses of both l t -and ly -cells are maximal at h, the l t -cells require l o x more pb @cd lg for maximal stimulation and their res onses decline much faster than those of ly cells. in addition, l t -cells are not stimulated by pb &cd f(ab)' fra ments and their responses t o @cd i are inhibitable by anti fcr as well as anti-lfa abs . re onsesof%oth l t -and ly -cells are !nhibita%le by @i and @i r abs but not by @l t , @ly or b l l . these experimentsdocument interesting differences in the triggering requirements of l t -and ly 'cells. supported by nih grants po ca , t m- and gm . the k glycoprotein encoded in the e region of ad and ad (gpl k) binds to class i mhc antigens in the endoplasmic reticulum and prevents their translocation to the cell surface. this has been proposed as a mechanism by which virus infected cells can avoid recognition by the host cytotoxic t lymphocyte (ctl) response. we have shown that gpl k can inhibit target cell lysis by adenovirus specific ctl, but the effectiveness of this inhibition varies greatly between different mouse strains. this is due in part to differences in the affinity of gpl k for different mhc class i molecules, but this cannot account for a l l the variation observed. i t has been shown that cd + + immature thymocytes fail to secrete il- or express il- receptors in response to activation signals. furthermore. they cannot induce il- gene transcription. several tumor lines have now been characterized which have a cd + + phenotype and fail to secrete i l - or express il- receptors in response to stimulation with ionomycin plus pma. these cells also do not express il- mrna after stimulation, as determined by northern blotting and rnase protection. to determine the molecular mechanism for this lack of transcriptional activity, nuclear extracts were analysed for the presence of the d n a binding factor nfat-i. this nuclear factor i s present only in ac- we have undertaken an mhc analysis using the polymerase chain-reaction (pcr) and dot blot analysis of the amplified lyme arthritis patients dna with allele specific oligonucleotide (aso) probes. genomic dna for the first domain of the dq beta chain and of the dr/pi from patients with lyme arthritis has been amplified and we are analyzing the distribution of dridrr and w a l l e l e s in this population to test the hypothesis that the mhc class i genes might be involved in presentation of selected spirochete epitopes whose recognition by t lymphocytes leads to lyme arthritis. most inbred strains of mice do not respond to porc insulin (pins). experiments were conducted to elucidate the mechanism of the non-responsiveness in h-zk mice: ) purification of cd- ' t c e l l s from pins-immune b o.mbr mice revealed pins-specific t helper (th) cells, ) these pins specific th cells could be activated by i-ak and i-ek expressing l -fibroblasts. therefore, both i-ak and i-ek molecules can present pins in an immunogenic manner and activate pins-specific th cells. by means of different cell-fractionation procedures, it was found that antigen-specific t suppressor (ts) cells regulated the pins immune response. these ts cells were of the fcr-, cd- -, cd- '. thy- ' phenotype, and they were present in normal mice. we believe that these experiments indicate that antigen-specific ts cells exist and are important regulators of immune and autoimmune responses. the possibility of functional inactivation of cd + clones by ts-cells was investigated. m leprae-responsive cd + clones were preincubated with ts cd + clones, apc and antigen for ; hours. after which the cd + cells were removed from culture. the cd + clones were then restimulated with e. leprae and apc. cd + clones incubated with cd + cells and antigen were unresponsive to restimulation by antigen, although they were not killed and could respond well to il- . addition of il- in the preor post-incubation culture neither prevented the induction of unresponsiveness nor reversed it. earlier models of tolerance have suggested that receptor occupancy in the absence of second signals induces tolerance in b-and t-cells. we would suggest that in the presence of ts-cells. a second signal may be negated leading to th-cell unresponsiveness. university of texas southwestern medical school, dallas, tx graft versus host disease (gvhd) poses a serious threat to the survival of patients with bone marrow transplants. the state of immunosuppression established in gvhd results in a variety of immunological abnormalities at the humoral and/or cellular level. we have developed a murine model of chronic gvhd across a minor histocompatibility (mh) barrier. in this model, immunosuppression develops. spleen cells from mice undergoing this type of gvhd are unable to respond to the polyclonal activators lipopolysaccharide and concanavalin a . however, the response against the b cell leukemia bcll remains intact. the protective immune response against bcll is directed towards the mh antigen h- and is mediated by cytotoxic t lymphocytes. thus, the specific t cell response against a mh antigen can occur in the presence of chronic gvhd despite the absence of a polyclonal b and t cell response lymphocytic choriomeningitis virus (lcmv), a member of the arenavirus family, has a biseqmented rna genome which encodes at least three polypeptides. the smaller rna segment encodes two virus structural proteins, the slycoprotein (gp) and the nucleoprotein (np). upon infection of mice with lcmv a cytotoxic t cell immune response directed against these proteins is measurable in vitro and in vivo. it can be demonstrated that depending on the haplotype of the mice, one or the other protein may play a major part in the immune response. in order to define the immunogenic epitope(s) of the nucleoprotein which are recognized specifically by the t cell receptors of cytotoxic t cells, stepwise ' truncated qene fraaments encodina the nucleoprotein were cloned and expressed in vaccinia virus. with these recombinant vaccinia viruses, protection experiments in mice aqainst lcmv infection were performed in parallel with in vitro studies, namely specific recoonition of target cells expressing truncated fragments of the nucleoprotein by lcmv primed spleen cells. cdna clones encoding the mouse and human t cell il- receptors have been isolated and expressed in mammalian cells. the recombinant receptor binds il- indistinguishably from the natural il- receptor, and is functional in signal transduction. deletion of the cytoplasmic portion of the receptor abolishes its signal transduction abilities. sequence and secondary structure analysis suggest that the cytoplasmic segment of the il- receptor binds a nucleotide. experiments designed to test this hypothesis and to examine the mode of signal transduction will be presented. also to be discussed are the mechanism of triggering of the receptor by il-i. and the nature of il- receptors expressed in other cell types such as b cells. it became evident that these subsets reflect different stages of helper t cell maturation before and after activation. therefore, these t cell subsets have been designated as naive t cells (cd r/ h +, cdw [ b ]-) and memory t cells (cd r/ h -, cdw [ b ]+). we analysed the expression of these antigens in dermal lymphohistiocytic infiltrates from different benign skin diseases and cutaneous t cell lymphomas (chronic contact dermatitis (n= ), parapsoriasis en plaques (n= ), lymphomatoid papulosis (n= ), mycosis fungoides (n= ), sezary's syndrome (n- ), pleomorphic t cell lymphoma (n= ) and high grade t cell lymphomas (~ )). in almost all cutaneous t cell infiltrates memory t cells were preferentially found whereas in the peripheral blood both subsets are equally distributed. this implicates, that t cells infiltrating the skin already have had contact with their respective antigen. where the switch from naive to memory t cells takes place can not be answerded by our findings, as we have investigated rather longstanding skin diseases. however, these memory t cells, which can be activated more easily, make diseased skin more e f f e c t i v e in the nmdlification of an immune resnonse. organs and after several days of stimulation with antigen or mitogen and lymphokines. we find that fresh th synthesize and secerte -iu,ifng,il and gmcsf but very little il or il. within - hours. this pattern resembles the pattern of lymphokines secreted by thl cell lines. the th responsible for this secretion are cd positive t cells which are long-lived since they disappear very slowly following adult thymectomy. they are also sensitive to the in vivo administration of ats(antithyrn cyte serum) and they express high levels of pgp- . the kinetics of lymphokine secretion and the phenotype of the cells suppon the hypothesis that lymphokine secretion from fresh lymphoid cells comes from a population of memory cells. in contrast we fmd that we can also stimulate a separate, ats-resistant population to become lymphokine-secreting cells after four days of in v i m priming.these primed cultures rapidly synthesize an secrete large amounts of i u and il in addition to ifng , il and gmcsf( a phenotype which could be combination of both thl and th helpers), when they are restimulated with ag or mitogen. the cells whic are responsible are cd positive and have a shorter liespan since they decline considerably after adult thymectomy. we suggest that the lymphokine secreting cells detected after priming come from a population(s) of helper t cell precufiofi which have differentiated to become effectors. this generation : effectors requires lymphokines, especially e- andlor ilz and apcs. thus the development of helper th appears to follow a similar pathway as that of cells of the b cell lineage developing into ab-secreting cells and cd positive t cells which develop into cytotoxic effectors from precursors. we have studied the secretion of lymphokines by helper t cells freshly obtained from lymphoid interleukin- production by t cells has been shown to be required for both humoral as well as cell-mediated imune responses. thus, il- production was measured in syphilitic rabbits as a function of their imune response. maximal il- production induced by con a at - days post-infection was only l/ that observed for uninfected rabbits and this correlated well with a decrease in t cell proliferation ( < % that of normal rabbits) upon stimulation with con a . this decrease in il- production in infected rabbits was restored upon removal of most of the adherent cells. furthermore, the il- production by - day infected spleens was restored above normal levels upon addition of indomethacin. this decrease in il- levels was not due to an increase in the ability of infected spleen cells to adsorb il- . finally, studies assessing il- production at various times postinfection indicated that at days post-infection il- levels were higher than normal, however as early as - days after infection il- levels decreased below normal levels and continued to be depressed as late as days post-infection. these results may explain why all organisms are not eliminated during primary infection w i t h ' . pallidum and why secondary and tertiary phases of the disease may develop. has been shown that especially the antigenic presentation of the fusion protein is important for eliciting a functional immune response. to study the immunolo ic properties of the f protein, we expressed the f gene in e.coli as a $galactosidase-f-fusion protein after insertion in a pex vector. we constructed deletion mutants with fragments generated with restriction enzymes and with the polymerase chain reaction method. using a panel of monoclonal antibodies a rough epitope mapping has been performed. two arears were found on the protein, with one area two monoclonal antibodies react and with an other area four monoclonal antibodies react. both area's were found in f , the c-terminal part of the protein. the pepscan method was used to fine map the epitopes of the monoclonal antibodies reacting with the second area on the primary sequence. in at least one viral system, cd + effector cells can be induced in animals lacking cd + t cells. since cd + effector cells are important in immunity to malaria sporozoites, we wished to know if they,too, could be generated without help from cd + cells. we depleted balb/c mice of their cd + t cells by injection of an anti-cd monoclonal antibody, and then tried to immunize them with irradiated plasmodium yoelii sporozoites. when challenged with infectious sporozoites, these mice were not protected against malaria infection. although they did not make antibodies to sporozoites, passive transfer of hyperimmune serum into these animals still did not protect them against a sporozoite infections. cd + t cells from these animals functioned normally in in vitro assays against tnp labelled targets. it appears that, unlike viral systems, the generation of cd + effectors in malaria requires cd + helper cells. thus both cd and cd epitopes should be included in any synthetic vaccine against malaria sporozoites. univ. pennsylvania, philadelphia, pa migrate from fetal liver or bone marrow. rearrange t cell receptor (tcr) genes, express tcr. undergo thymic selection and finally emerge as mature single positive t lymphocytes. most studies of thymic t cell development have been performed by using polyclonal populations of t lymphocytes, which have made the interpretation of the results complicated. cells ( clone) from nude mice by culturing nylon wool non-adherent cd -cd -spleen and lymph node cells in the presence of wehi supernatant and con a supernatant. clone was thy- -cd -cd -cd -il r(il receptor)-and they have been maintained more than months without changing phenotype. when the c clone was stimulated with il . ili/il . illnl . gm-csf, the cells were induced lo express thy-i, tcr and il r proteins. however, culture of the cells with gm-csffll did not induce the expression of these molecules. southern blotting of the dna isolakd from gm-csffll culture suggested that they have undergone partial db -jp rearrangement. the cultured cells were then recloned twice by limiting dilution. the cloned cells were again shown to induce expression of cd complex by the stimulation of il enriched medium. therefore. we have established a system in which to induce differentiation of cloned pre t cell line into tcr+ cells in vitro. the human lymphocyte differentiation antigen cd is encoded by a single gene which gives rise to a kda glycoprotein expressed on the cell surface as a dimer, and in higher molecular weight forms. we demonstrate that the mrna is alternatively spliced such that an exon encoding a transmembrane domain is deleted. that is secreted and exists primarily as a monomer. messenger rna corresponding to both forms is present in peripheral blood lymphocytes,(pbl), con a activated pbl and three cd + t cell lines with the membrane form being the major species. ratio of mrna for membrane cd (mcd ) and secreted cd (scd ) exist. in addition, the splicing pattern we observe differs from the pattern found for the mouse cd gene. this mrna is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted giving rise to a cell surface molecule which differs in its cytoplasmic tail from the protein encoded by the longer mrna. neither protein i s secreted. this is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. this represents one mechanism of generating diversity during speciation. cd ' t cells in the rat can be divided into two non-overlapping subsets by theii reactivity with the monoclonal antibody mrc ox- which binds some of the high molecular weight forms of the cd antigen. recent work, to be described has shown that the two subsets represent different stages of t cell maturation, with distinct t cell functions. the lymphokine repertoire of the memory t cell pool will be discussed with reference to the antigenic environment fmm which the cells are obtained. pancreatic islet allografts, gill, ronald g. and lafterty, kevin j., barbara davis center for childhood diabetes i univ. colo. health sciences center, e. th ave, box b- , denver, co. we studied the cellular interactions requkd for the rejeaion of cultured mhc class i-dispiuate islet allografts. this model was suitable for studying t-t collaboration in that islet allograft immunity is cd dependent but rejection of the cultured islet graft is mediated by the cd cell. recipient c by (b ) mice were grafted with mhc class idisparate b .c-h- bml (bml) islets beneath the mal capsule. islet grafts wen preaated for days in % oxygen culture to reduce immunogenicity. thirty days after grafting, recipient mice wen immunized with oe live spleen cells from the strains indicated below. rejection of the established graft was not trig-by challenge with donortype bml spleen cells, indicating that the mhc class i stimulus was insufficient to initiate all@ immunity. further, immunization with a mixture of m bml and oe mhc class ii-disparate b .c-h- bm (bml ) spleen cells failed to trigger host immunity. however, challenge with loe (bml x bml )fl spleen cells t r i g g d acute rejection of the established bml islet grafts. the requirement for l i i presmtatidrecognition of class i and class all* antigens to trigger allograft immunity indicates that the antigen-presenting (apc) plays an essential role for t-t collaboration in vivo. uature hla-dr complexes are purported to spontaneously internalize from and recycle to the plasma membrane of b but not t lymphocytes. using a neuraminidase protection assay, we have radiolabeled surface class i antigens on intact cells and cultured these cells under conditions which permit or prevent endocytosis; subsequently, surface glycoproteins on viable cells were desialylated and class i molecules were analyzed by iamunoprecipitation and two-dimensional gel electrophoresis. a panel of buman b lymphoblastoid cell lines and activated tonsillar b cell blasts failed to exhibit any internalization of class i complexes; control transferrin receptor molecules were endocytosed as ascertained by insensitivity to neuraminidase digestion. class ii+ pba blasts and sezary cells of the t lineage were also deficient in detectable hla-dr internalization. results did not vary regardless of the time allowed for efficient endocytosis ( ok ~saethionine). or the addition of anti-class i monoclonal antibody during the chase period for endocytosis. therefore. within the limits of sensitivity of this assay, class complexes do not appear to be internalized, either spontaneously or uben crosslinked by antibody. recycling represent a dynamic pathvay for regulating surface expression of class i antigens or a means of associating with and presenting foreign antigenic peptides. supported in part by usphs grants # t cao - and # eo a - . in previous studies of antigen-specific t cell responses in two distinct models of autoimmune tubulointerstitial nephritis (tin), viiia villosa lectin binding (vv+) t cells have been shown to be necessary for effector t cell expression and mediation of tin. in anti-tubular basement membrane disease, antigen-specifi vv+ t cells direct the phenotypic selection of cd + nephritogenic t cells in susceptible mouse strains (j. immunol. , nov. , ) . this function is mediated by an antigen-binding, i-js+ soluble protein factor. current studies investigate the role of the t cell glycoprotein which binds vv lectin in mediating w+ t cell function. using the previously described effector t cell induction assay, we found that n-acetyl-d-galactosamine (galnac) (at mm but not . mm) inhibits vv+ t cell function and cd + effector t cell selection. when cd + effector t cell differentiation occurs in the presence of soluble factors derived from antigen-primed vv+ t cells, galnac is not inhibitory. these studies suggested that soluble gal nac may competitively bind to a soluble protein which stimulates vv+ t cells, in part by binding to the w lectin receptor, to synthesize andlor secrete their biologically active soluble factor. as an additional test of this hypothesis, we prepared detergent solubilized membranes from vv+ t cells and purified vv lectin binding proteins by affinity chromatography. like galnac. these membrane derived lectin binding proteins also inhibit vv+ t cell function and cd + effector t cell selection. inhibition by soluble 'lectin receptors' is dose dependent and is demonstrable with lectin binding glycoproteins derived from - x lo cells, in an assay utilizing x vv+ cells. we are now further characterizing the lectin receptor and its endogenous ligand. elementary bodies and outer membranes of chlamydia trachomatis produce a high-titered igg response in rabbits and mice as measured by elisa and microscopic immunofluorescence assays. western blot analysis of total elementary body protein identifies a kd major outer membrane protein (momp) as the predominant antigen. to identify the cbmical structure of the epitope, purified momp was subjected to chemical and enzymatic fragmentation and the resulting peptides were purified by hplc and assayed for immunoreactivity. an immunoreactive kd cyanogen bromide peptide was amino-terminal sequenced and a series of overlapping synthetic peptides were synthesized and assayed for immunoreactivity. sequential single amino acid deletions at both the nhz and cooh termini allowed us to identify the precise epitope as a amino acid peptide spanning residues - of momp. two amino acid substitutions at positions (phe-gly) and (pro-gly) completely eliminated antibody binding. the -amino acid synthetic peptide is a potent immunogen producing high-titered antibody responses that are specific for the momp molecule. analysis of an independently derived mutant harboring the same defect has shorn that this trans-acting gene is not required for transport of class i molecules. class i heavy chain is synthesized in this cell line and associates with b m. transport of the class i appears to be blocked in the er or cis olgi as the majority of the class i glycoproteins are not processed to the endo b resistant form. the ability of the cell to significantly increase expression of surface class i when the incubation temperature is lowered from oc to oc suggests that this gene may function to stabilize a particular conformation of the protein. consistent with this is the increased sensitivity of class i molecules in the mutant as compared to the parent to degradation when cell lysates are incubated at elevated temperatures. the inability to immunoprecipitate class i antigens in the mutant is possibly due to the action of endogenous proteases present in these lysates. two complementing approaches are being employed to isolate this gene and further analyze its role in class i biosynthesis. the first involves inactivation of the trans-acting gene by insertion of a retroviral vector and subsequent pcr amplification of regions flanking the vector. in another approach a cdna library will be introduced into the mutant cell line and the cdna will be reisolated from cells reexpressing surface class i. houston. tx. we have induced a panel of highly immunogenic (imm+) vanants of the murine fibrosarcoma mca-f using i-methyl- niml-niuasoguanidine (mnng). -aza- '-deoxycytidine ( -azacdr). and uv radiation. these tumors grew m immunosuppressed mice. but were complelely rejecled by normal syngeneic hosts. mice thac had rejected large numbers of imm+ also developed a smng, tumor. specific immunity to the parental mor. lmmunizalion with low numbers of lmm+ engendered only variant-specific immunity. the frequency of imm+ variant g e n e d o n was similar for the three induction different protocols ( % to %), suggesling lhat generauon of imm+ was more closely relalcd to the cell line used than to the inducing agent however, the swngth of the imm+ phenaypc was related lo ihe agent used, since mnng induced clones had the m g e s t immunogeniciues and uv-b ihe weakest the smng neoantigens expressed by mnng induced imm+ were varunt-sppifc. while uv and s -d d r induced clones displayed significant cross-reactivilies not attributable to the parental t u n a antigen. increased or inappropriate expression of class-l mhc antigens did not correlate with the imm+ phenotype. we investigated the phenotypes of the spleen cells medrating tumor rejccuon using the local adopllve lransfer a s a y (lata). variant-specific immunity w mnng, -azacdr and uv induced imm+ were all m d a t c d by thy . +. l t +. lyc . -t cells. afrw immunization with high numbers of imm+ w engender both anti-lmm+ and anu-parental immunity. both cd + and cdw effectors rejecled the imm+ in lata, while only ihe c w + t cells could wnsfer resistance w the parent immunity the parenlal tumor anugen engendered by the imm+ suggeslcd associative recognition of the parental and neoantigem wgelher on ihe cell surface. this hypochfsis was supported by failure of lmm+ u) pmlect againu an antigenically disunct tumor (mca-d) admixed with it, either at the lime of immunization or at challenge. fusion of ihe h m * vanant with mca-d yielded a unique, hybrid parental umor antigen that was associatively recognized with rhc original imm+ neoanugen, demomirating the importance of antigen cocxpression. grant rr- - . w e have recently demonstrated and reported that substitution of anionic side chain carboxylic groups with aminoethylamide groups on protein antigens exhibits a pattern of enhanced immunogenicity both in vivo and in vitro. this enhanced immunogenicity was also observed in low responder strains of mice and we investigated the mechanism by which it is achieved. we examined antigen processing and presentation of native (nbsa) and modified bsa (mbsa) to t helper c e h isolated from c /bl low responder mice. a greatly reduced amount of mbsa than nbsa was required to activate both nbsa and mbsa primed th. proliferation of nbsa and mbsa primed t cells increased in proportion to the amount of time of exposure of the antigen presenting cells (apc) to nbsa, peaking at h. conversely, apc required less than min exposure to mbsa to achieve optimal activation, indicating rapid uptake of mbsa. paraformaldehyde fixed apc recognized mbsa without a lag phase processing, indicating that this event also occurred quite rapidly. apc processed nbsa w a s presented to primed t cells more effectively than the soluble antigen m shown by the increased rate of t cell proliferation. in contrast, mbsa was equally well presented to th cells by apc m in soluble unprocessed form. our data demonstrate that the reduced response in low responders is greatly enhanced by a modified antigen which is rapidly taken up and processed by apc. b cells which bear surface innunoglobulin (sig) receptors specific for a particular antigen are abile t o present fragments of that antigen very efficiently t o t cells. this i s due. in part, t o the high affinity of the receptor, which facilitates antigen binding at low concentrations. using tnp-abc and specific antigen, we have demonstrated that the tnp-abc process antigen very effectively. w e have compared specific antigen with i t s polyclonal analog, anti-ig, and demonstrated differences in the kinetics of degradation of anti-ig and tnp-antigens by tnp-aex. both antigen and anti-ig bound by tnp-abc are degraded into small fragments which are released into the supernatant. however, the following differences have been found: ) the rate of release of small fragments of tnp-antigen parallels the rate at which these cells become able to directly conjugate with t cells (a lneasure of antigen presentation), reaching a plateau between and hours. in contrast, the degradation of anti-ig and release of fragnents continues for hours. ) analysis of initial kinetics demonstrated that release of fragments of tnp-antigen begins minutes after binding; there i s no significant release of anti-ig fragrnents u n t i l about minutes. ) in contrast to anti-ig where there i s significant accumulation of degradation intermediates within the cells, there i s very l i t t l e intracellular accumulation of intennediate-size fragments of tnp-antigen. thus, we propose that the processing of antigen bound via specific sig may involve a specific intracellular pathway and that intracellular routing may be determined either by the degree of cross-linking of sig induced by antigen vs anti-ig or the mode of interaction of the various ligands with sig. alt*, departments of biochemistry (*) and medicine (+), college of physicians and surgeons of columbia univerity, new york, new york . we have recently analysed the structure of the / t cell receptor (tcr) expressed by the normal human thymocyte clone cii. cii expresses a c ' constant region that is a polymorphic form lacking a copy of an izternal exon; the sequence of this constant region accounts for the size of the chain and noncovalent linkage of and chains in the cii tcr. in order to elucidate its role, this / tcr will be reconstituted in immortalized t-cell lines. in addition, the productively rearranged human / receptor will be transgenically introduced into mice in order to assess the effect of the complete receptor on the development of t cells. the humoral immune response to human immunodeficiency virus has been shown to contain antibodies which act to mediate the uptake of virus through fc receptor mediated mechanisms. it is therefore possible that vaccination with the entire envelope polypeptide may present immunologic determinants that enhance infection. one means by which to generate an immune response to hiv that shall possess neutralizing activity in the absence of infection enhancing activity is to generate anti-idiotypic abs that bear the internal image of neutralizing human antibodies directed against hiv. we affinity purified human antibodies from hiv+ patients on a viral lysate column. we have produced monoclonal anti-idiotypic antibodies directed against these abi's. two of these monoclonals were shown to he ag inhibitable by their ability to inhibit the binding of p o l y e l o n a l human antisera to hiv viral lysate on ortho hiv ab t,est, wells. one monoclonal, b , when coupled t.o klh and used to immunize mice, produced an abs that. bound to viral lysate in an elisa assay. an affinity column containing rbs was used to purify an abi that was shown to bind to p and p hy western blot analysis. these data suggest that br may be a potential vaccine candidate. we have recently described a transgenic mouse model which co-expresses the tcr u and fl chains from the c cell line (recognized by the b anti-clonotype). t celh bearing the transgenic clonotype are positively selected by elements of the h-zb mhc for expression on cd ' cells. thus in the periphery of h-zb animals - % of the t cells are bz /cd *. the same peripheral expression is observed when the transgenes are expressed in f animals bearing a "neutral" mhc haplotype (eg. h-zb'*). however, when the transgene hi expressed in f animals which also express the h- ld gene product, negative selection occurs by clonal deletion. however, this deletion is functional rather than structural as the b clonotype is present on - % of peripheral t cells. these cells are unusual in that they express neither of the characteristic peripheral molecules cd or cd . the absence of cd expression on the b * cells appears to allow these potentially self-reactive clones to exist without evidence of autoimmunity. the original clone as well as b +/cd + cells from h- b animals are strongly inhibited by anti-cd reagents. in an effort to understand the process of negative selection and self-tolerance we have examined the capacity of these cells to be activated directly by the anti-clonotype rather than antigen (h- ld). the results demonstrate that the clonotype is fully functional on these double negative cells, indicating a normal maturation in the thymus. further examination of their surface phenotype also supports the conclusion that these are fully mature cells which are phenotypically distinct from double negative cells which exist in the thymus of h- b animals. of imunohematology. azl, leiden, the netherlands; praxis biologics, rochester, new york, usa and 'university of southampton, uk. immunity to disease caused by neisseria menineitidis is associated with the presence of bactericidal and opsonic antibodies to the capsular polysaccharide (cps), lipopolysaccharide and to outer membrane proteins (omps). the cps of group a and c meningococci are proven efficacious vaccines, although the immunogenicity in infants is poor and the immunity is of short duration. the combination with t-helper epitopes will certainly improve the immunogenic properties of these t-independent (ti,) antigens. the group b cps is poorly immunogenic in humans probably because of tolerance due to structural similarity to host glycopeptides and/or glycolipids. we have focused our research onto the class omps which show limited heterogeneity amongst meningococci. murine monoclonal antibodies to these proteins are highly bactericidal in vitro and will be used to map b-cell epitopes. t-epitopes have been identified by theoretical prediction of immunodominant sites by analysis of the amino acid sequence of the omp followed by their solid phase synthesis and subsequent testing for polyclonal activation of t-lymphocytes obtained from hl -typed volunteers immunized with the omp. in addition human t-cell clones are generated with omps and maintained with antigen, ebv-transformed b-cells, fresh feeders and ril . the clones are tested for antigen specificity, io vitro helper function, mhc restriction element, expression of surface markers and recognition of common meningococcal t-cell epitopes. c demonstration o f p-azobenzene-arsonate-l-tyrosine (aba-tyr) speclfic t cells in low responder h- mice by il- supported t cell proliferatlon previous studies have shown that h-zb mice immunized with aba-tyr fail to produce aba specific delayed-type hypersensitivity and show little or no t cell proliferation in vitro to aba-tyr. these observations suggest that h-zb mice are deficient in th cells that respond to aba-tyr. by contrast, immunization of h- b mice with tnp conjugates of aba-tyr revealed good cognate help, suggesting that these mice do possess aba-tyr specific th cells and that such cells are not revealed in conventional lymphoproliferative assays. because such assays are widely used to evaluate ir gene control and to map t cell epitopes, the databases generated from such studies may seriously under represent the total number of responder phenotypes and t cell epitopes. because of this concern, we established culture conditions that wlii support aba-tyr specific t cell proliferation in h- b mice. in these studies, c bu .l mice were immunized s.c. with aba-tyr and to days later the draining lymph nodes were cultured with varying doses of aba-tyr or with varying doses of aba-tyr and varying doses of recombinant il- alpha (rll-la), a known costimulator of th cells. culture with aba-tyr alone produced no proliferation. by contrast, culture with aba-tyr and rll-la revealed t cell proliferation that titrated with the dose of aba-tyr and the dose of rll-la specific for conalbumin presented on ryngeneic antigen presenting cells and dependent on il- for its proliferation, was used a s an indicator cell for the ability of neonatal murine spleen cells to present antigen and produce il- and il- .the antigen presenting capacity of neonatal spleen cells is low. during antigen presentation there is an augmentation of il- and il- production by the antigen presenting spleen cell population. however, neonatal spleen cells do not respond as well a s adult cells. the low levels o f il- can not be attributed t o a low potential for producing il- since neonatal cells produce high levels of il- after induction by a crude il- inducer factor (il- -if).the this impairment leads t o a decreased stimulus of the -helper cell to produce inducer factors which leads t o low levels of il- and il- production by the neonatal cells during antigen presentation. no suppressor mechanisms responsible for the l o w interleukin production were detected. human or murine class i genomic dna was transfected into a b-lymphohlastoid x t-lymphoblastoid hybrid cell line. this fusion hybrid has lost both t cell derived copies of chromosome six and contains deletions spanning the class i region on both copies of chromosome six derived from the b-cell parent. previous data have described a transacting factor within this region that is responsible for class i antigen expression. hla-bw and b glycoproteins, although synthesized, were not transported to the plasma membrane in the hybrid. were surface expressed. these data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport. the role of glycans in this transport dicotomy is currently under investigation. in addition, hybrid human-murine genes are being used to identify regions of the class i molecules involved in this transport phenomenon. we probed t h e means by which t h e a n t l g e n p r e s e n t l n g c e l l (apc) handles t h e a n t i g e n produce p e p t i d e s t h a t bind t o mhc-molecules. we propose t h e e x i s t a n c e o f a new type o f i n t e r n a l image i n which immunoglobulin v-region peptides. formed by processing, imitate peptides from conventional a n t i g e n s . w e r e f e r t o such denatured i n t e r n a l images as r e s i d u e internal images, since t h e y a r e a s s o c i a t e d w i t h t h e r e s i d u e of p e p t i d e s remaining a f t e r processing. in some cases, r e s i d u e internal images may be actual sequence images, i . e . , the v-region sequence m y be i d e n t i c a l t o the conventional a n t i g e n sequence.to be class i h- ka-restricted cytolytic t lymphocytes (ctl) are directed against two immunodominant sites on the a/jap/ / influenza hemagglutinin (ha) that can be mimicked by synthetic oligopeptides spanning residues - in the ha and - in the hydrophobic, transmembrane region. analysis of the fine specificity of hal-specific ctl clones demonstrated that these ctl clones can be subdivided into at least two group based on their patterns of recognition of closely related influenza h n field strains and a monoclonal antibody derived variant of a/guiyang/ / . using a series of nested synthetic peptides spanning the - region, the minimal amino acid residues necessary for recognition by the two groups of ctl clones were defined and found to consist of two separate but overlapping sites. sequence comparison of the ha of the a/jap/ / , the influenza field isolates and the monoclonal antibody derived variant has identified two amino acids, asn at position and gly at position , that are critical for t cell recognition. thus, animo acid substitutions induced either by antigenic drift or by monoclonal antibody selection can affect class i ctl recognition. pretreatment with a n t m e s reactive with class mhc anti-has previously been reported to be successful in exov~~kj a n t i g e n -w i r q dendritic cells (m) fran rodent tissue grafts. we have extended these exper-to inta? whole organ grafts. ilia pnmxses also demcnstrated a prolonged survival ( f days) ccapared to controls ( t days) (p < . ) tihen v l a n t e d into streprozatocin treat& da recipients. antigenic variation in the haemagglutinin (ha) of influenza a viruses frequently introduces new oligosacchekide attachment sites ( aan-x-serlthreo) and carbohydrate addition prevents antibody recognition by steric hindrance. acid substitution in mutant viruses of the h n subtype (ha asp+asn), that introduces an n-glycosylation site (hn gcys &thr ), abrogates antibody and cd + t recognition. infected with x virus recognise a synthetic peptide corresponding to antigenic site e, ha - , and are sensitive to a single substitution (ha asp-basn) in mutant viruses. virus infected target cells, thereby confirming that carbohydrate addition prevents cd ' t cell recognition. here ve show that an amino cell i-ad restricted, ha specific t cell clones f r m balblc mice-reviously recognition of mutant viruses is restored however by tunicamycin-treatment of key: cord- -echu zmf authors: aich, palok; potter, andrew a; griebel, philip j title: modern approaches to understanding stress and disease susceptibility: a review with special emphasis on respiratory disease date: - - journal: int j gen med doi: nan sha: doc_id: cord_uid: echu zmf studies in animals and humans link both physical and psychological stress with an increased incidence and severity of respiratory infections. for this manuscript we define stress as the physiological responses an individual undergoes while adjusting to a continually changing environment. it is known that stressors of various types (psychological/physical) can alter the physiological levels of certain hormones, chemokines and cytokines. these alterations send information to the central nervous system to take necessary action which then sends messages to appropriate organs/tissues/cells to respond. these messages can either activate or suppress the immune system as needed and failure to compensate for this by the body can lead to serious health-related problems. little is known how stress affects disease susceptibility, yet understanding this mechanism is important for developing effective treatments, and for improving health and food quality. the current review focuses on (a) the effects of psychological stressors in humans and animals, (b) various methodologies employed to understand stress responses and their outcomes, and (c) the current status of the attempts to correlate stress and disease with respiratory disease as model system. the methodologies included in this review span traditional epidemiological, behavioral and immunological studies to current high throughput genomic, proteomic, metabolomic/metabonomic approaches. with the advent of various newer omics and bioinformatics methodologies we postulate that it will become feasible to understand the mechanisms through which stress can influence disease onset. although the literature in this area is limited because of the infancy of this research area, the objective of this review is to illustrate the power of new approaches to address complex biological questions. these new approaches will also aid in our understanding how these processes are related to the dynamics and kinetics of changes in expression of multiple genes at various levels. from the preceding discussion it is clear that stress can be defi ned in many different ways depending upon the objectives or perspective of the researcher. all these defi nitions, however, share a common component of adaptive physiological responses following challenges to homeostasis. the adaptive reactions to stressors may involve mobilization of a wide variety of physiological responses including the immune response. stress responses usually include physical perturbations that can encompass either the entire body or specifi c cellular compartments. considering the volume of work in various areas of stressors and their effects the main objective of the current review is to focus on one type of stress, which includes psychological stressors. for our purposes, stress can be defi ned as a psychologically perturbing condition occurring in response to adverse external infl uences capable of affecting physical health. transportation, fear (ie, fright and fl ight response), overcrowding and weaning in the form of social reorganization are a few of the important types of psychological stressors identifi ed in the literature. these stressors have been linked to many conditions including immune suppression, disease susceptibility, hypertension and reproductive dysfunctions. [ ] [ ] [ ] stress is a major concern because it is ubiquitous, recurring in nature and has detrimental effects on health. in this review we will primarily deal with important psychological stressors and their infl uence on respiratory disease which is one of the most widely studied models. for many years, psychological stress has been shown to signifi cantly increase disease susceptibility. , until years ago, researchers investigating the psychological factors contributing to human disease focused primarily on coronary heart disease and cancer and neglected studies on infectious diseases. however, interest in this area started to shift with the publication of evidence that psychological factors infl uenced immune function. furthermore, there was an increasing awareness that stress and other psychological factors played a role in the onset and progression of acquired immunodefi ciency syndrome (aids). these studies demonstrated a signifi cant role for psychological stressors in compromising immunity and interest in the effects of stressors in other diseases was initiated. considerable emphasis has been placed on respiratory diseases in understanding the onset and severity of the disease as a result of psychological stress. increased risk of fatal bacterial respiratory infections following a primary viral infection has been observed in a wide variety of species. this phenomenon is called viral-bacterial synergy and was fi rst established following human infl uenza epidemics when a variety of secondary bacterial respiratory infections were associated with increased mortality. studies have also linked a variety of psychological stressors with an increased incidence and severity of respiratory infections in humans , and animals. , it is known that respiratory disease has a huge economic impact in the areas of human health care, animal welfare and the food industry. , to focus the review, we will restrict our discussion to research related to a comparative analysis of the effects of psychological stress on respiratory disease in humans and cattle. there are reports which have shown direct connections between stress and immune system function. similarly, other studies have shown that social stressors could also increase the risk for upper respiratory infection. a viral challenge study provides the strongest evidence for a link between stress and susceptibility to the common cold. other studies have extended these results by considering a wider range of psychosocial factors. the effects of stress on health are often mediated by a number of psychological factors. social support can often act as a buffer against the effects of stress as cohen and colleagues showed that social support reduced viral replication rate and increased mucociliary clearance of infection. , in another report, they examined the effects of stress and social support in a routine study of upper respiratory tract illnesses. under low levels of stress, high levels of social support were associated with a decreased risk of infection, whereas social support had no effect when levels of stress were high. a separate study was done to examine the associations between psychosocial factors (stress, social support, fl uctuations in mood) and viral exacerbations of asthma. the study involved naturally occurring illnesses rather than experimentally-induced infections but it maintained several important features of the methodology used by cohen and colleagues. , for example, stress was measured at the start of the study by measuring the immune responses in terms of leukocytes present in peripheral blood in order to determine the extent to which stress could predict subsequent illness. in addition, effects of personality, smoking status, and alcohol consumption were also studied as possible predictors of susceptibility to respiratory viral infections. before considering the effects of psychosocial factors on respiratory virus-induced exacerbations of asthma, it is essential to have strong evidence that these viruses play a direct role in asthma. until recently, it appeared to be only a weak association between asthma and upper respiratory tract infection in adults. , the absence of a stronger association in these epidemiological studies of adult asthmatics could, at least in part, have been due to diffi culties in isolating human rhinoviruses and coronaviruses. indeed, results from a study using enzyme-linked immunosorbent assays for antibodies to human coronavirus and semi-nested reverse transcriptase polymerase chain reactions for detections of rhinovirus suggested a stronger association between these viruses and asthma in adults. in summary, this study confirmed that psychosocial factors and health-related behaviors were associated with increased susceptibility to colds, which then led to an exacerbation of asthma. this conclusion was made in the context of a study where both diseases (cold and asthma) were verifi ed using objective measures. the well established buffering effect of social support was observed in the high stress group and possible confounders such as demographics, health-related behaviors or personality could not account for this effect. alcohol consumption, personality and demographic factors were also shown to be important predictors of susceptibility. in contrast smoking was related to illness severity but not disease susceptibility. these results show that one must consider a range of psychosocial factors, personality traits, demographic factors, and health-related behaviors in studies of individual differences in susceptibility and severity of upper respiratory tract infections. while studies have correlated stress-related behavior with disease susceptibility, research has also been performed to determine the immunological basis of the relationship between stress and disease. exposure to viral agents that cause upper respiratory disease provokes illness in some individuals but not others. moreover, the severity of clinical symptoms among those who develop illness can vary substantially. evidence from prospective epidemiological studies , , and from experimental viral-challenge studies , show that individuals reporting greater psychological stress have both a higher incidence and a greater severity of illness. however, past attempts to identify the behavioral patterns and biological responses linking psychological stress with upper respiratory viral illness have been unsuccessful. , , other studies reported that psychological stressors acutely activate the production of interleukin- (il- ), a pro-inflammatory cytokine. il- release in response to infection is thought to be mediated by glucocorticoids, thus providing a hypothetical pathway by which stressors (via the induction of glucocorticoid production) could control cytokine release. in addition, il- triggers additional release of glucocorticoids, possibly exacerbating the stress response through positive feedback. at least one source of il- is epithelial cells as evidenced by their production of il- in vitro and in vivo when exposed to rhinovirus. because a local increase in the concentration of this pro-infl ammatory cytokine precedes the development of acute signs and symptoms of illness, it has been implicated as a mediator in the pathway for symptom expression. in fact, il- concentrations in nasal secretions were associated with upper respiratory tract symptoms among persons infected with infl uenza a virus (a/texas and a/kawasaki) and rhinovirus (strain hanks' and type ). cohen and colleagues addressed the hypothesis that il- production in response to infl uenza a virus infection represents a viable pathway through which psychological stress infl uences the severity of illness. to achieve that goal, they measured levels of psychological stress in a group of adult subjects before experimentally infecting them with influenza a virus. self-reported respiratory symptoms, mucus weights, and local il- concentrations were then measured on the day before and for seven days after virus exposure. the data collected supported the conclusion that the level of psychological stress predicts the severity of illness and also the magnitude of the cytokine response. the data were then examined for evidence that il- mediated the association between stress and illness. these analyses suggested that most of the effects of psychological stress on clinical symptoms and mucus weights could be accounted for by changes in il- . however, it is possible that il- itself is not the causal link but rather just a marker (covariate) for other pro-infl ammatory cytokines which were elevated during the course of experimental infection. for example, in another related study reported that interferon α and il- levels (but not tumor necrosis factor [tnf], il- , il- , or il- ) increased early in the course of infection and both correlated with viral titers, increases in body temperature, mucus production and symptom scores. there is also an issue regarding the correlational nature of the mediational analysis. although consistent with the hypothesis that the association between stress and illness was mediated by il- , the data do not permit causal inference. for example, this pattern of data is also consistent with increases in il- occurring in response to tissue damage associated with illness symptoms. even with these reservations, this was the fi rst aich et al study to provide evidence consistent with the hypothetical model that psychological stress infl uences upper respiratory infections through a biological pathway. there are two parts to the stress response: sympatheticadrenal-medullary (sam) and the hypothalamic-pituitaryadrenal axis (hpa). the hpa is the core stress axis in mammals and together with the sam system co-ordinates response to the diverse range of stressors from psychological to physical. there is considerable interplay between both neuronal systems especially between the noradrenergic nucleus locus ceruleus which provides central regulation of the sam and the parvocellular neurones which regulate the hpa. the sam, by triggering catecholamine release, provides the acute stress response whilst the hpa governs longer term stress defence mechanisms. together those systems regulate energy utilisation and metabolic activity throughout the body. the sam system produces the immediate shock response by acting on the hypothalamus, which activates the adrenal medulla and the sympathetic autonomic nervous system (ans) (figure ). the sam produces the "fi ght-or-fl ight" response which increases alertness, blood fl ow to muscles, heart rate, blood pressure, respiration rate, etc. and might decrease activity in the digestive system. the hpa system regulates release of the hormone crf to activate the anterior pituitary and uses another hormone, adrenocorticotropic hormone (acth), to activate the adrenal cortex to release a group of hormones including cortisol ( figure ). cortisol and other glucocorticoid hormones have various effects such as conservation of glucose for neural tissues, elevation or stabilization of blood glucose levels, mobilization of protein reserves, conservation of salts and water, suppression of wound healing and the immune system. according to seyle's general adaptation syndrome (gas) theory the general adaptation syndrome divides the stress response into three stages: stage : alarm reaction (sam and hpa activity increases and result in the "fi ghtor-fl ight" response); stage : resistance (hpa activity takes over, bodily resources are at maximum and if the stress is experienced for short term the body returns to normalcy); stage : exhaustion (with very prolonged stress, bodily systems are ineffective. sympathetic ans action reappears. adrenal cortex damage causes parasympathetic action, omics approaches in understanding stress and disease susceptibility eg, energy storage failure. the immune system collapses, and stress-related diseases increase). while it has been assumed that psychological (psychogenic) and physical (neurogenic) stressors are most closely aligned with depression, the suspicion has arisen that systemic stressors, including immune alterations, may also act in such a provocative capacity. communication occurs between the immune, endocrine, autonomic and central nervous systems such that psychological events that affect central neurochemical processes may affect immune activity. conversely, immune activation may affect hormonal processes and the activity of central neurotransmitters. thus, by virtue of the neurochemical effects imparted, immune activation may affect behavioral outputs and may even be related to behavioral pathology such as depressive illness. the hypothesis that altered immune activation may occur as a result of various stressors emerged over time. the initial theory came from hans seyles' gas, which was derived from observation and experimentation on laboratory animals. using a variety of stressors (ie, pain, thermal extremes and starvation), seyle described a common nonspecifi c stress response pathway. after initial perception of a stressor, the animal mounts an emergency alarm or fi ght-or-fl ight response. this catecholamine-driven reaction results in increased cardiovascular function and an overall increase in metabolism. if the stressor persists, the resistance phase or 'conservation withdrawal reaction' is initiated, which is a physiological coping reaction to the increased demands of maintaining homeostasis. chronic stress leads to the exhaustion phase and may lead to pathology. seyle's theory unifi ed the stress phenomena because it provided a common response pathway to all the varied stressors encountered. this pathway, the hpa axis, involves perception in the brain with release of hypothalamic corticotropin-releasing factor (crf) and vasopressin which stimulates the anterior pituitary to secrete acth ( figure ). circulating acth causes the adrenal cortex to produce glucocorticoids (gc). glucocorticoids cause gluconeogenesis with conversion of lipid to glucose for the central nervous system (cns) and other functions. thus, gas allowed the identifi cation of stressors (and presumably the status of well being of the animal) by measurement of gc levels. it was an attractive theory because gc levels were relatively easy to measure. unfortunately the gas theory has proven to be too simplistic. mason's experiments with rhesus monkeys aich et al exposed to different types of stressors revealed a disparity in neuroendocrine responses provided by specifi c stressors. for instance, monkeys subjected to emotional stress had elevated serum gc levels but those subjected to a heat-stress regime failed to show gc elevation. in addition to gc, other neuroendocrine mediators were characteristically produced in response to specifi c stressors. emerging information on the response to stressors suggests that there are at least four different avenues of neuroendocrine responses. these involve the autonomic nervous system, the hpa axis, neuropeptides, neurotransmitters and neuroimmunological peptides and receptors. interactions between the immune and nervous systems play an important role in modulating host susceptibility and resistance to infl ammatory disease. neuroendocrine regulation of inflammatory and immune responses as well as onset of disease can occur at multiple levels: (a) systemically through the anti-infl ammatory action of gcs released via hpa axis stimulation, (b) regionally through production of gcs within the affected tissue as well as by sympathetic enervation of immune organs such as the thymus and (c) locally at sites of infl ammation. estrogens also play an important role in immune modulation and contribute to the approximately -to -fold higher incidence of autoimmune/infl ammatory diseases in females of all mammalian species. during infl ammation, cytokines from the periphery activate the central nervous system through multiple routes. this results in stimulation of the hpa axis which in turn, through the immunosuppressive effects of the glucocorticoids, generally inhibits infl ammation. recent studies indicate that physiological levels of glucocorticoids are immunomodulatory rather than solely immunosuppressive causing a shift in patterns of cytokine production from a th -to a th -type pattern. interruptions of this loop at any level and through multiple mechanisms, whether genetic, or through surgical or pharmacological interventions, can render an infl ammatory resistant host susceptible to infl ammatory disease. over-activation of this axis, as occurs during stress, can also affect severity of infectious diseases through the immunosuppressive effects of the glucocorticoids. these interactions have been clearly demonstrated in many animal models using a variety of species and infectious agents. the results from these models are also relevant to human infl ammatory, autoimmune and allergic illnesses including rheumatoid arthritis, systemic lupus erythematosus, sjogren's syndrome, allergic asthma and atopic skin disease. while many genes and environmental factors contribute to susceptibility and resistance to autoimmune/infl ammatory diseases, a full understanding of the molecular mechanisms by which a combination of neuropeptides, neurohormones and neurotransmitters can modulate immune responses is essential for effective design of future interventions. it is clear from the previous discussion and review of previous research that the relationship between stress and disease susceptibility is very complex and intertwined with cascades of events. in order to understand or characterize stress-induced disease susceptibility one needs to identify various biological or functional pathways and interaction networking involved at any given time. to follow the cascade of molecular events happening over time one needs to employ methodologies which are holistic in nature and can provide global information related to the kinetics of multiple changes in gene expression and multiple biomolecular interactions. with the advent of various high-throughput genomic approaches it has become possible to explore complex biological processes and in one step obtain information about gene expression at the transcriptional (genomics) and translational (proteomics) level, as well as to identify metabolites arising from these responses (metabolomics/metabonomics). these methodologies together are often referred to as 'omics' approaches. although these methodologies are still in their infancy they have started showing promise in understanding systems' biology and various disease processes. work employing omics approaches to understand mechanism of stress and disease susceptibility is limited at this time. there are reports which describe the effects of oxidative stress on cellular apoptosis but there are no reports on the effects of psychological stressor and disease susceptibility in animals using holistic methodologies such as various omics approaches. the literature in this area is very scarce because these methodologies are very new and few results are currently available. the main studies to correlate stress and disease susceptibility was done either in plant systems or a correlation between oxidative stress and infectious disease. the review by hirai and saito established, using liquid chromatogram based mass spectrometric proteomic and metabolomic and cdna microarray based transcriptomic analyses, that these omics studies can reveal the genomic networking involving several pathways related to oxidative stress response and key metabolic pathways. bioinformatics and statistical approaches specifi c for detailed transcriptomics or cluster analyses of expressed genes have been developed and are useful for extracting information on various functional genomic responses. [ ] [ ] [ ] it is important, however, to understand the translation of transcriptomic information into protein expression and modifi cations since it is the protein that acts as biomarkers for various disease processes or conditions. several proteomic tools have been developed. these include gel-based two-dimensional ( d) gel electrophoresis (ge), d fluorescence difference gel electrophoresis ( dige) or gel-free multidimensional protein identifi cation technology (mudpit), , isotope-coded affinity tag (icat™), surface-enhanced laser desorption-ionization time-of-flight (seldi-tof) ms , or isobaric tag for relative and absolute quantitation (itraq) methodologies. while these technologies are being matured more appropriate biological questions can be addressed. in d-ge whether traditional or newer dige system, proteins are fi rst focused (fi rst dimension) in terms of their pi values followed by sds-page (second dimension). in contrast to traditional d-ge, dige system utilizes the fl uorescence labeling of protein samples and two-to-three different samples can then be run on a single gel which makes it superior to traditional d-ge in terms of eliminating gel to gel variation and comparative analysis. although d-ge is a very good way of separating complex protein mixtures, it is limited in terms of high-throughput analysis and sensitivity. because of low sensitivity it is mostly limited to the analysis of high abundant proteins of a system. efforts have been made to improve the sensitivity and analysis capacity of proteomics techniques. as a result, other methodologies such as d-hplc, mudpit, icat, and itraq have been developed and are gaining popularity. although various bioinformatic and data analyses approaches have been developed to analyze gel images and mass spectrometric based protein characterization a highthroughput methodology in this area has yet to be developed. functional genomic techniques of transcriptomics and proteomics and available bioinformatic and statistical analyses promise unparalleled global information during the analyses of complex biological responses. however, if these technologies are used in isolation, the large multivariate data sets produced are often diffi cult to interpret and have the potential to ignore key metabolic events to understand the true biology. high resolution h nmr spectroscopy used in conjunction with pattern recognition provides one such tool for defi ning the dynamic phenotype of a cell, organ, or organism in terms of a metabolic phenotype. in a recent review the benefi ts of this metabonomics/metabolomics approach to problems in toxicology have been discussed. one of the major benefi ts of this approach is its high-throughput nature and cost-effectiveness on a per sample basis. using such a method, the consortium for metabonomic toxicology (comet) is currently investigating approximately model liver and kidney toxins. this investigation will allow the generation of expert systems where liver and kidney toxicity can be predicted for model drug compounds, providing a new research tool in the fi eld of drug metabolism. the review also included how metabonomics may be used to investigate co-responses with transcripts and proteins involved in metabolism and stress responses such as during drug-induced fatty liver disease. by using data integration to combine metabolite analysis and gene expression profi ling, key perturbed metabolic pathways can be identifi ed. detailed omics-based bioinformatics studies, to correlate stress-dependent disease susceptibility, have yet to analyze this complex biological response. a few preliminary studies have been reported in the literature. a recent study has shown the adverse effects of using mechanical ventilators for respiratory support. this acute lung injury because of mechanical stretch can lead to high mortality and this study has utilized recent advances in bioinformatic-intense candidate gene searches to correlate the lung injury and gene expression profi les in a rodent model analyzed with microarrays. the authors used , mouse/rat orthologous genes identifi ed on rg_u a and mg_u av arrays and the expression profi les were simultaneously analyzed by signifi cance analysis of microarrays. this combined ortholog and signifi cance analysis of microarrays approach identifi ed up-and downregulated ventilator-induced stress-related candidate genes. results were validated by comparable expression levels obtained by either real-time or relative rt-pcr for randomly selected genes. k-mean clustering of candidate genes clustered several well-known lung injury associated genes (il- , plasminogen activator inhibitor type , ccl- , cyclooxygenase- ) with a number of stressrelated genes (myc, cyr , socs ). the only unannotated member of this cluster (n = ) was riken_ f est, an ortholog of the stress-related gene /mig- gene. the authors speculated that the ortholog-signifi cant analysis of microarray approach is a useful, time-and resourceeffi cient tool for identifi cation of candidate genes in a variety of complex disease models such as ventilator induced lung injury. using microarray-based genomic approaches work has also been initiated to identify hypertension-related genes in rat model. though it is a common belief that the stressors mentioned earlier can compromise host immune responses and enhance susceptibility to various diseases, very little work has been done in this area to understand the mechanism of stress dependent disease susceptibility in mammals. kelley in identifi ed eight stressors that typically occur in modern livestock production units: heat, cold, crowding, mixing, weaning, controlled-feeding, noise and restraint. all these stressors were shown to alter components of the immune system in animals and these changes in immune function may ultimately explain the physiological basis of diseaseenvironment interactions. another study in showed that neurotransmitters and neuroendocrine hormones can modify the function of immune cells. conversely, cytokines produced by immune cells can alter brain homeostasis. this connection is further manifested by experimental studies showing a relationship between stress and resistance to infection. human subjects under high stress were shown to be more susceptible to infection with common cold viruses. furthermore, a diversity of experimental animal models confi rmed that laboratory stressors such as forced exercise, avoidance learning, restraint, isolation and cold exposure made animals more susceptible to primary infection with a variety of viruses and bacteria. the cellular and molecular basis for the observed modulation of host resistance is not fully understood but involves altered functioning of both t-lymphocytes and cells of the hypothalamic-pituitary adrenal axis. also involved is the altered production of cytokines and hormones produced by the immune system and brain. emau and colleagues in showed that the onset of immunodepression by stress or viral infection in the pathogenesis of bovine pneumonia permits super infection of the lungs with mannheimia haemolytica (formerly called pasteurella haemolytica) which results in exudative fi brinous pneumonia. although these studies provide a preliminary basis of stress-induced bovine respiratory disease (brd), there was a lack of necessary information to determine the molecular basis of such dependence, which can be found by studying changes at the level of gene expression (transcriptional and proteomic) and metabolites. a recent study compared proteomics of epithelial lining fl uid from lungs of weaned and nonweaned cattle. the study was done over a shorter period of time ( h) with a combination of stressors (weaning, transportation) and important questions such as to determine specifi city and duration (stability) of the protein biomarkers are yet to be addressed. the group also studied the proteomic profi le of bronchoalveolar lung lavage fl uid following treating the calves with dexamethasone to determine the effects of glucocorticoids, which is elevated following induction of stress. , more studies are however needed (a) to fi nd biomarkers associated with stressors and infection, (b) to determine the duration of the biomarkers so that these are sustainable enough to identify and predict stressed animals (which might be more susceptible to infection in real life situation such as in feedlot cattle). disease models have been developed to study the molecular mechanisms underlying the viral-bacterial synergy which results in fatal brd infections. [ ] [ ] [ ] we have confi rmed with the infectious brd model that the stress of weaning significantly enhances the viral-bacterial synergy leading to fatal bacterial respiratory infection. the major stressors young omics approaches in understanding stress and disease susceptibility cattle experience include maternal separation or weaning, dietary changes, transportation, social reorganization and other environmental effects (figure ). with this in mind, we focused our initial studies to understand the role of weaning on the clinical response to brd. fifteen suckling calves were removed from their mothers h prior to viral infection (abruptly weaned/stressed, aw). a second group of calves was weaned wks prior to viral infection (pre-conditioned/ control, pc); wks was chosen as an appropriate interval to eliminate psychological and physiological effects associated with breaking the maternal/nutritional bond and to adapt to the dietary change and social re-organization that follows weaning ( figure ). all calves were transported to vaccine and infections disease organization vido, and aerosol challenged with bovine herpesvirus- (bhv- ) followed by m. haemolytica; this combined viral-bacterial infection induces fatal pneumonia in %- % of calves. our clinical analyses revealed a signifi cant difference in brd clinical disease when comparing freshly weaned calves ( % mortality) versus pre-conditioned calves ( % mortality). , increased mortality associated with fresh weaning was characterized by a decrease in both survival time post-infection and, interestingly, decreased lung pathology which suggested a systemic reaction. contrary to expectations, transportation induced a signifi cant cortisolemia in pre-conditioned but not freshly weaned calves ( figure ). transportation is known as an important stressor which increases blood cortisol level. [ ] [ ] [ ] [ ] however, the stressor combination of weaning and transportation may have different physiological markers than commonly expected. cortisol is a potent regulator of pro-infl ammatory cytokine transcription by acting as a negative regulator of nfκb activation, and can be used as an indicator of stress. fatal bacterial respiratory infection in calves was usually associated with an accelerated decline in serum cortisol levels. therefore, we hypothesized that increased respiratory disease susceptibility was associated with enhanced pro-infl ammatory responses in freshly weaned calves. elevated body temperature and interferon-gamma (ifn-γ) levels in nasal secretions during bhv- infection of freshly weaned calves were similar to previous reports, , supporting this hypothesis. these immune responses were signifi cantly increased despite no signifi cant difference in virus shedding. furthermore, increased il- expression, during bhv- infection of pre-conditioned calves, was consistent with reduced pro-infl ammatory responses. we conducted bovine microarray analyses of rna isolated from blood mononuclear cells to determine if changes in gene expression correlated with either stress or the severity of brd infection; results support the conclusion that differential regulation of pro-infl ammatory responses is a major mechanism contributing to increased disease susceptibility. conserved responses included an enhanced potential to aich et al respond to pathogen-associated molecular patterns through increased expression of toll-like receptors (tlrs). tlr induction of innate immune responses is mediated primarily through activation of nuclear factor kappab (nfκb). we also observed differential expression of β-defensin (a host defense peptide induced by tlr signaling) in freshly weaned calves, consistent with reduced cortisol levels. in addition, when we compared expression of select innate immune genes (eg, tlr , tlr , ifn-γ and ' ' o-adenylate synthetase) between day (post bhv- infection) and day (prior to bhv- infection) for aw-and pc-groups, results revealed a trend of increased expression of the select genes on day irrespective of the stress situation (table ). this trend of activation of select immune genes clearly revealed that following bhv- infection the innate immune response was enhanced. this observation contradicts the current hypothesis that the primary viral enhances the secondary bacterial infection by compromising the immune system. , our studies revealed that the anti-infl ammatory gene il- was upregulated on day in pc groups while β-defensin was activated on day in aw groups (table ) . il- which acts as an anti-infl ammatory gene should be activated on day to control pro-infl ammatory responses as a result of bhv- infection in either aw or in both groups; instead it was only over expressed in control (pc) groups. on a similar token β-defensin on day should also be observed in both groups to control bhv- infection. these results are particularly interesting to explore further to understand the detailed mechanism of stress-induced disease correlation. although advances have been made in understanding various disease processes, successful intervention often depends upon the stage at which disease is detected. thus, identifying markers for disease or its cause, as well as understanding the mechanism of disease onset and susceptibility, are very important. systems biology approaches such as omics (genomic, proteomic, metabonomic) and multivariate analysis to understand mechanisms and to identify biomarkers provide potential tools to address these questions. , [ ] [ ] [ ] it is particularly important to employ a combination of molecular approaches such as omics to understand complex processes such as stress and its correlation with disease susceptibility. with such goals in mind, we identifi ed and characterized a group of protein, metabolite and elemental biomarkers using serum samples from bhv- infected as well as from stressed animals. the trend of each group of biomarkers distribution was analyzed to correlate with the groupings based on stress condition or infection. multivariate analysis revealed distinct differential trends in the distribution profi le of proteins, metabolites and elements following a stress response both before and after primary viral infection. a group of acute phase proteins, metabolites and elements could be specifi cally linked to either a stress response (decreased serum amyloid a and cu, increased apolipoprotein ciii, amino acids, low-density lipoprotein [ldl], p and mo) or a primary viral respiratory infection (increased apolipoprotein a , haptoglobin, glucose, amino acids, ldl and cu, decreased lipid and p). thus, combined omics analysis of serum samples revealed that multimethod analyses could be used to discriminate between the complex biological responses to stress and viral infection. there are reports which suggest that transport can be an important stressor which alters blood leukocyte populations and sets the stage for brd. , currently we are conducting experiments in which transportation of the animals is eliminated, such that stressors related to a new environment are removed. it was documented that abrupt weaning (separation of suckling calves from their dams) causes a prolonged psychological stress response. this stress response manifests as an increase in vocalization by both calves and cows and a significant increase in time spent walking by the calf. consequently, time spent eating and resting also decreases for abruptly weaned calves. statistically, these changes in behavior return to baseline values after four to fi ve days of weaning. as is evident from the review of the literature that traditionally effects of stress have been studied by attempting to understand behavioral patterns of the subject and cellular immune response of the host. it is, however, clear that amount of data to understand such a complex condition (such as stress and stress-induced diseases) is not adequate and enough. behavioral studies may not always be correlated to a particular stressor as there might have been other parameters affecting the observations. attempts have also been made to use cortisol as a unique biomarker for stress, but cortisol is diurnal hence cannot be a reliable marker. while it is important to understand cellular immunology to establish the mechanism of stress-induced disease susceptibility, however without identifying and establishing the immune markers specifi c for stressor it will not lead into any meaningful inference. systems biology approaches using various omics and bioinformatics methodologies can identify a group of biomarkers at various levels of host immune response, gene expression and metabolism. association of the identifi ed biomarkers and the patterns in changes in their expression level above or below a determined threshold level with specifi c stressor(s) could then be used to defi ne stress situation, identif ication of individuals susceptible to stressinduced disease. moreover, establishing a pattern in changes of biomarkers is more reliable than a single biomarker. duration of identifi ed biomarkers can further strengthen the methodology for applying in real life situation. the current review established the importance of psychological stress and its effects on infectious disease severity and susceptibility with emphasis on respiratory disease. as evident from the literature, initial work focused mainly on understanding the roles of various stressors on physiology and behavior. theories emerged as a consequence of these studies which implicated the hpa axis and the sympathetic nervous system (sns) in altered immunity. however, physiological responses studied in a laboratory may not necessarily be consistent with real life phenomena, especially when attempting to analyze heterogeneous populations such as cattle or humans. experiments with human subjects cannot always be properly controlled because of ethical concerns and therefore using appropriate animal models to mimic natural disease outcomes may be more informative. with the use of high-throughput omics approaches it should be possible to extract more detailed information out of these complex biological systems and understand the complex biology and mechanisms by which stress augments disease susceptibility. more studies are needed to understand the physiological differences between acute versus chronic stress as well as the combined effects of various stressors. it is becoming more apparent that stress not only makes an individual more susceptible to a disease but can also enhance disease severity. therefore, it is very important to understand the kinetics, specifi city and stability or duration of biomarkers associated with a specifi c stressor or combination of stressors before we can predict disease susceptibility in individual people or animals. the concept of stress and its 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and infections viral-bacterial pneumonia in calves: effects on plasma eicosanoids and long chain fatty acids stress alters the cellular and proteomic compartments of bovine bronchoalveolar lavage fl uid altered protein expression in neutrophils of calves treated with dexamethasone alterations in the bovine bronchoalveolar lavage proteome induced by dexamethasone effect of bovine alpha interferon on bovine herpesvirus type -induced respiratory disease use of recombinant bovine alpha interferon in reducing respiratory disease induced by bovine herpesvirus type prevention of experimental bovine pneumonic pasteurellosis with an extract of pasteurella haemolytica the effects of regrouping on behavioral and production parameters in finishing swine effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate infl ammation bovine herpesvirus- vaccination against experimental bovine herpesvirus- and pasteurella haemolytica respiratory tract infection: onset of protection effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate infl ammation comparative approaches to the investigation of responses to stress and viral infection in cattle transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress acute phase proteins in cattle after exposure to complex stress effects of road transportation on lymphocyte subsets in calves transportation stress alters the circulating steroid environment and neutrophil gene expression in beef bulls hypercortisolemia in acute stroke is related to the infl ammatory response induction of interferon activity in nasal mucus by association of levamisole by systemic route -inactivated virus by local route effect of stress on viral-bacterial synergy in bovine respiratory disease: a review of potential mechanisms immune mechanisms of pathogenetic synergy in concurrent bovine pulmonary infection with haemophilus somnus and bovine respiratory syncytial virus proteomic characterization of the interstitial fl uid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery unlocking the mysteries of virus-host interactions: does functional genomics hold the key? a comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in escherichia coli cells effect of transport stress on respiratory disease, serum antioxidant status, and serum concentrations of lipid peroxidation biomarkers in beef cattle effect of transportation on blood serum composition, disease incidence, and production traits in young calves. infl uence of the journey duration this project was funded by the saskatchewan agriculture and food rural revitalization (safrr), genome bc and genome prairie for the 'pathogenomics of innate immunity' research program and the ontario cattleman's association (oca). this manuscript is published with permission of the director of vaccine and infectious disease organization as article number . a.a.p. is the holder of an nserc senior industrial research chair. key: cord- -mn r x authors: hodgins, douglas c.; chattha, kuldeep; vlasova, anastasia; parreño, viviana; corbeil, lynette b.; renukaradhya, gourapura j.; saif, linda j. title: mucosal veterinary vaccines: comparative vaccinology date: - - journal: mucosal immunology doi: . /b - - - - . - sha: doc_id: cord_uid: mn r x infections of mucosal surfaces are major causes of morbidity, mortality, and economic loss in species of veterinary interest, and a concern for animal welfare. vaccines are used extensively in veterinary medicine, and innovative vaccine technologies such as recombinant dna-vectored and distinguishing infected from vaccinated animals (diva) vaccines and automated in ovo vaccination (of embryonated chicken eggs) have been rapidly adopted commercially. immunological research using outbred, nonrodent animal models has contributed to a broader understanding of mucosal defenses, and has provided the initial impetus for investigation of the common mucosal immune system. studies of the potential of novel adjuvants to improve vaccine efficacy against genetically unstable, immune-subverting rna viruses, such as porcine reproductive and respiratory syndrome virus in pigs, should assist in the control of pathogens with similar characteristics in other species. successful development of vaccines to prevent and treat ascending infections of the reproductive tract of cattle set a precedent for applications in other species including humans. studies of mucosal adjuvants and delivery systems continue at the interface between passive and active immunity, with the goal of inducing the earliest possible protection against enteric and respiratory pathogens of neonates. despite advances in nutrition, genetics, housing, and therapeutics, diseases of the respiratory, reproductive, and gastrointestinal tracts of domestic animals and poultry continue to be major causes of morbidity and mortality. although vaccines have been developed and licensed for prevention of many of these diseases, there is a need for improvement in vaccine efficacy and for new vaccines. reasons for low vaccine efficacy include inappropriate, unstable, or outdated antigens in vaccine preparations (e.g., in vitro expressed antigens instead of in vivo expressed antigens), inappropriate immune responses (e.g., systemic instead of mucosal, or th type instead of th type or vise versa), and inappropriate use of otherwise efficacious vaccines (e.g., inappropriate timing of vaccination) (tizard, ). an overview of some of the vaccines currently licensed for vaccination of domesticated animals and wildlife by mucosal routes is provided in table . many of these vaccines consist of pathogens attenuated by traditional methods, but engineered virus-vectored vaccines are now used extensively (by both mucosal and parenteral routes) in veterinary medicine. the majority of current poultry vaccines are attenuated agents delivered in ovo, orally, intranasally (in) or by other mucosal routes, for reasons of ease of administration, economy, and protective efficacy. in comparison, fewer vaccines for domestic mammals are delivered by mucosal routes. management practices for mammals differ from those for poultry; mass vaccination techniques for mucosal delivery have been developed for poultry, but have not been pursued as zealously for mammals. recently however, a number of attenuated live vaccines for in administration have been developed for respiratory tract infections, using traditional methodologies. improved protective efficacy and rapid onset of immunity compared to killed vaccine products have led to widespread acceptance. attenuated live oral vaccines for enteric diseases have also been marketed, but in many cases efficacy has been disappointing due to lack of potency in adults or interference by maternal antibodies in suckling animals. better strategies for induction of immunity in the gastrointestinal tract are needed, especially for neonates in the presence of maternal antibodies. in contrast, effective parenteral vaccines for the most common diseases of the reproductive tract in veterinary species have been available for years, and there has been little motivation to develop mucosal vaccines. many of the diseases of the respiratory and gastrointestinal tracts are most devastating in the neonatal period. for these diseases active immunization may not provide protection before natural exposure to the pathogen. maternal vaccination to enhance passive immunity has been widely used in veterinary medicine, especially for control of enteric diseases. practical difficulties arise, however, with diseases such as parvovirus enteritis in puppies in which a smooth transition must be made from protection by passive maternal antibodies to protection by active immunity, without permitting a window in between of disease susceptibility. this transition is difficult to achieve because induction of active immunity is commonly inhibited by maternal antibodies. various strategies are used to address this problem, but improved vaccines, adjuvants, and antigen delivery systems would improve the reliability of neonatal immunization. although progress is being made in disease prevention in veterinary species, ever changing management practices (e.g., earlier weaning of piglets, larger animal operations) generate new patterns of disease, requiring new control strategies. the emergence of new pathogens (e.g., porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus- ) provides new challenges for vaccine research before some of the older challenges have been met. in this chapter, we focus on mucosal veterinary vaccines and vaccine concepts related to selected pathogens of economic importance. our intent is to highlight progress, to review existing and future vaccination strategies, and to acknowledge the unique contributions of this research to our understanding of mucosal vaccines and immunology. respiratory tract infections are a major cause of morbidity and mortality in farm animals, poultry, and pets. disease conditions are intensified by current management practices such as mixing of recently weaned, stressed beef calves from multiple sources in auction barns. certain disease conditions, such as atrophic rhinitis of pigs, result from the interplay of several pathogens, and multiple agents must be represented in vaccines. some respiratory pathogens such as mycoplasma hyopneumoniae in young pigs are causing new patterns of disease as management practices change (e.g., weaning at an earlier age), requiring changes in vaccine strategies. other pathogens such as prrsv have only recently emerged and improved vaccines await advances in understanding of the agent and of disease pathogenesis. a discussion of respiratory vaccines for even the major pathogens of veterinary species is beyond the scope of the present review. this section will focus on vaccines for prrsv infections in pigs and influenza in horses to illustrate general principles. porcine reproductive and respiratory syndrome (prrs) is a chronic reproductive and respiratory disease of pigs caused by prrsv, a member of the family arteriviridae (benfield et al., ) . signs associated with prrs are anorexia, fever, lethargy, pneumonia, red/blue discoloration of the ears and vulva, delayed return of sows to estrus after weaning, abortion, fetal mummification, stillborn or weak born piglets, and high preweaning mortality (mengeling et al., ) . prrsv is prevalent in swine-producing countries worldwide. annual economic losses to the pork industry in the united states due to prrs have been estimated at $ million (holtkamp and kliebenstein, ) . according to the american animal and plant health inspection service (aphis) report of january , . % of unvaccinated pigs in the united states are seropositive to prrsv. infected pigs excrete prrsv in saliva, nasal secretions, urine, milk, colostrum, and feces at low levels or intermittently, and also in semen of infected boars (rossow et al., ) . in the absence of control measures, prrsv is spread by aerosols, fomites, and personnel. prrsv is divided into two distinct genotypes, type i (european) and type ii (north american). each genotype contains subtypes and strains, which are genetically diverse and vary in virulence and pathogenicity (kim et al., ) . immunity to one genotype of prrsv may provide partial or no protection against reinfection, reflecting the complexity of prrsv genetics and immunological variation among strains (botner et al., ) . swine are the only known species susceptible to prrsv. alveolar macrophages (mΦs) are the primary permissive cells to prrsv; mΦs present in pulmonary intravascular spaces, lymph nodes, thymus, heart, spleen, placenta, and umbilical cord may also be infected by the virus (halbur et al., ) . the most efficient and rapid host response against viruses consists of production of type i interferons (ifns) (ifnα and ifnβ). in prrsv-infected pigs, innate ifnα secretion is significantly suppressed (albina et al., ) and the virus dampens innate nk cell-mediated cytotoxicity as early as day postinfection (renukaradhya et al., ; dwivedi et al., ) . in pigs, a significant correlation has been observed between the prrsv infection and an increased expression of il- , associated with reduced expression of ifnα, ifnγ, il- , and tnfα (gómez-laguna et al., ) . induction of il- is mediated by interaction between prrsv proteins and mΦs/dendritic cells (dcs). the prrsv nucleocapsid protein induces il- production by peripheral blood mononuclear cells (pbmcs) and alveolar mΦs (wongyanin et al., ) , delaying the onset of protective cell-mediated immunity (cmi) to prrsv (dwivedi et al., a . dysregulated immune responses in infected pigs affect viral pathogenesis, disease severity, and susceptibility to secondary microbial infections (thanawongnuwech et al., ; renukaradhya et al., ) . immune responses against prrsv are inadequate to completely clear the virus. viremia disappears in - weeks, but the virus persists in the tonsils and lymphoid tissues for several months (moyron-quiroz et al., ) . prrsv infection in both germ-free and conventionally raised pigs is associated with polyclonal b cell activation, hypergammaglobulinemia, lymphoid adenopathy, renal lesions, and lymphoid hyperplasia (cooper et al., ) . polyclonal lymphoplasia also occurs in mice infected with lactate dehydrogenase-elevating virus, a related virus (grossmann et al., ) . primary antibody responses occur promptly after prrsv infection (loemba et al., ) , but the majority of secreted antibodies are autoantibodies or prrsv nonneutralizing antibodies (lemke et al., ) . the virusneutralizing antibody (na) response is weak, and delayed (mateu et al., ) . both killed and modified live virus (mlv) prrsv vaccines are available commercially for intramuscular or intradermal administration (charerntantanakul, ) . mlv vaccines confer protection against genetically homologous prrsv, but incomplete protection against heterologous viruses (mengeling et al., ) . in both prrsv-infected and mlv-prrsv-vaccinated pigs, virus-specific cell-mediated immune responses are either delayed or dampened . reversion to virulence is a major concern with modified live vaccines. there are numerous reports of reversion of prrsv vaccine virus to virulence and of the presence of reverted vaccine strain prrsv in unvaccinated sows and pigs . vaccine-derived virus has been isolated from fetuses, stillborn, and dead piglets, indicating the spread of disease from vaccinated pigs. identification of mutations in multiple vaccine-derived isolates at identical positions of the viral genome suggests a strong selective pressure on critical viral proteins in field situations. available killed prrsv vaccines fail to protect even against homologous virus, do not elicit na, and induce weak cell-mediated immune responses (bassaganya-riera et al., ) . pregnant sows and gilts and breeding boars are not protected from prrsv due to a lack of safe and protective vaccines. control of prrsv in breeding stock is critical in preventing vertical and horizontal transmission of the virus. early attempts to develop killed prrsv vaccines using recombinant prrsv proteins, plasmids expressing viral genes, and inactivated prrsv administered with or without adjuvants have been unsuccessful ( charerntantanakul, ) . however, recent studies show promise. killed prrsv vaccine coadministered in with cpg oligodeoxynucleotide adjuvant (a tlr- ligand) induced antibodies, virus-specific t cells, and secretion of ifnγ and il- (zhang et al., ) . in another study, prrsv inactivated by uv light or binary ethylenimine induced na and protected pigs against homologous viral challenge. the suppressive effect of live prrsv on ifnα production was lost when the virus was inactivated by uv irradiation (vanhee et al., ). an oral immunization strategy using prrsv nucleocapsid protein genetically fused to cholera toxin (ct) stimulated prrsv antibody responses in the intestines, but no detectable response in vaginal secretions (hyland et al., ) . lack of success in developing protective killed prrsv vaccines may reflect an inability to present killed prrsv antigens effectively to the pig immune system, or (like live prrsv) killed virus may induce immunosuppressive effects. in addition, the antigenic mass used in killed vaccines may be insufficient, suggesting the need for potent adjuvants or novel delivery systems. attempts have been made to improve mlv-prrsv vaccines using adjuvants. in one study, pigs were injected with recombinant porcine il- , il- , il- , or ct within week of intramuscular administration of mlv-prrsv. il- and ct enhanced ifnγ and gp antibody responses, respectively (foss et al., ) . however, use of these adjuvants did not reduce the severity of viremia. unfortunately, prrsv (na) responses were not assayed, and protection against heterologous challenge was not assessed in this study. prrsv gains entry through respiratory and reproductive mucosal surfaces and causes disease primarily at mucosal sites. therefore, a mucosal vaccine against prrsv may be an effective strategy for controlling the disease. until recently, attempts to elicit protective mucosal immunity against prrsv have been unsuccessful, probably due to a lack of appropriate adjuvants. because prrsv infection rapidly subverts the host immune responses, an effective adjuvant must overcome immunosuppression caused by vaccine virus and simultaneously potentiate virus-specific adaptive immunity. a panel of bacterial preparations derived from mycobacterium, vibrio, and streptococcus species, which were potent adjuvants in rodent models, were tested in with mlv-prrsv (bonavida et al., ; barral and brenner, ) . based on mucosal and systemic immune responses, three of the preparations, mycobacterium tuberculosis whole cell lysate (m. tb wcl), ct b subunit, and ok- (a product of streptococcus pyogenes), were selected for viral challenge trials. only m. tb wcl significantly dampened prrsv immunosuppressive effects, and enhanced virus-specific adaptive responses (renukaradhya et al., ; dwivedi et al., b) . historically, heat-killed mycobacteria are recognized to have as potent adjuvant effects as components of freund's complete adjuvant (fca) and have been used extensively in experimental animals. the use of fca in humans and food animals is unacceptable due to severe granulomatous inflammatory reactions to toxic cell wall components ( bekierkunst, ). however, a nontoxic water-soluble purified fraction of m. tb wcl (werner et al., ) has adjuvant properties in rodents, guinea pigs, and rabbits. pigs inoculated in with m. tb wcl have no detectable signs of toxicity locally or systemically (dwivedi et al., a,b) . a recombinant vaccine (prrsv gp and m expressed in bacillus calmette-guerin (bcg)) is reported to reduce clinical signs of prrs with decreased viremia and viral load in the tissues (bastos et al., ) . cross-protective immunity against prrsv has been evaluated in pigs receiving mlv-prrsv and m. tb wcl in, by challenging with virulent heterologous prrsv virus (kim et al., ) . reduced clinical disease, viremia, and virus-mediated immunosuppression were noted. in addition, secretion of ifnγ and il- by lung and blood lymphocytes in response to prrsv m and nucleocapsid proteins was enhanced (dwivedi et al., a) . guillonneau et al. ( ) reported similar findings in mice vaccinated in with adjuvanted influenza vaccines. enhanced virus-specific cytotoxic t cell and memory responses to internal viral proteins were noted, as well as cross-protective immunity. studies of passive protection of pigs by prrsv na have indicated that modest na titers (≤ / ) protect pregnant sows against reproductive failure, block placental transmission of infection, prevent viremia in piglets, and provide sterilizing immunity (osorio et al., ) . pigs immunized in with mlv-prrsv alone or with m. tb wcl developed na titers < / and / , respectively, postimmunization (dwivedi et al., b) . in pigs immunized (mlv-prrsv + m. tb wcl) and challenged with prrsv mn , an na titer > / persisted until pid (dwivedi et al., a) . relatively low numbers of circulating virus-specific ifnγ secreting cells have been reported for pigs vaccinated in with mlv-prrsv without adjuvant, ( - cells per million pbmcs) (dwivedi et al., a) . in contrast, meier et al. ( ) have reported - ifnγ secreting cells per million pbmcs in pigs vaccinated against aujeszky's disease virus. pigs immunized in with mlv-prrsv with m. tb wcl and challenged with prrsv had greater than ifnγ secreting cells per million pbmcs, and more than a twofold higher frequency of cd + cd + t cells (dwivedi et al., a) . the literature concerning mucosal immune responses in the pig respiratory tract is limited compared to studies of rodents and humans. recent research highlights the advantages of activating the mucosal immune system using vaccines delivered with potent adjuvants. induction of adequate immune responses in the respiratory and reproductive tract will be essential for control of prrs in swine. in-delivered, potent mucosal vaccines generate better cross-protective immunity against genetically variable prrsv field viruses. efforts to control prrs outbreaks using conventional parenterally delivered vaccines have had limited success; development of an alternative approach of generating immunity in the respiratory tract should be a priority. in particular, mucosal adjuvants based on components of mycobacterial species show promise. respiratory tract disease affects virtually every aspect of equine husbandry, including working, pleasure, and race horses. considerable efforts are expended to prevent epizootics of respiratory disease in stables, fairs, shows, and race tracks. equine influenza virus will be discussed in detail as an example of past, current, and future vaccine approaches. equine influenza virus causes epizootics of upper and lower respiratory tract disease almost worldwide. until equine influenza was excluded from the continent of australia through import restrictions and quarantine. in august of that year, importation of an infected horse led to an explosive epizootic of equine influenza with an estimated , horses infected over a -month period (callinan, ) , demonstrating the potential of the virus to spread in a naïve, unvaccinated population. infection can occur in horses of all ages, but epizootics often involve younger animals (van maanen and cullinane, ) . clinical signs include high fever, a persistent dry cough, nasal discharge, anorexia, and depression. secondary bacterial pneumonia may complicate the clinical picture. equine influenza viruses are classified as type a influenza. antigenic differences in the hemagglutinin (h) and neuraminidase (n) glycoproteins define the two recognized subtypes, a/equine/ (h n ) and a/equine/ (h n ) (wilson, ) . two lineages of a/equine/ , american and european, have been identified; multiple virus strains are included in vaccines since protection against heterologous strains is incomplete (yates and mumford, ) . antigenic drift is sufficient to require regular reappraisal of strains included in vaccines (mumford and wood, ) . natural infection induces iga antibodies in nasal secretions, igga and iggb antibodies in serum nelson et al., ) , and circulating cytotoxic t lymphocytes . protection against reinfection persists for at least a year (hannant et al., ) . vaccination with inactivated virus vaccines induces serum igg (t) antibodies without detectable iga in nasal secretions (nelson et al., ) and without cytotoxic t cell activity (van maanen and cullinane, ) . two or three doses of vaccine are typically administered in the primary series, with booster doses at least once a year thereafter. more frequent vaccination is advised for horses at high risk of infection (wilson, ) . protection is typically incomplete and of limited duration. improved adjuvants can enhance the level and duration of antibody responses to inactivated virus vaccines (mumford et al., c) . suppressive effects of maternal antibodies on responses to inactivated vaccines have led to recommendations not to vaccinate foals before months of age (van oirschot et al., ) . a subunit equine influenza vaccine based on the immune stimulating complex (iscom) adjuvant technology has been licensed and marketed in europe since (newmark, ) . antibody responses to iscom-based vaccines typically are of higher titer and are more persistent than for conventional inactivated vaccines (mumford et al., a) . protection has been demonstrated against experimental challenge, months after a three dose vaccination series (mumford et al., b) . protection may be due in part to the ability of iscom-adjuvanted vaccines to induce cytotoxic t lymphocytes (morein et al., ) . although iscom-based vaccines can induce iga antibody responses following in administration (hu et al., ) , the commercial influenza iscom vaccine is administered parenterally. in administration of inactivated equine influenza virus with ct b has been reported to induce local iga antibodies, and protection against experimental challenge (hannant et al., ) . a cold-adapted, temperature-sensitive live vaccine for in administration is available commercially . protection against experimental challenge has been demonstrated months after a single vaccination . this is a notable improvement in efficacy and practicality over conventional killed vaccines. experimental plasmid vaccines encoding the hemagglutinin gene of equine influenza have been examined in horses. three doses of plasmid administered to the skin and mucosal sites (tongue, conjunctiva, and third eyelid) induced protection against clinical disease and partial protection against viral shedding. protection against clinical disease was reduced if plasmid was administered only to the skin (lunn et al., ) . a canarypox-vectored equine influenza vaccine, expressing hemagglutinins from two strains of h n equine influenza, has been available commercially (intramuscular administration) since (toulemonde et al., ) . in the australian epizootic of , this vaccine was used extensively during the government program to eradicate equine influenza. the vectored vaccine was chosen for this program because of its diva (distinguishing infected from vaccinated animals) characteristics ( kirkland and delbridge, ) . infected horses mount antibody responses to the viral nucleoprotein in addition to the viral hemagglutinin, whereas vaccinated horses respond only to the hemagglutinins expressed by the vaccine vector. a recent report indicates that adequate antibody responses are generated with as little as days between primary and secondary vaccination (el-hage et al., ) . protection against experimental challenge has also been noted weeks after a single dose of vaccine in ponies ( toulemonde et al., ) . for some respiratory pathogens (e.g., m. hyopneumoniae in pigs) the critical antigens associated with protective immune responses have not been identified. for other pathogens (e.g., prrsv) there is also a need to identify the appropriate type of immune response (th or th ) needed for protection. for some complex disease conditions (e.g., bovine respiratory disease complex) there is continuing uncertainty whether all of the relevant contributing pathogens have been identified. although many parenteral vaccines are efficacious in reducing lower respiratory tract disease, there is a need to investigate whether induction of mucosal immunity in the upper airways, in combination with systemic immunity, can further reduce infection rates, transmission of pathogens, and economic losses. finally, there is a need to devise and implement changes in management procedures to reduce disease exposure (by nonimmunological methods), and to optimize immune interventions by improved timing of vaccinations. vaccines to prevent reproductive tract disease have received much emphasis in veterinary medicine. this is especially true of food-producing animals because reproductive failure is a major economic problem. although vaccines to prevent reproduction are of interest for abandoned pets or deer in areas of overpopulation, this section will deal only with vaccines designed to prevent infectious disease of the reproductive mucosa. infections causing adverse pregnancy outcome can be classified by route of infection: hematogenous or ascending. several hematogenous infections have a predilection for the gravid uterus resulting in early to late abortions . these include leptospirosis, chlamydial infection, and brucellosis in several animal species, histophilus somni infection in cattle and sheep and neospora caninum in cattle. although vaccines are available for several of these infections, the vaccine for brucellosis has been available since the s and is the most well known. several brucella species cause abortion or epididymitis/orchitis in the primary hosts ( brucella abortus in cattle, brucella suis in swine, brucella melitensis in goats, brucella ovis in sheep, brucella canis in dogs, and brucella marinum (or brucella delphini) in marine mammals). infection may be acquired via the gut mucosa or the conjunctiva/upper respiratory mucosa and localizes in the reticuloendothelial system and endometrium/placenta by systemic spread. thus, systemic vaccines are effective. a modified live b. abortus vaccine, along with a "test and slaughter" eradication program, has been successful in controlling bovine brucellosis in north america. the modified live vaccine (b. abortus strain ) is very effective in stimulating cmi that is critical for protection against this facultative intracellular pathogen. strain now has been largely replaced by a new attenuated strain, rb , which does not stimulate an antibody response known to interfere with diagnostic serologic assays. there is considerable information on mechanisms of systemic immunity to brucellosis. neospora caninum also causes abortion by a hematogenous route and a vaccine is available (weston et al., ) . histophilus somni can infect either by the hematogenous route or by an ascending genital route to cause abortion or infertility and vaccines are available. since the focus of this volume is mucosal immunity, no more will be said concerning protection against hematogenous infections of the genital tract. ascending local infections of the reproductive tract are usually transmitted sexually. the two best examples of vaccines for sexually transmitted infections of animals are campylobacter fetus subsp. venerealis (formerly vibrio fetus subsp. venerealis) and tritrichomonas foetus. both are host-specific bovine sexually transmitted diseases (stds) that only infect the reproductive mucosa. both are extracellular pathogens that do not invade the mucosa of the reproductive tract but may be found in the placenta and fetus. the localized nature of these infections and transmission limited to coitus suggest that mucosal immunity must be important. because a vaccine has been available for c. fetus subsp. venerealis for several decades and its use has controlled the disease in developed countries, that vaccine will be discussed first. vibriosis (or campylobacteriosis) is a chronic bacterial genital infection with no overt clinical signs other than reproductive failure (corbeil et al., ) . after months of infection, the uterus is cleared first, followed by the vagina. convalescent immunity is partially protective for a limited time. antibody is effective in protection against this extracellular pathogen as demonstrated by systemic passive immunization (berg et al., ) . the antibody response to infection is primarily iga in the vagina and igg in the uterus (corbeil et al., ) . systemic immunization with a whole cell vaccine results in both igg and igg antibody responses to surface antigen in serum, and uterine and vaginal secretions (corbeil et al., ) . this response prevents infection and can rapidly clear infected cows (corbeil and winter, ) . that is, the vaccine can be used prophylactically and even therapeutically. immunization is efficacious even though surface antigenic variation occurs in the face of a local immune response (corbeil and winter, ) . presumably, immune clearance occurs when the dynamic interaction between protection and evasion is shifted in the favor of the host. this appears to occur earlier when the response is primarily igg than when iga predominates (corbeil et al., ) . this may be related to the ability of the igg antibodies to mediate opsonization and intracellular killing of the bacterium, an ability which iga antibodies lack (corbeil and winter, ) . although this work was done many years ago, it sets a precedent for systemic immunization for prophylaxis and therapy of reproductive mucosal infections. trichomoniasis is a similar chronic genital mucosal infection of cattle. it is caused by the protozoan, t. foetus, and results in pregnancy loss. trichomonas vaginalis causes a human std also associated with adverse pregnancy outcome. thus, bovine trichomoniasis serves as a model for immune prevention of both bovine and human reproductive mucosal infections. because of the economic significance of bovine trichomoniasis and because no chemotherapy is approved, investigations have focused on immunoprophylaxis and immunotherapy. t. foetus colonizes the vaginal and uterine or preputial surfaces for months, like c. fetus subsp. venerealis infection. in fact, mature bulls are often infected for life whereas young bulls may clear the infection with time (cobo et al., ) . this is probably related to innate immunity. trichomonads are anaerobic parasites and are found deep in uterine glands and epithelial crypts of the penis and prepuce (rhyan et al., ) where the oxygen tension is probably lowest. older bulls have deeper epithelial crypts, which may have lower oxygen tension. in order to understand protective acquired immune responses, monoclonal antibodies (mabs) with putative protective functions were chosen for immunoaffinity purification of a highly glycosylated surface antigen (bondurant et al., ; corbeil et al., ) . analysis of many isolates of t. foetus indicated that two mabs recognized different epitopes of the same antigen, which was conserved in all isolates tested. this glycosylated surface antigen was later shown to be a lipophosphoglycan (lpg)/protein complex. systemic immunization with the immunoaffinity-purified lpg-containing surface antigen, followed by intravaginal challenge with t. foetus, resulted in significantly earlier clearance of the parasite from vaccinated animals than from controls (bondurant et al., ; corbeil et al., ) . more importantly, clearance of immunized animals most often occurred before weeks of infection. parsonson et al. ( ) showed that significant inflammation accompanied by reproductive failure did not occur until after weeks of infection, so the vaccine should protect against fetal loss. analysis of vaccine-induced antibody responses demonstrated predominantly igg responses in the serum and iga plus igg antibodies in secretions (corbeil et al., , . ige antibodies were also increased during infection. as ige antibodies increased, mast cells degranulated and clearance of t. foetus occurred. these studies raised several questions. first, why is the systemic response to the lpg/protein complex skewed toward igg (a th response in cattle) and not igg (a th response)? this question is under investigation. second, are igg or iga antibodies to the lpg-containing antigen most protective and how can that ig isotype be enriched to enhance protection? to address the latter questions, preliminary studies were done in mice to determine the best routes and adjuvants to enrich for igg or iga in genital secretions . subcutaneous priming with the immunoaffinity-purified lpg-containing surface antigen (called tf . antigen) in quil a adjuvant and subcutaneous boosting with whole cells enriched for igg anti-tf . antibodies in genital secretions whereas subcutaneous priming and intravaginal boosting greatly enriched for iga antibodies in genital secretions. when cattle immunized by these two methods were challenged intravaginally with t. foetus, those with predominantly iga or predominantly igg anti-tf . antibodies in genital secretions were equally protected. later studies with similar in immunizations showed that stimulation of the common mucosal immune system gave results similar to those of intravaginal immunization . this raised the question of inductive sites for local immune responses in the genital tract. others have suggested that the genital tract is not an inductive site because m cells and mucosa-associated lymphoid tissue (malt) are not present. this is true of cattle as well as mice and people. however, even though control cows did not have histologically demonstrable malt in the uterus and vagina, cows experimentally infected with t. foetus did . similar lymphoid nodules and follicles under a modified epithelium were detected in preputial and penial surfaces of bulls infected with t. foetus (rhyan et al., ) . immunostaining of parallel sections with mab to tf . antigen indicated uptake of antigen by epithelial cells and large mΦ or dc under the basement membrane near the lymphoid follicles (rhyan et al., ) . similar antigen uptake has been detected in the infected female uterine and vaginal mucosa (corbeil et al., ) . thus, even though the parasite is noninvasive, released tf . antigen appears to be taken up by epithelial cells. rat uterine epithelial cells can present antigen to t helper cells (wira and rossol, ) . also, mΦ/ dc positive for antigen should be antigen-presenting cells (apcs). detection of igg and iga antibodies in genital secretions of infected bulls (cobo et al., ; rhyan et al., ) and cows and the histologic demonstration of follicles and putative apcs suggests that inductive sites in the genital tract are formed in response to antigenic stimulation. like c. fetus subsp. venerealis, t. foetus has mechanisms for evasion of immune responses. these include coating of the surface with ig nonspecifically , epitope variation (ikeda et al., ) , and cleavage of igg , igg , and complement component by extracellular cysteine proteinase (talbot et al., ; kania et al., ) . studies with cysteine proteinase inhibitors demonstrated decreased cytotoxicity of t. foetus for bovine trophoblast cells and decreased infectivity in a mouse model, confirming the likely role of cysteine proteinases in pathogenesis (cobo et al., ) . even with these mechanisms for immune evasion, as with c. fetus, it is clear that the dynamic interaction between host and parasite can be made to favor the host by systemic or local immunization. the usefulness of whole cell vaccines in preventing t. foetus infection and reproductive failure in cows has been demonstrated in clinical trials (kvasnicka et al., ) . first-generation whole cell vaccines are now commercially available for prevention of trichomoniasis in cows. earlier, clark et al. ( ) demonstrated efficacious immunization of bulls with whole t. foetus cells or crude membrane glycoproteins that probably contained tf . antigen. this study indicated that vaccination of bulls could both prevent infection and clear already established infection. so vaccines for bovine trichomoniasis are both immunoprophylactic and immunotherapeutic. recent studies showed that vaccination of bulls with a commercially available whole cell killed t. foetus vaccine in oil adjuvant resulted in protection against challenge and high levels of igg antibodies in serum and preputial secretions (cobo et al., ) . igg antibodies to whole cell t. foetus antigens predominated but fairly high levels of igg antibodies were also detected. the above studies with c. fetus subsp. venerealis and t. foetus show that: . stds can be prevented or even cured by systemic vaccination of both males and females. . at least for one std, igg and iga of the same antigenic specificity are equally protective at the mucosal surface. either systemic immunization or mucosal (in or intravaginal) immunization with systemic boosting is protective. . inductive sites are formed in the mucosa of infected male and female genital tracts even with noninvasive pathogens. . strong and appropriate immune responses will clear microbial infection from the genital tract even when the microbe has multiple immune evasive mechanisms. . the fact that protection against two stds has been demonstrated in the natural outbred host (cattle) has advantages over murine models of std vaccines. in the latter, the human pathogen is usually inoculated into the unnatural murine host and the disease does not mimic the human infection. furthermore, although inbred mice provide a homogeneous experimental animal, they do not represent the variation in immune responses seen in the outbred human population. the work on bovine vibriosis and trichomoniasis demonstrates protection under field conditions for two stds that cause adverse pregnancy outcome in an outbred host. this is an encouraging precedent for control of human stds and related adverse pregnancy outcomes. future needs include identification of the protective antigens for most stds. for antibody-mediated protection of the genital mucosa, several questions have not yet been addressed. as far as we know, the role of ige in the genital tract is largely unstudied even though it does seem to be involved in clearance of trichomonads from the bovine genital tract. lastly, manipulating genital immune responses to enhance th or th type responses is an unexplored research area. enteric disease is a major cause of mortality and morbidity in animals. agents causing diarrhea in animals include viruses (e.g., adenoviruses, pestiviruses, caliciviruses, coronaviruses, parvoviruses, rotaviruses, toroviruses), bacteria (e.g., campylobacter spp., clostridium spp., diarrheagenic escherichia coli, salmonella spp., yersinia spp.), and parasites (e.g., coccidia spp., cryptosporidium parvum). these infections occur most commonly in suckling animals or in poultry under weeks of age, but may also be common postweaning and in susceptible seronegative or stressed adult animals (saif and jackwood, ) . induction of local secretory iga (s-iga) antibodies (prevent attachment, invasion, and neutralize toxins or infectious agent) and mucosal cellular immune responses (against intracellular bacteria and viruses) by vaccines are essential for protection from enteric pathogens. peyer's patches (pp) and mesenteric lymph nodes are two important organized gut-associated lymphoid tissues (galt) in domestic animals and serve as the induction site for gut immune responses. ileal pp in some domestic animals (sheep, cattle, pigs, dogs) differ from jejunal pp, serving as a primary lymphoid organ similar to the bursa of fabricius in chickens, unlike in humans and rodents where pp are secondary lymphoid organs (chu and liu, ; yasuda et al., ) . cryptopatches or clusters of lymphoid cells in the basal lamina propria occur in mice, but are absent in pigs (pabst et al., ) . mucosal lymphoid tissues and lymphoid cells in domestic animals and humans differ in toll-like receptor (tlr) expression and function (after binding their ligand) when compared to mice (iwasaki and medzhitov, ; tohno et al., ; guzylack-piriou et al., ) . these differences in immune components suggest that vaccine studies carried out in mice may fail to translate to domestic animals or humans. enteric pathogens have different characteristics related to their intestinal tropism and replication, requiring different vaccination strategies. enteric viruses have predilections for replication in distinct vertical and longitudinal regions of the small or large intestines. cytolytic infection of enterocytes leads to varying degrees of villous loss and fusion, reduced small intestinal absorptive capacity, and a malabsorptive, maldigestive diarrhea (saif, a) . secretory diarrhea is also induced by some viruses such as rotavirus (rv), which involves the viral enterotoxin, nonstructural protein (nsp ), and/or stimulation of the enteric nervous system (ball et al., ; lundgren et al., ) . thus, viral diarrheas can be of variable severity and act via multiple mechanisms that differ from those of enteric bacteria, most of which cause secretory diarrhea mediated by enterotoxins (fairbrother and gyles, ) . enteropathogenic viruses can be divided into three types (types i, ii, and iii) according to their preferred site of replication in the intestine (reviewed by saif ( a) ). type i (transmissible gastroenteritis virus (tgev) and rv) and ii virus infections can be prevented by inducing local intestinal immunity, whereas type iii viruses (parvovirus), which infect crypt enterocytes basolaterally, can be prevented by inducing systemic immunity. the enteropathogenicity of bacteria is determined by their virulence factors including adhesion factors (fimbriae or pili) and enterotoxins; therefore, bacterial vaccines generally need to prevent attachment and toxin action within the intestine (fairbrother and gyles, ) . in the following sections, we will review vaccine strategies for these different types of enteric infections. tgev and rv vaccines in pigs will be reviewed to illustrate findings using domestic outbred animals instead of inbred laboratory rodent models. neonates can be protected from enteric infections by providing passive lactogenic immunity, which can be achieved by immunizing mothers preparturition. pioneering work done by bohl and saif in the early s with live oral virulent tgev in pigs was the foundation for the gut-mammary gland-s-iga immunologic axis, which later became the basis for the concept of the common mucosal immune system. their studies showed that in tgev seronegative sows, only oral immunization with virulent tgev induced high rates of protection in suckling neonates, which was associated with high titers of s-iga antibodies in colostrum and milk, whereas systemic immunization induced mainly igg antibodies saif et al., ) . rotavirus is a major cause of dehydrating diarrhea in young livestock, infants, and poultry (saif and fernandez, ) . multiple rv serogroups, based on common inner capsid vp antigens (a-h), and multiple g (vp , glycoprotein) and p (vp , protease-sensitive) serotypes (based on neutralizing epitopes) or genotypes (based on sequence analysis) for group a rvs have been detected in humans, sheep, swine, cattle, horses, dogs, cats, poultry, and wildlife (estes and kapikian, ; martella et al., ) . among the distinct rv serogroups and g/p serotypes/genotypes, cross-protection is limited. the antigenic divergence among different sero/genotypes of rvs presents a challenge for the design of vaccines that are capable of inducing heterotypic protection. commercial modified live and killed rv vaccines for rv diarrhea in livestock and poultry are limited to group a rvs (saif and fernandez, ) . mebus et al. ( ) developed the first oral rv vaccine for calves in ( year prior to the discovery of human rv) using a cell cultureadapted neonatal calf diarrhea rv strain. although a significant reduction in morbidity and mortality was observed in a field trial among vaccinated calves, in the majority of herds (compared to previous years), subsequent field studies revealed variable efficacy. experimental studies suggested that maternal antibodies interfered with live oral vaccine replication and suppressed development of active immunity (saif and fernandez, ) . the neonatal gnotobiotic pig model has been used to investigate immune responses to rv vaccines and infection for nearly three decades (saif et al., , yuan and saif, ; gonzalez et al., ) . gnotobiotic pigs are free of maternal antibodies (placental transfer of igs does not occur in swine), but they are immunocompetent at birth. they are maintained aseptically and are free of exposure to extraneous wildtype rvs, assuring that exposure to a single pathogen can be analyzed. initial studies were conducted to mimic natural rv infection (bohl et al., ) and to study immune correlates of protection gonzalez et al., ; yuan et al., ) . understanding the sequence and kinetics of immune responses stimulated by virulent rvs allows for the determination of markers of protective immunity and pathogenicity, which can then be used to design vaccines that will stimulate protective immunity without inducing pathology. gnotobiotic pigs orally inoculated with virulent porcine or human rvs were completely protected from homotypic but not heterotypic (distinct g/p types) rv challenge (hoshino et al., ; saif et al., ) . significant correlations were observed between the protection to rv-induced diarrhea and shedding and the following immune parameters: the number of intestinal iga rv antibody-secreting cells (ascs), serum and intestinal iga rv antibody titers, and the frequency of intestinal rv-specific ifnγ producing cd + and cd + t cells yuan et al., ; to et al., ; yuan and saif, ; yuan et al., ) . these immune parameters were significantly higher in virulent rv inoculated pigs than in those inoculated with attenuated or inactivated rv (ward et al., b) . pigs inoculated with two or three doses of attenuated rv were moderately protected against diarrhea ( %- doses, %- doses) and virus shedding ( %- doses and %- doses) after homotypic challenge, suggesting a need for multiple doses and suitable mucosal adjuvants to enhance the efficacy of rv vaccines yuan et al., yuan et al., , yuan and saif, ) . gnotobiotic pigs inoculated with virulent rv developed an acute proinflammatory serum cytokine profile (il- , tnfα) coinciding with peak diarrhea and viremia, followed immediately by increased th (il- , ifnγ) cytokines and convalescent th (il ) and tr (il- ) cytokine responses. gnotobiotic pigs inoculated with one dose of attenuated rv showed lower early ifnγ and proinflammatory cytokine responses compared to virulent rv inoculated pigs. both attenuated and virulent rv inoculated pigs developed th /th /tr cytokine-secreting cell (csc) responses at - weeks postinoculation; however, attenuated rv inoculated pigs developed lower intestinal ifnγ and higher intestinal and splenic il- cscs compared to virulent hrv inoculated pigs (azevedo et al., ) . virulent rv inoculated pigs had significantly higher protection rates against rv challenge ( % and % against diarrhea and shedding, respectively) compared to one dose of attenuated rv inoculated pigs ( % and % against diarrhea and shedding, respectively) (azevedo et al., ) . these findings suggest that higher protection rates are associated with early proinflammatory and th cytokine responses, which promote cytotoxic t cell activity and viral clearance, and later th induced cytokines, which are important for protective s-iga antibody responses. thus, protection against rv in pigs requires balanced th /th /treg responses (azevedo et al., ; gonzalez et al., ) . infection of gnotobiotic pigs with virulent rv causes early recruitment of innate apcs (monocytes/mΦ and dcs) and γδ t cells into the ileum, and enhanced tlr , tlr , and tlr expression among apcs in spleen (zhang et al., c; wen et al., wen et al., , . in virulent rv-infected pigs, plasmacytoid dcs are major producers of serum ifnα and, along with other innate immune cells (γδ t cells and apcs) and cytokines (tnfα, ifnγ and il- ), are important for controlling early rv viral replication and subsequent development of protective adaptive immune responses . development of attenuated rv vaccines with mucosal adjuvants that mimic immune responses to virulent rv may improve existing vaccines. immunogenicity and protective efficacy of rv vaccine formulations (attenuated replicating virus, inactivated virus, and recombinant baculovirus-expressed virus-like particles (vlp)), administration routes, and adjuvants have also been evaluated using the gnotobiotic pig model (bohl et al., ; yuan and saif, ; gonzalez et al., ) . inactivated oral or intramuscular rv vaccines failed to protect against virulent rv challenge, despite high igg antibody responses induced in serum and systemic lymphoid tissues. serum igg antibodies did not correlate with protection. however, a recent study showed that an inactivated reassortant rv strain (cdc- strain) formulated with aluminum phosphate and administered systemically in gnotobiotic pigs resulted in induction of serum igg antibody titers, coinciding with partial protection against shedding and diarrhea, suggesting that adjuvant may have stimulated local specific (gut iga antibodies) or nonspecific immune responses, which were not assessed in this study (wang et al., ) . rotavirus subunit vaccines consisting of double-layered vlp composed of rv inner capsid proteins vp and vp ( / -vlps) administered in or orally with mutant heat-labile toxin of e. coli (mlt) or iscoms as adjuvants (yuan and saif, ) induced igg asc responses in systemic lymphoid tissues and low or no iga asc responses in intestinal lymphoid tissues and also failed to mediate protection, contrary to results in adult mouse studies (yuan and saif, ) . the failure of in or oral / -vlp vaccines suggests that protective immunity to rv diarrhea in neonatal pigs requires mainly the presence of systemic or intestinal iga antibodies to the outer capsid rv proteins, vp and vp , each of which induces na. however, when / -vlps adjuvanted with mlt or iscom were used as in or oral booster doses in pigs orally primed with attenuated rv, the protective efficacy increased significantly. an oral attenuated rv prime and in / -vlp-iscom boost regimen (attrv/ / -vlp) induced the highest numbers of intestinal iga ascs and serum and intestinal iga antibody titers, and protection rates were similar to or higher than those induced by three oral doses of attenuated rv (gonzalez et al., azevedo et al., ) . interestingly, priming with two doses of / -vlp (in or oral) followed by live attenuated rv was ineffective for inducing iga antibodies or protection. thus the use of a replicating vaccine to prime at one inductive site (galt) followed by boosting with a nonreplicating vaccine at a second mucosal inductive site (nasal-associated lymphoid tissue, nalt) is an effective strategy for stimulating protective mucosal immune responses, which can be applied to other enteric viral vaccines. furthermore, efficacy of the prime/boost strategy (replicating/nonreplicating vaccine, attrv/ / -vlp) was examined in the presence of high and low titers of passive antibodies to mimic neonatal pigs receiving maternal antibodies (nguyen et al., a,b) . similar to attrv/ / -vlp prime/boost vaccine studies, plasmid dna containing vp was used as a booster subsequent to oral attenuated rv vaccine priming. this regimen showed high protection against shedding, but poor efficacy against rv diarrhea (yuan et al., ) . collectively these findings suggest that mucosal boosters are effective in enhancing iga antibody titers to rvs in orally primed animals . studies have shown that immunogenicity and efficacy of mucosal vaccines can be improved by the use of appropriate strains of probiotics that modulate mucosal and systemic immune responses, by interaction with epithelial cells and the underlying intestinal immune cells (fukushima et al., ; sanz and de palma, ) . certain probiotic strains are reported to enhance immune responses to rv vaccines in children; others reduce rv diarrhea severity, but the mechanisms are not well defined (fang et al., ; holscher et al., ) . supplementation with lactobacillus acidophilus in attenuated rv vaccinated pigs is reported to enhance intestinal ifnγ-producing cd + t cells, intestinal iga and igg rv ascs, and serum iga and igg rv antibody titers (zhang et al., b) . these findings suggest that probiotics are an alternative, cheap, and safe adjuvant for enhancing efficacy of oral attenuated rv vaccines in animals and potentially in humans. colonization of pigs by two different strains of lactic acid bacteria (lab, l. acidophilus and lactobacillus reuteri) and subsequent virulent rv infection resulted in higher and balanced th /th /treg cytokine responses (il- , ifnγ, il- , il- , tgfβ), higher total intestinal iga-secreting cells, total serum igm, and intestinal igm and igg titers, although the numbers of iga rv ascs and serum and intestinal rv antibody titers did not differ compared to virulent rv-only infected pigs. no overall difference in protection rates was noted when compared to virulent rv-only inoculated pigs, which was likely because of the short interval (only days) between lab colonization and virulent rv challenge (zhang et al., a; azevedo et al., ) . dual colonization of the aforementioned lab strains also modulated innate immune components in virulent rv inoculated pigs as follows: ( ) enhanced frequency of γδ t cells in the intestine and their distribution; ( ) enhanced tlr and tlr -expressing cd + apc, and tlr -expressing cd − apcs in the blood, but reduced tlr -and tlr expressing cd − apcs in the spleen, and ( ) reduced frequency of mΦ and cdcs in the spleen. collectively, these findings suggest that probiotics may reduce systemic inflammatory responses induced by virulent rv. effects on tlr expression on apcs in the ileum were not determined (zhang et al., c; wen et al., wen et al., , . probiotics may not only modulate immune responses to rvs or other enteric vaccines, but may also directly ameliorate diarrhea/infection by enhancing gut barrier integrity and maintaining intestinal permeability, by stimulating nonspecific immune responses, by changing gut microbial populations, and by aiding in the regulation and prevention of apoptosis ( madsen et al., ; yan and polk, ; preidis et al., ) . using the tgev model for evaluation of active protection against diarrhea in pigs, researchers also delineated compartmentalization in the common mucosal immune system and its impact on mucosal vaccine strategies and protection (vancott et al., (vancott et al., , . the natural occurrence of a deletion mutant of tgev with exclusive respiratory tropism, referred to as porcine respiratory coronavirus (prcv), provided a unique opportunity to study asc responses and protective immunity to two antigenically related porcine coronaviruses with enteric (tgev) versus respiratory (prcv) tropism. oral immunization of pigs with tgev induced high numbers of iga asc in the intestine and provided complete protection against tgev challenge, whereas in immunization of pigs with prcv induced mainly systemic responses (igg asc) and provided only partial protection against tgev challenge. thus, the in prcv alone failed to elicit sufficient intestinal iga asc to provide full protection against the enteric pathogen, tgev. findings from this study and rv vaccine studies suggest that the use of multiple mucosal inductive sites in a prime/boost vaccination regimen may be an effective approach for overcoming compartmentalization in the common mucosal immune system. canine parvovirus (cpv) infects crypt enterocytes causing hemorrhagic gastroenteritis in pups (saif and jackwood, ) . because cpv is likely disseminated to the basolateral surface of crypts by the hematogenous route, serum na (derived maternally or actively produced) is protective against the disease. pollock and carmichael ( ) demonstrated that pups with hemagglutination inhibition (hi) serum antibody titers of > : were immune to oronasal cpv type challenge. cpv is highly stable in the environment and pups were susceptible to infection as soon as maternal antibodies declined to hi titers of : - : . a maternal hi antibody titer as low as : severely affected the efficacy of a live low titer cpv vaccine ( - th passage in culture) in generating active immune responses (carmichael et al., ) , which resulted in a window of susceptibility for pups. others have shown that an hi titer of less than : in cpv- (low passage, high titer) vaccinated pups did not severely affect active antibody responses (burtonboy et al., ; hoare et al., ) , suggesting that increasing the dose or reducing the attenuation of the virus may help to overcome inhibitory effects of maternal antibodies. studies with modified live variant cpv- b strain (low titer) have shown that these vaccines, when given either parenterally ( th passage) or in ( th tissue culture passage), elicited almost % protection in pups with maternal antibody titers of : to : and even % protection in pups with antibody titers of : . higher efficacy of these vaccines can be attributed to either strong inherent immunogenicity of these new vaccines or antigenic differences among cpv- and cpv- a and cpv- b (pratelli et al., ) . overall, during the past four decades of cpv vaccine development, modified live viruses have proven superior to inactivated intramuscular (im) vaccines (appel, ) . in brief, various strategies such as the use of less attenuated virus (low serial passage), high titer vaccines, multiple doses, or the use of the in route of immunization have been attempted to overcome inhibitory influences of maternal antibodies and to reduce the window of susceptibility in pups. recently new variant cpv- c has emerged, which is highly pathogenic and causes more severe diarrhea in dogs. currently used vaccines (cpv- or cpv- b strains) are effective in protecting dogs against challenge with cpv- c virus under experimental conditions (spibey et al., ) , although efficacy in the field is unknown (reviewed by decaro and buonavoglia ( ) ). antigenic differences between original cpv- and its variants may reduce the efficacy of current cpv vaccines, which is supported by in vitro virus-neutralization tests conducted on vaccinated animals that showed low cross-reactivity between heterologous cpv variants (cavalli et al., ) . however, this may not represent actual cross-protection in clinical cases. there is a need not only to make current vaccines effective in the presence of maternal antibodies, but also to update them based on continuous epidemiological surveillance studies. dna plasmids expressing vp (jiang et al., ) , replicon-based cpv dna vaccine expressing vp (dahiya et al., ) , b cell epitope ( l peptide of vp ) fused to ct b subunits expressed in transgenic tobacco chloroplasts (molina et al., ) , chimeric virus particles expressing cpv peptide (different prime/boost strategies) (nicholas et al., ) , and recombinant vlps formed by baculovirusexpressed vp (casal, ) have been evaluated in dogs or rodents without maternal antibodies and have demonstrated good immunogenicity and/or protective efficacy. further efficacy tests in pups with maternal antibodies are needed to assess their commercial potential. oral vaccines for the induction of active immunity against bacterial diarrhea are not commonly used in livestock, although e. coli diarrhea is an important problem in neonatal and postweaning calves and pigs. f (k ) and f are the major fimbrial adhesins present on swine enterotoxigenic e. coli (etec) and are major targets for e. coli vaccines (fairbrother et al., ) . whole bacteria vaccines are routinely administered parenterally to pregnant cattle, sheep, and swine to protect their suckling neonates against etec infections (moon and bunn, ) . such vaccines are practical and effective because: ( ) fimbriae are required for the adhesion-colonization of bacteria early in the pathogenesis of the disease; ( ) most fatal etec infections in farm animals occur in the neonatal period; ( ) more than % of the etec in farm animals belong to a small family of fimbrial antigen types, ( ) and mothers are seropositive to etec so booster responses are elicited. recent vaccine studies have focused on administration of purified bacterial subunits (transgenically expressed adhesin of f fimbriae in plants) and mucosal adjuvants (verdonck et al., ; joensuu et al., ) and have shown promising results. overall, the vaccine strategy used, parenteral vaccination of field-exposed seropositive mothers, to induce lactogenic immunity is the same as that shown to be effective for parenteral application of rv vaccines in rv seropositive mothers (bohl et al., ; saif and jackwood, ; saif and fernandez, ) . studies of live oral enteric vaccines in animals have clarified the mechanisms of induction of protective immunity against enteric disease and contributed to our understanding of the common mucosal immune system. however, commercial live oral vaccines often have shown inadequate or inconsistent efficacy under field conditions (saif and jackwood, ; saif and fernandez, ) . major obstacles to improved efficacy of oral vaccines include: ( ) maternal antibodies in the intestine of neonates (mainly colostrum and milk antibodies), which interfere with live vaccine replication; ( ) qualitative and quantitative limitations in the neonatal immune system, although neonates are immunocompetent; ( ) the inability of attenuated vaccine strains to adequately infect (too high attenuation) or stimulate s-iga antibodies in the intestine; ( ) the use of inappropriate (or unstable) antigens or route for subunit vaccines; ( ) the lack of oral delivery vehicles or mucosal adjuvants for subunit vaccines; and ( ) infection by pathogens prior to vaccination. studies of neonatal pigs indicate that protection rates against rv diarrhea upon challenge correlate with the magnitude of iga asc and memory b cell responses in intestinal lymphoid tissues gonzalez et al., ; yuan and saif, ) . studies conducted in immunodeficient or specific gene knockout adult mice have shown that neither cd + or cd + t cells nor antibodies were essential for induction of protective immunity to rv infection, but usually one of these effectors (t or b cells) was necessary for elimination of primary rv infection (mcneal et al., (mcneal et al., , . in pigs, recent studies have shown that cd + and cd + ifnγ producing t cells play a role in protection against rv diarrhea and infection (yuan et al., ) , but it is difficult to create genetically modified pigs similar to knockout mice, to assess the contribution of b cells and t cells. the redundant nature of immune responses to rv in mice, the multiple immunologic and possibly nonimmunologic pathways to resolve rv infections (franco and greenberg, ; ward, ) , the age factor and host differences in rv pathogenesis in mice and pigs (saif et al., , ward, ) , and the use of inbred mouse strains contribute to the discrepancies seen between the adult mouse and the neonatal pig models. the majority of pathogens enter and initiate infection at mucosal surfaces, making mucosal sites relevant targets for vaccines to prevent infection. to develop effective mucosal vaccines, it is important to determine correlates of protection against enteric pathogens. generally for localized gut infections, s-iga antibodies and intestinal t cells play an important role. improved vaccines that induce high levels of intestinal iga antibodies against the appropriate microbial antigens can be achieved by choosing the proper vaccine formulation and delivery method. vaccines should also induce: ( ) heterotypic protection; ( ) active immunity in the presence of maternal antibodies; and, ( ) long-lasting immunological memory. novel vaccines (e.g., vlps, transgenic plants), adjuvants (e.g., mlt, iscoms, tlr ligands (e.g., cpg), mycobacterial extracts, α -dihydroxyvitamin d , retinoic acid, probiotics, and cytokines), and vaccine delivery systems (e.g., recombinant plant or animal viruses or bacterial vectors, genetically engineered probiotics, iscoms, liposomes, and nanoparticles) should be explored and evaluated in relevant animal models to further enhance the efficacy of current or new vaccines. recent studies have shown that the targeting of antigens directly to apcs (via surface receptors) is an effective way to tailor immune responses to optimize protection and should be explored further in large animals (trumpfheller et al., ) . the passive transfer of maternal immunity provides essential protection in newborns. although most neonatal immune systems are competent to mount primary immune responses against pathogens, primary responses often do not develop quickly enough to prevent disease. maternal immunologic transfer provides a critical (though temporary) aid to survival for neonates. the enhancement of passive immunity through vaccination of the mother has been a successful disease prevention strategy in domesticated animals. vaccinated mothers develop higher levels of specific antibodies in colostrum and milk and increased levels of immunity in their offspring (glezen, ; saif and fernandez, ) . passive immunity can also be enhanced by oral administration of immune milk or heterologous antibody preparations (e.g., chicken egg yolk igy (ikemori et al., ; kuroki et al., ) or monoclonal antibodies) or by parenteral administration of hyperimmune plasma (becu et al., ) . recent studies using monoclonal, single-chain antibodies (variable heavy domain (vhh) nanoantibodies) of llama origin open new possibilities for providing passive immunity to humans and animals (garaicoechea et al., ) . immunoglobulins of the igg and igg isotypes of camelids consist of heavy chains without associated light chains (hamers-casterman et al., ) . the distinctive biochemical characteristics and binding qualities of vhh antibodies overcome some key limitations of conventional antibodies (see below). unfortunately, passive antibodies often interfere with active immunization of young animals and birds. various vaccination strategies have been developed to minimize the suppressive effects of maternal antibodies, but improved adjuvants and antigen-delivery systems are needed to facilitate efficient induction of active immunity in the presence of maternal antibodies. this section will address past, current, and future approaches for enhancing passive immunity in veterinary species. the transfer of systemic passive immunity from mother to offspring can occur prenatally, via the placenta or yolk sac, or postnasally via ingestion of colostrum and milk, depending on the species. the main ig isotype transferred in most mammalian species is igg. in avian species igy, the functional equivalent of mammalian igg, is transferred to the yolk to passively protect the developing chick (kovacs-nolan and mine, ) . in primates and rabbits, maternal igg is transferred across the placenta to the fetus. in rodents, transplacental transfer occurs in combination with prolonged ( - days) postnatal transfer by means of colostrum and milk (husband, ) . in dogs and cats transfer of igg occurs by a combination of prenatal and postnatal mechanisms, with % to % of total transfer occurring before birth (tizard, ) . in ruminants, horses, and pigs, offspring are born virtually agammaglobulinemic and transmission of ig occurs only via colostrum for a limited time after birth (tizard, ) . after the transition from production of colostrum to milk, ig are no longer absorbed from the intestines and only act locally in the gastrointestinal tract. immunoglobulin absorption in neonates of large domestic species is facilitated by the presence of protease inhibitors in colostrum and its efficiency declines rapidly after birth, with maximal absorption occurring in the first h. the cessation of absorption of intact macromolecules is termed "gut closure," and occurs at different ages in different species. in calves and pigs closure normally occurs by - h after birth. absorption of colostrum ig can be highly effective, supplying the newborn with serum ig at concentrations similar to those of the dam. failure of passive transfer (fpt) is a common problem, however, in newborn calves and foals (besser and gay, ; tyler-mcgowan et al., ) . fpt may occur because of the production of low quantities of colostrum, because of production of colostrum with low concentrations of maternal antibodies, because of ingestion of low quantities of colostrum, or because of inefficient absorption (quigley and drewry, ) . colostrum supplements, colostrum replacers, and plasma products have been developed commercially to address this problem, with variable success. vaccination of the dam in late pregnancy can enhance antibody titers in colostrum and after suckling in the serum of the offspring (saif and fernandez, ) . the benefits of vaccination of the dam for enhancing passive immunity can be lost if colostrum is of low quality or if absorption of colostral ig is inefficient. the half-life of ig varies considerably among species of domestic animals and with the ig isotype. neonatal receptor for fc of igg (fcrn) is involved in homeostasis of serum levels of igg in general, but distinct mechanisms may function in neonates. the main route of clearance of passively acquired maternal igg in calves is transfer from the serum to the intestine (besser et al., ) . approximately % of passively acquired igg is eliminated by this route. if titers of passive circulating antibodies are high enough, the transfer of antibodies from the circulation to the intestinal lumen can mediate short-term protection against rotavirus diarrhea (besser et al., ) . the same mechanism may be functional in piglets (saif and wesley, ; parreño et al., ; ward et al., a) . the persistence of titers of circulating maternal antibodies is generally considered in designing vaccination strategies for young animals because of suppressive effects of maternal antibodies on active immune responses. induction of immune memory can occur in the absence of a detectable serum antibody response (boersema et al., ; parreño et al., ) . the presence of passive antibodies in the intestines can interfere with mucosal immune responses to natural infection as well as to vaccination parreño et al., ; nguyen et al., a,b) . experiments in colostrum-deprived lambs (jones et al., ) and calves (mosier et al., ) have demonstrated the ability of parenterally administered immune antisera to mediate protection following experimental challenge with mannheimia haemolytica. parenteral administration of hyperimmune plasma raised against rhodococcus equi has been shown to protect against pneumonia in young foals in experimental (hooper-mcgrevy et al., ) and field studies (becu et al., ) . hyperimmune plasma is available commercially for use in foals. prepartum vaccination of beef (van donkersgoed et al., ) and dairy (hodgins and shewen, ) cows has been demonstrated to increase antibody titers to m. haemolytica in their colostrum and in the serum of their calves. vaccination of broiler breeder chickens can be used to provide passive protection against respiratory/septicemic disease caused by avian pathogenic e. coli (kariyawasam et al., ) . rodents have been a popular model for the study of passive protection by milk antibodies. however, rats and mice actively transport igg from the gut into the circulation during the first - weeks of life. thus, antibodies in ingested milk contribute to both local and systemic immunity in rodents, in contrast to the strictly local effects occurring in humans and most domestic animals. in pigs, horses, dogs, and cats, igg is the most abundant ig in colostrum but iga predominates in milk. parenteral vaccination, by enhancing serum igg antibody titers, contributes to igg antibodies in colostrum but has limited effects on iga antibodies in milk. milk antibodies provide passive protection to the neonatal intestinal tract by immune exclusion preventing the attachment of viruses, bacteria, and parasites and by neutralizing viruses or enterotoxins. s-iga antibodies, because of their resistance to cleavage by digestive enzymes, and higher levels in milk are more efficient in mediating protection in the gut of pigs and other monogastrics (saif and jackwood, ) , but high persisting levels of passive igg antibodies are also protective. in ruminants igg antibodies, relatively resistant to proteolytic enzymes (brock et al., ) and predominant in milk, have functions similar to those of s-iga. numerous vaccines are marketed for vaccination of cows and sows to provide lactogenic immunity to rotavirus, coronavirus, and e. coli in suckling offspring. vaccine efficacy has been variable. ideally, suckling animals become subclinically infected with enteric pathogens while receiving adequate passive antibodies to prevent disease, and develop active immunity to prevent subsequent diarrhea. this balance between passive immunity and disease can be disrupted in intensive animal production systems by exposing animals to pathogens in confined, contaminated environments. earlier weaning practices reduce intake of milk antibodies. maternal enteric vaccines are commonly used in two populations of pregnant animals. to control epidemic infections, they are used in naïve, seronegative animals to induce primary immune responses. to control endemic infections (such as rotavirus and e. coli), booster vaccines are used in seropositive, field-exposed animals to stimulate anamnestic memory responses. virulent tgev given to pregnant sows stimulates high levels of iga antibodies in milk and passive protection (saif and jackwood, ) . oral attenuated tgev vaccines, which replicate poorly in sows, induce lower iga milk antibody titers and low or variable efficacy in the field (moxley and olson, ) . parenteral killed tgev vaccines induce only low igg milk antibody titers and have the lowest protection rates. attempts to develop maternal tgev recombinant subunit vaccines based on the surface tgev spike (s) protein that induces na, or live vector vaccines expressing the s protein, have also been of limited success in tgev seronegative swine (saif and wesley, ) . however, prime/boost strategies such as im administration of tgev s protein following oral/in priming with attenuated tgev have shown promise as a means of enhancing iga milk antibody titers (park et al., ) . booster vaccination strategies are required to enhance lactogenic immunity to endemic enteric pathogens, such as rotavirus and e. coli, because antibody titers in milk decline dramatically during lactation. breast milk iga antibodies are increased in women endemically exposed to cholera following parenteral boosting with a cholera vaccine (svennerholm et al., ) . in pigs infected with rotavirus, iga memory b cells initially reside in the ileal pps but are subsequently present in substantial numbers in spleen (yuan et al., ) . systemic stimulation of such iga memory b cells by parenteral booster vaccines can yield dimeric iga antibodies in serum for transport to mucosal secretions via the polymeric ig receptor. under field conditions, antibodies to endemic intestinal pathogens are also common in bovine colostrum and milk, but without the boosting effect of highly immunogenic vaccines, antibody titers are often too low to protect calves (besser and gay, ; saif and fernandez, ) . thus vaccines are marketed for prepartum vaccination of cows against rotavirus, coronavirus, and e. coli to enhance passive immunity in their calves, but the field efficacy of these vaccines has been questioned (waltner-toews et al., ) . vaccination of pregnant dairy cows with modified live or binary ethyleneamine inactivated rotavirus or recombinant / / / vlps has been shown to increase igg and virus na titers to rotavirus in colostrum and milk and mediate passive protection in calves against oral rotavirus challenge (saif and fernandez, ; kim et al., ) . prepartum vaccination of cows and sows with bacterins prepared from enteropathogenic (epec) e. coli for prevention of diarrhea in their offspring is also commonly practiced. under modern farming practices, dairy and veal calves rarely are fed whole milk from their dams for more than or days. thus vaccine efficacy is based on antibodies absorbed from colostrum or retained temporarily in the gut, rather than on a continuing supply of immune milk. piglets, in contrast, continue to receive immune milk until weaning at - weeks of age. the importance of a continuous supply of passive antibodies for protection against tgev has been demonstrated (saif and wesley, ) . numerous commercial ig preparations with antibody activity against specific enteric pathogens have been marketed. products intended for prevention of e. coli enteritis in calves include dried bovine colostrum and whey, hyperimmune sera raised in horses, and mouse monoclonal antibodies to the k (f ) antigen of e. coli. these products are administered orally in the first h of life to prevent adhesion of epec e. coli. orally administered bovine colostral whey containing rotavirus antibodies also passively protects piglets against rotavirus (schaller et al., ) . immunization of chickens shows promise as an efficient method for producing polyclonal antibodies for passive protection. specific antibodies of the igy isotype are induced by vaccination and are concentrated in egg yolk. laying hens can produce about g of igy per year. yolk antibodies with virus na provide calves with partial protection against diarrhea caused by rotavirus (kuroki et al., ; vega et al., ) , and etec e. coli (ikemori et al., ) . in a recent study, supplementation of the milk ration of neonatal calves with egg yolk containing igy antibodies to rotavirus for days provided % protection against rotavirus diarrhea after challenge, and also enhanced mucosal asc numbers in the duodenum (vega et al., ) . egg yolk lacking rotavirus specific igy did not provide clinical protection, but did enhance asc responses, suggesting the presence of immune modulators in egg yolk. protective effects of yolk antibodies are dependent on antibody titers in oral preparations (marquardt, ) . development of better means to protect yolk antibodies from digestive processes will improve both the efficacy and the economic viability of yolk antibodies for clinical applications (kovacs-nolan and mine, ). for many diseases of newborns and neonates, passive immunity is the only practical means of providing timely protection. unfortunately, it is well documented that maternal antibodies can suppress active immune responses following vaccination. this effect has been observed with both live and nonreplicating vaccines, and for both systemic and mucosal responses (siegrist et al., ; hodgins et al., ; parreño et al., ; nguyen et al., a,b) . antibody responses especially are affected; t-lymphocyte responses may not be suppressed (siegrist et al., ) . titers of maternal antibodies are maximal for most species of interest in the first week of life and then decline gradually over the next few months, but variability of titers among individuals is high. with many vaccines, a "window of disease susceptibility" of variable duration occurs when titers of maternal antibodies are too low to mediate protection, but are too high to permit effective vaccination. a number of strategies are used to cope with this problem. some veterinary vaccines for cattle are sold with the disclaimer that "animals vaccinated before months of age should receive a booster dose of vaccine at months of age." this provides little solace for the many diseases of cattle occurring in the first weeks or months of life. a common strategy for vaccines of dogs and cats is to administer a series of doses of vaccine from an early age (at which time only a few individuals will be responsive) and to continue vaccinating until an age at which virtually all can respond to vaccination. this strategy has economic disadvantages for the pet owner. some manufacturers produce low passage, high virus titer vaccines especially for use in situations where high titers of maternal antibodies and high pathogen exposure are anticipated. this is similar to a strategy once (but no longer) approved by the world health organization for vaccination of children in developing countries against measles (gellin and katz, ) . preliminary evidence suggests that incorporation of vaccine antigens in highly structured iscoms or in application of vaccines can enhance immune responses in the presence of maternal antibodies (van binnendijk et al., ; brockmeier et al., ) . immunoglobulin g in most mammalian species is composed of two heavy chains, covalently linked by disulfide bonds, and two light chains. "conventional" heavy chains consist of one variable domain and three constant domains (ch , ch , and ch ); light chains are composed of a variable and constant domain. in contrast camelid species produce "heavy chain immunoglobulins" that lack light chains and the first constant heavy domain (hamers-casterman et al., ) . the antigen-binding site of these unusual heavy chain antibodies is formed by a single domain, designated vhh in camelids. vhh are easily produced as recombinant proteins, designated single domain nanoantibodies or nanobodies ® and represent the smallest molecule in nature capable of binding a specific antigen. the cdr region of these nanobodies has the capacity to form long loops that can extend into cavities on antigens, e.g., the active site crevice of enzymes. other advantageous features of nanobodies include their high solubility, thermal stability (resist pasteurization), refolding capacity, and optimal tissue penetration in vivo (reviewed by vanlandschoot et al. ( ) ). nanobodies have demonstrated efficacy as agents of passive immunity for infectious diseases. vhh specific for rotavirus inner capsid protein vp are able to broadly neutralize rotaviruses independently of serotype, and in mouse experiments provide passive protection against challenge with human rotavirus (pant et al., ; garaicoechea et al., ) . nanobodies against other viral diseases with veterinary impact have been developed (foot-and-mouth disease, influenza, rabies (vanlandschoot et al., ) ) and represent a promising next-generation biologic platform for passive immunity. maternal vaccination to enhance passive immunity is already widely used in veterinary medicine. some of these vaccines, especially vaccines against enteric viruses, have limited efficacy. new approaches to enhance immunogenicity are promising, but await commercial development. a clearer understanding of protective mechanisms and immune modulation mediated by passive antibodies would contribute to more effective interventions. there is an urgent need for adjuvants and delivery systems capable of reliably inducing active immunity in neonates despite the presence of maternal antibodies. the ability to provide continuity of immune protection from birth, by combining passive immunity with active immunization, would have a major impact on neonatal morbidity and mortality. the rapid expansion of commercial fish farming (aquaculture) in many countries in recent decades has been accompanied by an urgent need to develop vaccines to prevent (infectious) fish diseases that were previously unknown or obscure. added to the difficulties involved in identifying the pathogens responsible, there has been the challenge of delivering vaccine efficiently with minimal stress to very large numbers of fish. some commercial vaccines for fish are administered by intraperitoneal or im injection, but mucosal vaccines are also widely used (reviewed by brudeseth et al. ( ) ). fish are routinely vaccinated by immersion in tanks of diluted bacterin, modified live bacteria or viruses, with exposure times ranging from s up to min, depending on the vaccine and age of the fish. it is unclear whether the main route of antigen uptake is oral or via the mucosal surface of the gills. several commercial vaccines are now available that consist of recombinant viral proteins for mixing into the feed (evensen and leong, ) ; days of feeding is recommended by the manufacturer. research on mucosal veterinary vaccines has contributed new concepts to the field of mucosal immunity. investigations of pathogen-host interactions in outbred animals have illustrated the complexity of these interactions. early studies of an enteric coronavirus infection of swine (tgev) led to the concept of the gut-mammary gland s-iga immunologic axis and provided part of the basic tenet for a common mucosal immune system. later studies of tgev and a deletion mutant of tgev with respiratory tropism (prcv) revealed functional compartmentalization within the common mucosal immune system whereby in inoculation of pigs with prcv failed to elicit sufficient intestinal iga antibody to fully protect against the enteric pathogen tgev (vancott et al., (vancott et al., , . subsequent studies have explored new prime/boost mucosal immunization strategies to elicit intestinal immunity to rotavirus in naïve pigs (saif, b; yuan and saif, ; gonzalez et al., ) . in these studies only oral priming with attenuated virus led to successful in booster responses using nonreplicating (vlp) vaccines combined with mucosal adjuvants such as iscom or mlt. thus use of a replicating vaccine to prime lymphocytes at a major mucosal inductive site (galt) followed by boosting with a nonreplicating vaccine at a second inductive site (nalt) effectively stimulated intestinal iga antibodies and induced active protection against rotavirus diarrhea. although there is progress in developing safe and effective nonreplicating vaccines to boost mucosal immune responses (saif and fernandez, ) , the dilemma remains to develop effective vaccines to prime for mucosal immunity. mucosal adjuvants (mlt, iscom, cpg, cytokines) and new delivery systems (replicating vectors, microparticles) have shown promise in animal studies reviewed in this chapter. however, their economical production and final evaluation under field conditions, including in the presence of maternal antibodies as relevant, are needed. considerable research effort has been devoted to development of vaccines for respiratory diseases of domestic animals. in some instances attenuated organisms delivered by mucosal routes have demonstrated improved efficacy over nonreplicating antigens given by systemic routes. for many respiratory diseases, however, further progress in the development of mucosal vaccines will have to await advances in understanding of disease pathogenesis and identification of protective antigens. in contrast, studies 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antibody responses and protection induced by a prime/boost rotavirus-dna vaccine in a gnotobiotic pig model short-term immunoglobulin a b-cell memory resides in intestinal lymphoid tissues but not in bone marrow of gnotobiotic pigs inoculated with wa human rotavirus systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease virus-specific intestinal ifn-γ producing t cell responses induced by human rotavirus infection and vaccines are correlated with protection against rotavirus diarrhea in gnotobiotic pigs intranasal administration of cpg oligonucleotides induces mucosal and systemic type immune responses and adjuvant activity to porcine reproductive and respiratory syndrome killed virus vaccine in piglets in vivo influence of probiotic lactobacilli colonization on neonatal b cell responses in a gnotobiotic pig model of human rotavirus infection and disease probiotic lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs key: cord- -spt oea authors: bhardwaj, prateek; bhatia, eshant; sharma, shivam; ahamad, nadim; banerjee, rinti title: advancements in prophylactic and therapeutic nanovaccines date: - - journal: acta biomater doi: . /j.actbio. . . sha: doc_id: cord_uid: spt oea vaccines activate suitable immune responses to fight against diseases but can possess limitations such as compromised efficacy and immunogenic responses, poor stability, and requirement of adherence to multiple doses. ‘nanovaccines’ have been explored to elicit a strong immune response with the advantages of nano-sized range, high antigen loading, enhanced immunogenicity, controlled antigen presentation, more retention in lymph nodes and promote patient compliance by a lower frequency of dosing. various types of nanoparticles with diverse pathogenic or foreign antigens can help to overcome immunotolerance and alleviate the need of booster doses as required with conventional vaccines. nanovaccines have the potential to induce both cell-mediated and antibody-mediated immunity and can render long-lasting immunogenic memory. with such properties, nanovaccines have shown high potential for the prevention of infectious diseases such as acquired immunodeficiency syndrome (aids), malaria, tuberculosis, influenza, and cancer. their therapeutic potential has also been explored in the treatment of cancer. the various kinds of nanomaterials used for vaccine development and their effects on immune system activation have been discussed with special relevance to their implications in various pathological conditions. statement of significance: interaction of nanoparticles with the immune system has opened multiple avenues to combat a variety of infectious and non-infectious pathological conditions. limitations of conventional vaccines have paved the path for nanomedicine associated benefits with a hope of producing effective nanovaccines. this review highlights the role of different types of nanovaccines and the role of nanoparticles in modulating the immune response of vaccines. the applications of nanovaccines in infectious and non-infectious diseases like malaria, tuberculosis, aids, influenza, and cancers have been discussed. it will help the readers develop an understanding of mechanisms of immune activation by nanovaccines and design appropriate strategies for novel nanovaccines. since the advent of the first vaccine centuries ago, researchers have been trying to find the answer of most common pandemic diseases through immunotherapy. vaccination has successfully managed to eradicate a lot of prevalent diseases, but associated off-target responses, lack of prolonged protection across variable pathogenic strains and allergy, limits its horizon for the prevention or treatment of other globally prevalent diseases like tuberculosis, aids, malaria, influenza and cancer. though prophylactic vaccines use live-attenuated or inactivated pathogens, lack of response and protection against diverse pathogenic strains [ ] . in addition, nanoparticles have the ability to impart more stability with high antigen loading and less proteasomal degradation of antigenic subunits. small and more specific subunits often tend to be less immunogenic, which can be overcome by the use of adjuvants that produce co-immunostimulatory or immunomodulatory signals. side effects associated with such conventional adjuvants with individual-specific response, immunotolerance to the target antigen and unwanted reactions towards self-antigens limit their use [ ] . therefore, biocompatible nanoparticles can also be directly used as adjuvants and may mitigate the need of strong conventional adjuvants (e.g., alum). another reason associated with low subunit immunogenicity is their low cellular internalization and rapid clearance from the body. nanoparticles with tunable physicochemical properties can overcome this limitation by enhancing circulation time, bio-accumulation in lymphoid organs and efficient targeting of immune cells. they can also increase crosspresentation by antigen-presenting cells (apcs) and activate the immune system at much lower doses. for instance, poly (lactic-coglycolic acid) (plga)-based nanoparticles enhanced the cross presentation of ovalbumin through major histocompatibility complex-i (mhc-i) in bone marrow derived primary dendritic cells due to which t cells were stimulated at -fold lower concentrations as compared to soluble antigen [ ] . different kinds of nanoparticles (lipid-based, polymeric, inorganic and protein/peptide-based) have been extensively used as adjuvants, immunogens and antigen delivery vehicles for immune activation [ ] . most of the synthetic nanoparticles interact with apcs structurally to increase internalization rather than functionally as they lack specific binding locations for cell receptors. on the contrary, protein-based nanoparticles can interact both structurally and functionally as they can carry antigens as well as interact with the pattern associated receptors on apcs [ ] . nanovaccines have the ability to induce both the components of human adaptive immunity i.e. cell-mediated as well as antibody-mediated immunity with induction of memory response. prolonged release of antigens from nanoparticle depots can cause enhanced stimulation for a long period of time, alleviating the need for frequent booster doses and therefore, nanovaccines can be used as both prophylactic or therapeutic which involves its administration before or after the occurrence of the disease respectively. however, the safety, biodistribution and residence of the nanoparticles must be optimized to obtain preferred immune responses towards nanoparticle-based vaccines. in this review, the potential and pros and cons of nanoparticlebased vaccines are explored. the role of different nanovaccines in activating various arms of immunity with an intent to abate the use of frequent booster doses as vaccines for tuberculosis, malaria, hiv (human immunodeficiency virus), influenza, and cancer are discussed. the applications and advantages of nanovaccines against various infectious and non-infectious diseases are also further delineated in this article. the ultimate goal for vaccination is to generate safe and efficient primary as well as secondary immune responses in the body. in a simplified way, primary immune responses protect the body from potential damages that might occur on first exposure of the pathogen or antigen. on the other hand, secondary immune responses, which are comparatively rapid and stronger, are elicited depending on the immunological memory generated during first exposure and protect the body from future encounters of the same epitope [ ] . nanoparticle-based vaccine delivery emerged as an appealing platform for boosting both primary as well as secondary immune responses in the body [ , ] . there are multiple mechanisms by which nanovaccines help enhancing immune responses. nanovaccines facilitate uptake of antigens in dendritic cells (dcs), which can further be enhanced by decorating the particle surface with ligands that selectively recognize receptors on dcs (refer fig. ). for example, ligands including fc fragment, mannose and anti-dec- (mab) have been widely explored which selectively binds fc receptors, mannose receptors and dec- receptors respectively on dcs [ , ] . in addition to efficient internalization, ability to co-deliver multiple-antigens and adjuvants, inherent adjuvant property, rapid lymph node drainage and efficient antigen presentation on dc's surface are other predominant mechanisms by which nanovaccines help increase the duration as well as the magnitude of the immune responses in immunized animals [ , ] . interestingly, these properties of nanoparticles have been substantially engineered to achieve desirable immune response for developing either prophylactic or therapeutic vaccines [ ] . for designing a safe and effective nanovaccine, it is imperative to understand the mechanism by which the nanovaccine activates both innate and adaptive immunity in the body [ ] . prophylactic nanovaccines are generally administered subcutaneously, intramuscularly, or intranasally whereas therapeutic vaccines (e.g. for cancer treatment) are injected intravenously. depending on the route of immunization, nanovaccines first encounter immune cells including neutrophils, macrophages, dcs and natural killer (nk) cells which quickly recognize the nanovaccine (foreign particle) based on the pathogen associated molecular patterns (pamps) associated with them [ ] . pamps (e.g. bacterial lps, teichoic acid, lectins, oligonucleotides or material from the carrier nanoparticle etc.) serve as the ligands for prrs [e.g. toll-like receptors (tlrs), c-type lectin receptors (clrs), retinoic acid-inducible gene-i -like receptors (rlrs) etc.] abundantly present on these cells [ , ] . thus, interaction of pamps with prr trigger endocytosis of larger particles (usually > nm) preferentially by macrophages and smaller particles by dcs. engulfment of nanovaccines by macrophages results in gradual degradation of nanovaccine particles and the encapsulated antigens. to prevent this, nanovaccine particles have been engineered to survive the attack by macrophages and facilitate direct delivery to apcs [ , ] . additionally, depending on the type of pamps, neutrophils and macrophage secrete a variety of cytokines and chemokines which further activate apcs. besides pamps, there are several unknown mechanisms which can activate innate immunity for example, induction of immune responses (potential adjuvant effect) by glycolic acid component of plga co-polymer [ ] or by cationic liposomes [ ] . eventually, the secreted cytokines and chemokines stimulate maturation and activation of apcs (mainly dcs) which initiate strong adaptive immune response (refer fig. ). therapeutic nanovaccines developed for treatment of metastatic cancer directly target dcs for strengthening adaptive immune responses in body [ ] . activated dcs serve as the link between innate and adaptive immunity. adaptive immunity generally has two arms; cell-mediated and antibody-mediated immunity. adaptive immune responses are generated within few days to several weeks and last longer. essentially, both types of immunity are necessary for a protective and prolonged immune response. however, their requirement is different in both prophylactic and therapeutic nanovaccines. for instance, strong cell-mediated cytotoxic responses are more desirable in case of therapeutic nanovaccine in order to effectively kill the metastatic cancer cells [ ] . vaccination provokes cell-mediated immunity generated by b cells and t cells which serve to neutralize the pathogen/antigen and simultaneously stimulating the body for developing immunological memory [ ] . the cmi responses are triggered by migration of activated dcs (following uptake of nanovaccine particle) to lymphatic organs (spleen and lymph noted). activated dcs present their antigens via mhc class i receptor to cd + cytotoxic t lymphocytes (ctls) (refer fig. ). activated ctls elicit strong cmi and selectively kill target cells (infected altered cells or cancerous cell) by inducing apoptosis in them [ ] . killing target cells is a complex immunological process that attracts several compliment systems (cs) proteins and co-stimulatory molecules. therapeutic cancer nanovaccines are preferentially designed to strengthen ctls responses by ex vivo educating dcs via adoptive transfer of antigen (for example, sipuleucel-t, a therapeutic vaccine approved for treatment of metastatic contraction resistant prostate cancer) [ ] . on other hand, activated dcs also present antigens to cd + t-helper cells (th cells) via mhc-ii receptor. subsequently, effector th cells undergo activation and maturation leading to secretion of variety of cytokines signals (refer fig. ). depending on the type of cytokine secreted, th populations are divided into th cells and th cells subsets [ ] . th subset majorly secretes proinflammatory cytokines (e.g. inf-γ , tnf-α, il- ) which stimulates proliferation of ctls and strengthen the function of cmi. th subset secrete class of cytokines (e.g. il- , il- , il- , il- , il- and il- ) which stimulate b cell proliferation during antibody-mediated immune responses [ , ] . a subtle balance between th /th responses determines overall therapeutic or prophylactic vaccine potential of candidate nanovaccine [ ] . in some cases, nanovaccines also work by suppressing t-regulatory (treg) cells, which naturally suppress activation and proliferation of effector t cells in the body [ ] . depending on the potential of nanovaccines to generate strong th cytokine response, naïve b cells located in spleen and lymph nodes get stimulated and bind to soluble antigen via b cell receptors (bcrs). activated b cell population undergoes proliferation in the germinal center. further, by somatic hyper mutation, b cells becomes specific to particular epitope of an antigen and then only epitope-specific b cells are selectively (clonal selection) proliferated. at this stage, activated b cells either differentiate into antibody-secreting plasma cells and secrete soluble antibodies against target antigen or remain dormant as memory cells for a future encounter of the same antigen (refer fig. ) [ ] . plasma cells are short-lived and their number gradually declines over time, as do the corresponding antibody titers [ ] . in that case, memory cells (b cells and t cells) which were generated and stored in the lymphatic organs or bone marrow for several months, come into play and protect the body from reinfection of the same antigen. in such situations, memory b cells quickly proliferate and turn to antibody-secreting (mainly igg) plasma cells to neutralize the antigen. on the other hand, memory t cells (both types cd + and cd + ) serve to produce more cytokine and chemokine signals to stimulate enhanced cmi and antibody-mediated responses. however, in case the structure of the antigen (epitope) has undergone significant changes due to cleavage, aggregation or re-folding, even memory cells cannot provide the required protection [ ] . although, in some cases, primary immunization can offer up to % protection, however, as even the rest % can be disastrous, therefore booster doses are scheduled to increase protection up to % [ ] . booster doses promote generating of more population and a variety of memory cells. generally, booster doses are scheduled after the antibody titer from plasma cells has dramatically declined to allow more competitive binding of memory cells to the injected antigen [ , ] . in a re-lated context, the use of nanoparticles help recalling more robust immunological memory even at a low dose [ ] . nanoparticles (e.g. plga based nanovaccine) facilitate prolonged availability of intact antigen in blood due to sustained release that leads to a higher proliferation of b cells and hence more memory cells [ , ] . also due to small size, nanoparticles can travel through the narrow lymphatic system to lymph nodes inducing differentiation of more memory cells [ ] . for example, demento et al., reported that over alum-based (free antigen) or liposome-based (burst release) carriers, mice immunized with ova-expressing listeria monocytogenes (lm-ova) encapsulated in plga particles (sustained-release) showed the highest protection against a lethal dose of l. monocytogenes even after weeks post immunization. they observed that mice immunized with antigen in plga nanoparticles had a robust population of cd + t memory cells [ ] . similarly, kanchan et al., showed that encapsulation of tetanus toxoid (tt) inside poly(lactic acid) (pla) particles generated long lasting primary antibody response in mice and interestingly even after months re-exposing the animal using very low antigen dose induced heightened and robust b cell responses [ ] . nanomaterials can be efficiently used for designing nanovaccines for the enhancement of immune-modulation. based on their potential use, they can be employed as adjuvants, immunogens or nanocarriers for enhanced and prolonged antigenic delivery. these properties of a nanoparticle are strongly governed by its size, shape, composition, surface chemistry and route of administration. it plays an important role in deciding its capability to induce inflammatory response or expression of specific defenserelated genes. different types of nanoparticles like protein, lipid, polymer, or inorganic particles (such as gold, iron, carbon and silica nanoparticles) can induce immune stimulation and have been discussed below [ ] . adjuvants are used with vaccines to enhance their potency by combination of mechanisms. adjuvants activate innate immune responses that shape and trigger the adaptive immune responses to vaccines [ ] . traditionally used adjuvants such as alum, complete freund's adjuvant and as (combination of alum and monophosphoryl lipid a) are known to form local depots that induce secretion of cytokines, chemokines and recruitment of immune cells. absorption of antigen on surface of adjuvants enhances antigen uptake and presentation, thus promoting antigen transport to draining lymph nodes [ ] . activation of tlrs has been potentially used for designing vaccine adjuvants. tlr ligands or agonists have served as potential immunostimulatory molecules as tlrs activate innate immune responses by identifying pamps and down signaling the transcription factors responsible for immune responses such as nf-κb, irfs and lymphocyte activation [ ] . the co-delivery of antigen along with tlr ligands promotes antigen presentation to cd + t cells via mhc-ii signaling and crosspresentation to activate cd + t cells [ ] . tlr agonist such as cpg up-regulates expression of cd , cd , cd , cd , and mhc class ii molecules thus enhancing antigen processing and presentation in plasmacytoid dcs [ ] . nanovaccines generate amplified, specific and robust responses as compared to traditional vaccines despite having similar mechanism of immune activation. the exaggerated immune response generated by nanoparticle associated antigen is due to their ability to specifically target dendritic cells with enhanced cellular uptake and antigen presentation. moreover, nanoparticles can be used as adjuvants with specific antigens or as immunogen themselves. vaccine antigens are either encapsulated inside or surface decorated over nanoparticles to get delivered efficiently (refer fig. ). there are chances that free antigens may degrade and elicit local immune responses at the site of administration, therefore, encapsulating free antigen inside nanoparticles prevents their degradation and provides prolonged and controlled release at the target site. the controlled release of antigen prevents burst responses and eliminates the need of booster doses. surface decoration of nanoparticles with antigen also confers the advantage of presenting the antigen to cells in a similar way as presented by pathogens [ ] . polyanhydride-based nanoparticles encapsulating f -v antigen when administered intranasally induced an immune response that persisted for weeks and elicited a high anti-f -v igg antibody response post-vaccination and conferred long-lived protective immunity against yersinia pestis infections compared to recombinant f -v antigen [ ] . similar nanoparticles were functionalized with di-mannose targeted macrophage mannose receptor (mmr). these carbohydrate functionalized nanoparticles enabled sustained release of immunogen for days and enhanced the uptake of antigen by dendritic cells simultaneously upregulating the co-stimulatory molecule clr, cd , and cd in cells as compared to non-functionalized nanoparticles [ ] . apart from encapsulation, several other strategies have been exploited for the co-delivery of antigens and nanoparticle such as surface absorption, conjugation, and mixture (refer fig. ). nevagi et al., observed enhanced immune activation against gas (group a streptococcus ) antigen using tri-methyl chitosan (tmc) nanoparticle conjugated with b cell and t cell epitope as compared to antigen mixed with tmc [ ] . nanoparticles have also shown potential to be used as adjuvants because of their immunomodulating activities such as inflammasome activation, complement system activation and recruitment of immune cells [ ] . due to above-mentioned effects, nanoparticles act as a better adjuvant in comparison to complete freund's adjuvant and conventional aluminum containing salts by inducing higher immunogenic responses at low antigen doses [ ] . also, their small size facilitates internalization by dendritic cells and induces adaptive immune responses. studies by swaminathan et al., have proved enhanced immune responses by lipid nanoparticle (lnp) adjuvant formulations against ovalbumin (ova) and hepatitis b virus surface antigen (hbsag) in balb/c and c bl/ mice injected with hbsag along with lnps as compared to hbsag alone [ ] . co-administration of quarternized chitosan nanoparticles as adjuvants along with ovalbumin intranasally in female balb/c mice induced activation of apcs followed by enhanced lymphocyte proliferation and differentiation as compared to ova mixed with aluminum hydroxide gel [ ] . asgary et al., also reported that silver nanoparticles showed increased antibodymediated responses against cvs- rabies and reduced toxicity as compared to conventionally used adjuvants such as alum [ ] . although nanoparticles possess great adjuvant properties, their codelivery along with generally used adjuvants such as tlr agonists can provide very robust immune protection. the use of tlr agonists is restricted because of their instability, particularly while using rna as tlr agonists. nugyen et al., have designed lipidoids to effectively deliver immunostimulatory rna to activate innate and adaptive immune responses. these lipid-rna nanoparticles enhanced ifn-γ responses and anti-ova cell-mediated and antibodymediated responses. these lipidoids were superior to the conventionally used n-[ -( , -dioleoyloxy) propyl]-n,n,n-tri-methyl ammonium methyl sulfate-rna delivery system [ ] . similarly unmethylated cpg-rich oligodeoxynucleotides (cpg), a tlr agonist. these nanoparticles induced pro inflammatory responses and antigen-specific antibody and cell-mediated immunity. colocalization of mpla and cpg also induced functional memory cd + cells. these findings clearly demonstrated the ability of bacterial elements and tlr agonists towards developing a more effective vaccine design [ ] . in addition, tlr agonists have been used in designing disease-specific nanovaccines, which are discussed later in this review. layer by layer protein nanoclusters synthesized from recombinant trimeric hemagglutinin were immunogenic by themselves and induced protective antibody-mediated responses without the use of adjuvants. such high immunogenic potential of peptide nanoclusters is because of the high antigen density available on the surface of nanoparticles [ ] . various types of nanoparticles have been used for nanovaccine formulation and each type serves a different function, which has been elaborated in the next section. protein-based nanovaccine can serve both structural as well as functional purpose by carrying antigens and engaging pattern recognition receptors of apcs. the protein-based nanovaccine can be categorized as biomimetic (virus-like particles, cages, vaults), rationally designed (nanofibrils, self-assembled protein nanoparticles, nanoclusters) and micelles. biomimetic protein-based nanovaccines : biomimetic proteinbased nanovaccines are of viral origin with self-assembled viral capsid proteins devoid of genetic material and are categorized as virus-like particles (vlps) (refer fig. (e) ). on the other hand, those of prokaryotic or eukaryotic origin are called protein cages and vaults. cages and vaults are generally used for biocatalysis or molecular transport. they share common properties with vlps like nanoscale, symmetry and container like geometries. all these structures can be modified chemically or biologically with antigens for immune activation [ ] . vlps ( - nm) of salmonella typhimurium bacteriophage p were engineered to encapsulate two respiratory syncytial virus (rsv) protein antigens, matrix (m) and matrix (m ). mice vaccination and intranasal boosting showed antigen-specific cd + and cd + t cell responses. upon subsequent rsv challenge, -fold reduction in viral load and increased antigen-specific cd + t cells were observed in the lungs of vaccinated mice as compared to empty p vaccinated and unvaccinated animals [ ] . cages like nm e cage derived from the pyruvate dehydrogenase complex of bacillus stearothermophilus [ ] , nm cage of human heavy chain ferritin (an iron storage protein) [ ] , ~ nm encapsulins (class of icosahedral cage structures) of bacteria and archaea are used for the presentation of different antigens to elicit immune response after immunization [ ] . a recent study on nonviral e protein-based biomimetic nanoparticle-containing acidlabile cpg (dc activating molecule) and siinfekl peptide epitope conjugate has shown the potential to enhance the cpg mediated dc activation by -fold in vitro as compared to unbound cpg. interestingly, co-delivery of siinfekl peptide conjugated with cpg has shown elevated cross-presentation and cd + t cell activation by inducing a -fold greater display of siinfekl on mhc-i by dcs as compared to unbound peptide or peptide bound directly to e protein. this shows the ability of biomimetic protein-based nanoparticles to facilitate enhanced immune response by dc activation and cross-presentation [ ] . vaults are eukaryotic ribonucleoproteins assembly of a cage-like barrel-shaped structure (approximately nm × nm × nm) made from the major vault protein (mvp) [ ] . it has been shown that increased antigen-specific cd + t cell response with a reduction in genital bacterial load and inflammation was observed after chlamydia muridarum polymorphic membrane protein g- (pmpg) encapsulating vaults were administered intranasally [ ] . rationally designed protein-based nanovaccines: nanofibrils are generally made of engineered β-sheets or α-helical coiled coils. β-sheet nanofibrils can be formed with peptide sequences like q (qqkfqfqfeqq) and kfe (fkfefkfe) appended to c terminus of a peptide antigen with a short linker sequence (refer fig. (d)). similarly, α-helical coiled-coil can also be formed into fibrils from α-helical peptide-like coil [ ] . such peptide sequences undergo assembly into fibrils with the antigenic epitope sticking outward from the fibril surface [ ] . these fibrils have been assembled with various epitopes of plasmodium falciparum [ ] , mycobacterium tuberculosis [ ] , streptococcus aureus [ ] , t-helper cells epitope padre [ ] and ova epitopes [ ] . conjugation of e ep immunogenic epitope of chikungunya virus e glycoprotein with β-sheet self-adjuvanted amyloid assemblies showed cytocompatibility with enhanced macrophage uptake and robust igg response against e ep epitope [ ] . self-assembling protein nanoparticles (sapns) are - nm icosahedrons containing pentameric and trimeric helical coiledcoils conjugated with (glycine) linker (refer fig. (f)). antigens and adjuvants can be inserted depending on the expected structure from in silico modeling [ ] . subunit malaria [ ] , hiv [ ] , toxoplasma [ ] and severe acute respiratory syndrome [ ] . wahome et al., encapsulated two hiv protein epitopes ( e and f ) onto sapn surface and observed enhanced production of epitope-specific neutralizing antibodies. this indicates the potential of sapn as nanovaccine to activate immune response against hiv [ ] . nanoclusters have also emerged as a prominent vaccine platform prepared from desolvation of proteins and peptides. they are generally made up of antigens and crosslinkers with an intent to increase antigen loading and eliminate off-target sequences. intranasal vaccination of influenza matrix protein (m e) containing self-assembled protein nanoclusters elicited enhanced immune response and complete protection after lethal challenge with hetero and homo-subtypic virus [ ] . since the preparation of nanoclusters requires organic solvents, it may alter the secondary or quaternary structure of full protein antigens [ ] . micelles: antigenic molecules can be conjugated to micelles in two different ways. either they can be covalently linked onto the micellar surface or can be conjugated to the variety of alkyl tails to form amphiphilic peptide micelles (pams). micelles of different sizes and shapes affect their immune activation potential [ ] . studies have shown that cylindrical or spherical pams are efficiently able to reach lymph nodes and carry the required amount of antigen and adjuvant [ ] . studies have reported pam mediated increased immunogenicity of t cells [ ] and b cells epitopes [ ] . such peptide lipid conjugates also form nano-disc vaccines (refer fig. (g)). subcutaneous vaccination with neo-antigen adpgk nanodiscs showed enhanced cd + cellular response, apc uptake and increase survival after b f melanoma challenge as compared to intramuscular vaccination [ ] . peptide-polymeric micelles have also been explored as a vaccine platform by conjugating hydrophilic peptide antigen with dendrimer polymers for immunotherapy [ ] . the most common lipid-based nanovaccines getting explored from the past few decades are liposomal nanovaccines (refer fig. (b) ). they offer a distinct advantage over other nanovaccine systems in terms of being biodegradable, tolerable, less immunogenic and having tunable physicochemical properties. they provide a wide repertoire to load antigens of different philicity inside and outside of liposomes with easy surface conjugation [ ] . different physicochemical properties (like size [ ] , surface charge [ ] , surface modifications (peg) [ ] , bilayer composition and fluidity [ ] ) affect the immunological outcome of liposomal nanovaccines. size-dependent differential activation of immune response indicated the activation of th response by nm sized liposomes as compared to th response by liposomes ≥ nm [ ] . transdermal immunization of mice with dissolving microneedles loaded with opposite charged transferosomes indicated the enhancement of th immune response by cationic transferosomes as compared to anionic counterparts by exhibiting strong endocytic escape property with fecilitating antigen processing via mhc-i pathway and larger accumulation in lymph nodes [ ] . kaur et al., showed the effect of bilayer fluidity on immune activation by varying cholesterol concentrations. relatively lesser in vivo igg production and ifn-γ was observed by liposomal formulations containing high cholesterol [ ] . similarly, the effect of another important factor affecting bilayer fluidity on liposomal mediated immune activation was observed by preparing liposomes with phospholipids having different transition temperatures (tm). high tm phospholipids containing liposomes stimulated th immune response as compared to low tm phospholipids that induced th response in mice immunized with leishmaniasis rgp antigen [ ] . numerous clinical studies showed the use of liposomes as adjuvants in therapeutic vaccines. liposome encapsulated d. pteronyssinus vaccination reduced allergen bronchial provocation induced inflammatory changes and improved condition of asthma patient after sustained mite exposure [ ] . a phase iii study of tecetomide vaccine, containing blp lipopeptide antigen incorporated into dmpg:dppc:chol:mpl-liposomes was performed for stage iii non-small cell lung cancer after subcutaneous immunization (nct ) [ ] . however, the study was terminated due to no effect on survival and primary or secondary endpoint in a phase ii study [ ] . a phase i trial (nct ) showed safety and immunogenicity of caf (liposomal adjuvant system) given with a subunit vaccine against m. tuberculosis antigen h . this adjuvanted vaccine showed long-lasting t cell response with no antibody response observed in healthy adults [ ] . viral liposomes called "virosomes" have also been studied in the literature. virosome exhibits viral capsid proteins onto the li- posomal surface for effective fusing with the target cell membrane. recently, virosomes and liposomes mediated immune response was investigated on human respiratory tract triple culture model. both virosomes and liposomes were prepared from the same neutral phospholipids, dopc ( , -dioleoyl-sn-glycero- -phosphocholine) and pope ( -palmitoyl- -oleoyl-sn-glycero- phosphoethanolamine), but virosomes were also fused with influenza membrane protein. results showed enhanced internalization of virosomes in epithelial cells of triple culture as compared to liposomes of similar sizes [ ] . a recent study has shown the use of lipid-based - nm particles (iscomatrix particles) as adjuvant with chimera peptide vaccine containing gp , tax, gp and gag epitopes of human t cell lymphotropic virus type when given subcutaneously in mice. enhanced production of mucosal iga and igg a antibody titers along with ifn-γ and il- cytokines were observed as compared to vaccine formulation [ ] . another class of lipid-based nanocarrier includes 'hexosomes' and 'cubosomes' that can provide a high membrane surface area for antigen and membrane protein loading. hexosomes are generally made up of lipids known to form non-lamellar structure in hydrated systems with inverse hexagonal liquid crystalline phase. comparative adjuvant activity of mono mycoloyl glycerol- (mmg- ) immunopotentiator containing hexosomes and cationic liposomes (caf ) loaded with chlamydia trachomatis major outer membrane protein (momp) antigen in cb /f mice after vaccination was studied by rodrigues et al. mmg- hexosomes made up of lipid phytantriol induced a strong momp-specific antibodymediated immune response whereas mmg- cationic liposomes elicited much stronger momp-specific cell-mediated response. it indicated the immune activation by two lipid-based adjuvants via different mechanisms [ ] . in addition, cubosomes are generally made up of lipid bilayers forming continuous periodic cubic lattice structures with interwoven water channels and a stabilizing polymeric outer corona [ ] . phytantriol, pluronic f and propylene glycol-based cubosome formulation containing (imiquimod and monophosphoryl lipid a (mpl)) adjuvants and model ova antigen elicited robust ifn-γ production and cd + and cd + t cell proliferation as compared to similar adjuvant containing liposomal formulation and alum [ ] . such lipid-based self-assembled structures can provide high antigen and adjuvant loading with enhanced immunoactivation in comparison to conventional lipidbased carriers like liposomes. polymeric particles showed a great possibility to be used as nanovaccines due to their ease of preparation, biocompatibility and biodegradability, fine-tuning of surface properties and controlled release kinetics (refer fig. (a) ) [ ] . the most commonly used polymeric nanoparticle for antigen delivery is poly (lactic-coglycolic acid) plga or poly (lactic acid) pla that has been used to deliver a broad range of antigens like hepatitis b virus antigen [ ] , tetanus toxoid [ ] , bacillus anthracis antigen [ ] , mycobacterium tuberculosis antigen [ ] and ovalbumin [ ] . other natural polymers like chitosan [ ] , alginate [ ] , pullulans [ ] , and inulin [ ] have also been used as adjuvants and antigen carriers. chitosan alginate-based nanovaccines for hepatitis b virus [ ] and dna-based chitosan vaccines for mycobacterium tuberculosis ( mtb h rv ) showed a marked increase in immunologic and protective efficacy in immunized mice [ ] . hyperbranched polyglycerol (hbpg) globular polymer can be a potential candidate for inert dendrimer based multi-valent vaccine coupled with b cell epitope for tumor-associated antigen (muc ) and t cell epitope for tetanus toxin p . it showed enhanced immune response and igg antibodies against breast cancer cells. hence polymeric nanoparticles can be used for potential multivalent vaccines [ ] . several inorganic nanoparticles (gold, iron, carbon, silica nanoparticles) are being explored for vaccine delivery in different disease models (refer fig. (c) ). ease of antigen functionalization onto easily accessible surface groups, robust properties with reproducibility outweighs their low biodegradability and gives them an edge to be used as nanovaccines. they also increase antigen stability by preventing premature proteolytic degradation. however, there are reports showing dose and size-dependent clinical toxicities of these nanoparticles [ ] . amongst all the inorganic nanoparticles, gold nanoparticles have gained maximum attention in vaccine delivery. these particles have been used for the epitope delivery against hiv [ ] , influenza [ ] , malaria [ ] and tumor [ ] . effect of shape of gold nanovaccine on antibody production against wnv envelope e protein showed that rods induced only % of the antibody response as compared to spherical nanoparticles [ ] . multicopy multivalent nano-glycoconjugates of gold nanoparticles decorated with tn-antigen glycan generated strong long-lasting antibodies against breast cancer expressing aberrant mucin glycan [ ] . inorganic carbon spherical nanoparticles and nanotubes were used as adjuvants to increase immunogenicity and act as delivery vehicles for peptides and protein against various viral infections [ , ] . oral immunization of bovine serum albumin (bsa) loaded carbon nanoparticles showed effective stimulation of mucosal iga antibodies in intestinal, vaginal and salivary mucosa along with th and th responses [ ] . abundance of silanol groups over silica nanoparticle surface provides an advantage for easy conjugation of targeting moiety for various viral infections [ ] . silica vesicles adsorbing e glycoprotein of bovine viral diarrhea virus (bvdv) showed -month antibody-mediated and cellmediated immune response with no histopathological changes at the site of infection [ ] . the use of magnetic iron oxide nanoparticles for tumor-associated carbohydrate antigen (taca) through phospholipid functionalization of taca glycopeptide showed enhanced antibody titers against both human and mouse tumor cells expressing that glycopeptide [ ] . calcium nanoparticles have also been reported for their superior adjuvant property with dna vaccines and to confer mucosal immunity. they can also induce enhanced antibody production against viral antigens as compared to aluminum adjuvants [ ] . since different types of nanoparticles impart different properties to the developed nanovaccine, their vast use in immune activation against a variety of antigens have been observed. though clear demarcation or direct comparison across nanocarriers has not been studied but there are unique properties and advantages associated with each nanocarrier that gives them an edge over each other. biomimetic carriers like vlps are expected to possess inherent immunogenicity that can elicit antibody reaction against carrier antigens itself. this could lead to the competition with the target antigen and alter antibody repertoire or memory response against it. reactive toxicities or neutralization upon repeated dosing can also limit their multiple use in a patient [ ] . such problems can be avoided by the use of nanoclusters that are solely made up of single type of antigen. these protein-based biomimetic and rationally designed nanocarriers are most suitable for the incorporation of a highly hydrophobic antigens of varied sizes with an intent to preserve its native structure, whereas hydrophilic peptides can be well delivered using amphiphilic micelles [ ] . carrier specific immune response has also been observed for certain polymeric and lipid-based carriers but it can be avoided using natural or biomimetic components (e.g., cellular lipid isolates or extracellular matrix polymers) [ ] . control over spatiotempo- table . different type of nanovaccines can escalate the immunogenic potential of antigens by boosting the immunogenic responses and memory generated against antigens at low doses. immunogenic responses generated against certain antigens can be used in two different scenarios by varying the use of vaccines as prophylactic or therapeutic (refer fig. ) . prophylactic vaccines aim to develop protective immunity and are administered before the onset of disease. the goal of prophylactic vaccines is to develop immunogenic memory against certain infections. major challenge of prophylaxis is obtaining sufficiently long-lasting immunogenic memory. for instance, single-dose immunization of mice with polyanhydride nanoparticle containing pneumococcal surface protein a (pspa), a virulence factor of s. pneumonia activated protective immunity and enhanced the survival of animals upon challenge even at -fold reduction in dose. pspa based nanovaccine formulation performed comparable to protein adjuvanted with alum, with much less tissue reactivity at the site of immunization [ ] . prophylactic nanovaccines confer protective immunity mostly against infectious conditions at low doses of immunostimulating antigen and reduced need for adjuvants, thus mitigating associated toxicities. therapeutic vaccines are administered after the onset of diseases to alter the course of disease by encouraging the immune system to fight harder against the prevailing conditions. unique immune responses are generated against disease-specific antigens. generally, therapeutic vaccines are used for cancer treatment, but there are compelling evidences suggesting their potential use in autoimmune disorders also. the advantage of using therapeutic vaccines instead of conventional pharmaceutical treatment lies in their specificity [ ] . mono-specific, disease- phase ii [ ] relevant pmhc complexes when coated on nanoparticle surfaces triggered the selective expansion of memory-like autoregulatory cd + t cells that arise only in affected individuals. such engineered nanoparticles have the potential to become suitable vaccines with the capacity of resolving organ and disease-specific autoimmune responses. systemic delivery of type-i diabetes relevant peptide-mhc-nanoparticle complex triggered expansion of memory autoregulatory t cells and suppressed the autoimmune attack against insulin-producing beta cells, thus restoring glucose balance [ ] . in certain cases, nanovaccines can be formulated to achieve both prophylactic and therapeutic responses. poly ( γ -glutamic acid) based synthetic vaccine nanoparticles (svnps) loaded with ovalbumin and toll-like receptor agonist (poly (i:c)) were synthesized by kim et al., both of them enhanced the uptake of antigens by apcs and enhanced the secretion of inflammatory cytokines (tnf-α, il- ) and type i interferon (ifn-α, ifn-β). simultaneous injection of both svnp-ova and svnp-ic conferred both the protective as well as therapeutic immunities by inhibiting tumor growth in eg -ova tumor-bearing mice [ ] . it is clear from the above-mentioned examples that nanovaccines modulate the immune system either for providing prophylaxis or therapeutic responses against pathological conditions. majorly prophylactic vaccines are used to prevent infections ( table ) and therapeutic vaccines have been used for cancer ( table ) treatment. infections that can be prevented by vaccination still contribute to approximately half of the child morbidity each year [ ] .treatment and immunization of such infectious conditions is a greater challenge due to the rapid emergence of new pathogenic variants. in spite of advancements in vaccine development over the years, many vaccines are associated with the risk of regaining their pathogenicity under certain immuno-compromised conditions. so, there is an imperative need for the development of risk-free and effective vaccines that confers desired antibody-mediated and cellmediated immunity against infectious diseases [ ] . subunit nanovaccine against enterohemorrhagic escherichia coli (ehec) was developed using gold nanoparticle conjugated with ehec antigens and induced high titers of serum igg upon subcutaneous administration in mice. it has also prevented colonization of ehec in both the cecum and large intestine at days post-immunization [ ] . mice immunized against anthrax with a single dose of pad -np (plga-based protective antigen domain, nanoparticles) elicited robust antibody-mediated and cell-mediated responses with mixed igg /igg a subtypes and th /th response. the survival rate of mice immunized with pad -np was more than for pad immunized mice after a lethal challenge with bacillus anthracis spores [ ] . nanovaccines have also shown great prospects in mitigating parasitic invasion. chimeric peptides containing leishmania infantum epitopes (histone h , kinetoplastid membrane protein and cysteine peptidase a) were encapsulated in plga nanoparticles. stimulation with the peptide-based nanovaccine induced maturation of dcs and il- production subsequently promoting allogeneic t cell proliferation and production of ifn-γ by cd + and cd + t cell. on immunization of transgenic mice with this peptide-based nanovaccine induced peptide-specific ifn-γ producing cd + t cells and conferred protection against l. infantum infection [ ] . multiple self-assembling peptide-based nanovaccines containing epitopes of toxoplasma gondii specific antigens have been designed. immunization with self-assembled protein nanoparticles activated cd + t cells to produce ifn-γ and protected transgenic mice against subsequent challenge with type ii parasites [ , ] . some more example of nanovaccines for highly prevalent infectious diseases have been discussed below in detail. tuberculosis being the deadliest infectious disease, is the greatest cause of mortality worldwide. % of infected individuals develop the disease within one or two years of infection and the rest develop the disease in later stages of life when immune functions are compromised. co-infection of hiv patients with mtb ( mycobacterium tuberculosis ), further complicates the situation with % mortality each year [ ] . bcg (bacille calmette-guerin) is a widely accepted vaccine against tb (tuberculosis), but its protective efficacy is limited by age due to the absence of consensus genomic loci that are present in most of the virulent strains of mtb. it induces short term and variable immune responses from % to % [ , ] . there is an urgent requirement of booster vaccines that augment t cell immunity in the lungs of bcg-vaccinated individuals. to address this, ansari et al., have synthesized archeosomes encapsulating a t cell antigen rv c, that upon immunization elicited type- cytokine response in mice. also, it increased the production of igg a antibodies, antigen-specific t lymphocytes and enhanced jid: actbio [m g; april , ; : ] expression of co-stimulatory molecules on the surface of apcs. by the virtue of all these properties, vaccination with archeosomes containing antigen reduced mycobacterial burden in the lungs and spleen of animals upon challenging with mtb [ ] . similarly, selfassembled peptide nanofibers, when loaded with t cell epitope, also showed similar responses by inducing corresponding effector memory t cells and increasing cytotoxic t cell population in the lungs upon intranasal immunization. mice vaccinated with coassembly of nanofibers containing cd + t cell epitopes and tlr agonists in the lungs increased the expansion of multi-functional cytotoxic t cell population. on further challenge with mtb, significant reduction in lung bacterial load was observed in comparison to bcg primed mice [ ] . in another study, mtb lipid-based nanovaccines were used wherein chitosan nanoparticles bound with mtb lipids induced cell-mediated and antibody-mediated immunity by secretion of immunoglobulins (igg, igm) and prominent th and th cytokines in lymph node and spleen cells [ ] . the higher potency of nanoparticle encapsulated antigen preparation is because of their ability to form depots with a slow and steady release in the surrounding milieu and enhanced uptake of antigen by antigen-presenting cells [ ] [ ] [ ] . as shown in a study by diogo et al., liposome-encapsulated antigens elicited more profound antibody-mediated and cell-mediated responses than free antigens. mice were immunized with free antigens and liposomeencapsulated antigen, further challenging mice weeks post immunization with mtb, it was observed that mtb burden in the lungs and spleen was significantly less in mice immunized with liposome-encapsulated antigen as compared to free bcg vaccine [ ] . the potential of nanoparticles in augmenting the prophylactic activity of antigens against tb was well understood and human trials were initiated. first-in-man trial on novel liposome caf as an adjuvant along with the tuberculosis vaccine ag b-esat- (h ) is reported in [ ] . human volunteers were vaccinated with escalating doses of caf at and weeks. after immunization strong antigen-specific t cell responses persisted for weeks and did not cause local or systemic adverse effects [ ] . above discussed preclinical and clinical assessment of various antigen-loaded nanoparticles indicates the potential of nanovaccines in inducing robust and long-lasting cellular immunity against mtb and shows hope for the development of a different and more effective prophylactic regime against tb. it is clear that with the advent of nanotechnology, adjuvant and delivery vehicle associated problems have been reduced, but the effect of the host environment and lack of consensus genetic loci still remains an inevitable challenge while developing efficient vaccines against tb. malaria is known to affect almost million people annually, with half a million deaths worldwide [ ] . fighting malaria is challenging because of its multistage life cycle. various efforts have been made in designing vaccines against the pre-erythrocytic and blood stage of malaria [ , ] . similarly, nanovaccines have been used to target multiple stages of malaria. iron oxide (io) nanoparticles have been used for the delivery of merozoite surface proteins (rmsp ). immunization of mice with rmsp- loaded nanoparticles induced parasite inhibitory antibodies with high titers. macrophages and dendritic cells showed more than % internalization of io nanoparticles and enhanced the expression of cytokines and chemokines [ ] . immune responses generated against sexual stage antigens of malaria impairs its transmission and reduces the disease burden. pfs is one such malaria transmission-blocking vaccine antigen, but its immunogenicity is limited in humans. hence, pfs has been used along with nanoformulations to achieve desired im-mune responses. for example, codon harmonized pfs (chrpfs ) has been used with gold nanoparticles as adjuvants for the induction of immunity against transmission. strong transmissionblocking antibodies elicited by simultaneous delivery of chrpfs with gold nanoparticles could be due to the co-ingestion of nanoparticles and antigen by immune cells [ ] . similarly, recombinant polyhistidine-tagged (his-tagged) pfs when mixed with preformed co-porphyrin containing liposomes at the time of immunization resulted in spontaneous nanoliposome antigen particularization (snap). immunization of mice and rabbits with snap resulted in higher functional antibody generation as compared with other 'mix-and-inject' adjuvants. they have also been used for developing multi-antigen vaccines (apical membrane antigen- , pfg , pfs and the nanp peptide), targeting multiple stages of the plasmodium life cycle using liposomes [ ] . ovalbumin loaded carboxylated polystyrene nanoparticles acted as an adjuvant and induced il- and granulocyte colonystimulating factor, that affects the migratory and homing ability of dendritic cells. mice challenged with malaria two weeks after immunization with nanoparticles produced anti-malaria antibodies and create the state of immune readiness to a subsequent infectious challenge [ ] . stable nanomimic, which are polymersomes with receptors for parasite attachment were synthesized. they bind to host cells and block re-invasion of the parasite after their release from host cells in vitro . this strategy can be used in the future to modulate immune responses against malaria and in the efficient designing of the vaccine [ ] . thus, the increased role of nanovaccines in targeting multiple life cycle stages of malaria shows new avenues in building a robust vaccination regime for prevention against malaria infection. three types of influenza viruses a, b and c have different viral nucleoproteins and matrix proteins giving rise to variable antigenic differences among them. influenza virus a and b cause epidemics attacking both adults and children, causing around million infections annually [ ] . presently, two types of vaccines are used against influenza based on strain a and strain b that target and produce antibody-mediated protective responses against hemagglutinin and neuraminidase. these antigens are highly prone to antigenic shift and drift due to error-prone rna dependent rna replication in influenza [ , ] . due to environmental selection and antigenic variations, chances of influenza epidemic still persists [ ] . although animal influenza virus is distinct from the human virus, zoonotic animal viruses can still occasionally infect humans through direct/indirect contact. avian and swine influenza viruses have been known to infect humans in some countries. thus, there is an unmet need for producing more efficient vaccines that can provide both antibody-mediated and cell-mediated immunity to confront homologous and heterologous variants [ ] . association of various influenza antigens with nanoparticles have shown promising preliminary results in providing enhanced immune responses against variable influenza antigens. as the antigenic variability limits the use of a single vaccine against influenza a virus (iav), there are effort s to develop nanovaccines conferring protection against more than one serotype by using most conserved ectodomain of influenza matrix protein (m e). nanoparticles containing self-assembling recombinant cage of human heavy chain ferritin presented with -sequential repeats of m e protein was observed to elicit elevated levels of m especific igg and mucosal secretory iga antibodies with enhanced t cell response against h n and h n lethal infection after intranasal administration in mice [ ] . nanovaccines against the conserved ectodomains can confer protection against inter-species viral infection. self-assembled virus-like particles (vlps) containing multiple copies of m e protein inserted into capsid (cap) protein of porcine circovirus type (pcv ) have shown dual protection against iav and pcv lethal challenge in mice and pigs after generating m e specific and pcv neutralizing antibodies [ ] . hemagglutinin trimer has also been exploited as a potential antigen for inducing protective immunity against influenza virus. pham et al., immunized balb/c mice with nanodiamond conjugated trimeric h and observed a significant higher h specific igg production as compared to immunization with h trimeric antigen alone [ ] . similarly, ross et al., have shown that intranasal or subcutaneous immunization of balb/c mice with polyanhydride nanoparticles encapsulated h induced high titer neutralizing antibodies. also, significantly higher cd + t cell expansion was observed in vaccinated mice as compared to non-vaccinated mice, thus providing cell-mediated and antibody-mediated protective immunity to mice. mice were further challenged intranasally with a/h n vnh n -pr cdc-rg influenza virus at days post immunization and viral load was significantly reduced as compared to saline-administered mice [ ] . it has also been demonstrated that the polyanhydride nanoparticles give equivalent responses at -fold lower doses as compared to free antigen [ ] . also, encapsulation of antigen in polyanhydride nanovaccines exhibited stability of antigen for one year at room temperature, thus providing a major benefit for maintaining stocks of pandemic vaccines [ ] . as discussed above, the nanovaccines have been designed by various research groups as combinations of nanomaterials and different antigens, but they provide protection from a particular strain of the virus. due to antigenic variations, multiple strains of influenza virus have emerged over the years posing serious public threats, indicating the need for a universal vaccine, which includes broad cross-presentation against multiple strains of influenza to reduce community threats. insights into the structural properties of antigens or peptides can be further explored for the rational designing of broad-spectrum nanovaccines using multiple antigens. on similar lines, to increase the breadth and potency of nanovaccines, deng et al., have designed double-layered protein nanoparticles. the core of the protein nanoparticles ( mtg) contains four types of m e from human (hum e), swine (swnm e), avian (avim e), and domestic fowl (fwlm e) viral consensus sequences coated with ha variants hrh and hrh (refer fig. ). mice were immunized intramuscularly with uni mc (pnp), uni c (hrh -coated double-layer pnp) and uni c (hrh -coated doublelayer pnp) and uni c (cocktail of uni c and uni c ). robust antibody-mediated responses were generated and induced m e antibodies were strongly cross-reactive to diverse antigens. also, uni c group produced broad cellular responses by significantly inducing more ifn-γ secreting splenocytes after stimulation with diverse antigen peptides. naïve mice were vaccinated with sera from pre-immunized mice with dpbs, uni mc, or uni c and then challenged intranasally with pr or aic after hours. sera from uni c immunized mice prevented the mice from a viral infection, indicating the role of serum antibodies. also, antibodies specific to human m e, pr , and aic were viable for four months after the immunization in mice and implied long-lasting protection [ ] . from the examples discussed above, it is evident that with the help of structurally important antigens, smartly designed protein nanomaterials will have the potential of inducing long-lasting immunity against broad spectrum of antigens, thus providing platforms for producing efficient nanovaccines to combat pandemic viruses. hiv is the sixth leading cause of death worldwide. hiv infection causes systemic depletion of cd + t cells, hence compromising the activity of the immune system and the development of aids. immune functions can be preserved in the early stages of hiv by using antiretroviral therapy (art). it targets the hiv life cycle, but the major challenge is strict adherence to complex art regimens. this necessitates the urgent need for the development of a prophylactic vaccine against hiv. the most recent and promising hiv vaccine rv is in the phase iii trial [ ] . igg antibodies raised against v v loop were inversely correlated with the risk of infection in the clinical trial of rv [ ] . sapn-based vaccine reduces the need for essential glycosylation of the hiv- env v v loop to form a native-like structure to act as potential immunogen [ ] . although self-assembling vlps as vectors have shown great potential in the development of immunity against hiv, anti-vector immunity is a significant concern. here, synthetic nanoparticles offer versatile platform technologies that can induce strong adaptive immune responses while avoiding anti vector immunity and toxicity issues [ ] . plga based nanoparticles have been used for co-utilization of tlr agonist and the antigen. intradermal immunization of mice with hiv- p -nef/flic/plga (antigen/agonist/nanoparticle) enhanced igg production even at -fold lesser dose of antigen. nanovaccines shifted the immune response towards th polarization and enhanced the th cytokine pattern [ ] . recently, protease cleavage sites (pcs) has been utilized as a strategy to hamper the maturation and infectivity of hiv. due to low immunogenicity of peptide antigens, they have been crosslinked with chitosan and hyaluronic based nano-formulation. nanoparticles containing pcs showed enhanced activation of apcs and the production of anti-pcs antibody. also, these nanoparticles generated memory t cells at longer time points after the last booster dose, indicating their capacity to elicit a good immune response upon infection [ ] . silver nanoparticles coated with amantadine triggered the production of hiv specific ctls in the spleen of mice with -fold stronger tnf-α production in vivo . the co-culture of these hiv specific ctls with hiv infected cells in vitro enhanced the death of infected cells and reduced the production of hiv [ ] . similarly, hiv- peptide and oligosaccharide loaded aunps enhanced antigen presentation to isolated t cells from hiv- patients. treatment with aunp formulated vaccine increased proliferation of hiv-specific cd + and cd + t cells with increased secretion of pro-th cytokines and pro-inflammatory cytokines in vitro [ ] . malik et al., have shown the prophylactic potential of intravaginally delivered nanogold loaded formulation. poloxomer and hyaluronic based thermogels were loaded with gold niosomes and mannosylated gold niosomes along with efavirenz (efv). efv and gnps inhibited viral dissemination in pbmcs (peripheral blood mononuclear cells) host cells, but their individual potential in inhibiting pre-interaction viral dissemination was lower than that of the combination. a substantial . % increase in activity was seen when efv was given with gnps. both the moieties had a dual-faced attack wherein gnps inhibited hiv entry into the cell by causing denaturation of gp glycoprotein and efv inhibited transcriptase enzyme. the mannosylated efv-gnps (man(egnz)) showed further potential activity in inhibiting viral dissemination when the host was pre-exposed to them before viral infection. thermogels containing efv-gnp and man(egnz) showed . ± . % and . ± . % inhibition respectively in p antigen during anti-hiv prophylactic challenged study [ ] . various such formulations have been developed over the years that enhanced the prophylactic action of raltegravir and efavir [ ] , phthalate and efavirenz [ ] or efavirenz and saquinavir [ ] . bayon et al., have also shown the capacity of nano-lipid complexes (nlc) to induce p specific immune responses against hiv in non-human primates (nhp). four intradermal immunization over a period of months showed that nlc-loaded p- antigen elicited significantly higher p -specific antibodies as compared to the combination of free p antigen and cpg adjuvant [ ] . there have been advancements in understanding the role of nanovaccines in fighting hiv and development of nanovaccine loaded formulations for prophylaxis of hiv. with the emerging use of nanotechnology development of an efficient vaccine system for hiv prevention is not that far. despite constant efforts to develop effective cancer vaccines, their efficacy in clinics has always been limited. nonetheless, two prophylactic vaccines for hepatitis b virus-associated liver cancer and human papillomavirus virus-associated cervical cancer have been approved by us fda till now [ ] . the prophylactic vaccine development for cancer eradication is a challenge due to inefficient identification of tumor-specific antigens (neoantigens), rapid clearance with less accumulation in the lymphoid organs and generation of weak memory immune response. therefore, the use of nanoparticles to overcome these limitations would be an advantageous strategy. prolonged and sustained delivery of antigen would be extremely important for the generation of immunologic memory and booster dose reduction. immunization of mice with protein nanoparticles conjugated to melanoma-associated epitope gp and cpg adjuvant enhanced the production of melanoma-specific cd + t cells. pre-immunization with nanoparticles delayed onset of tumor growth and increased the survival rate to %, showing prophylactic potential of protein nanoparticles [ ] . luo et al., showed strong cytotoxic t cell response by a physical mixture of pc a polymeric nanoparticle and ova antigen. this was achieved by enhanced cytosolic delivery and antigen presentation in apcs of draining lymph node by pc a nps and activation of type-i interferon stimulating genes. nanovaccines showed tumor growth inhibition in melanoma, human papilloma virus e /e tumor and colon cancer models. the combination of anti-pd with pc a nps showed synergism with % survival in a tc- model over days. further, re-challenging of tumor-free mice with tc- cells after immunization with pc a nanoparticles and anti-pd showed complete tumor growth inhibition. this indicates the prophylactic response of nanovaccine by generating long-term antitumor memory [ ] . liu et al., developed a unique prophylactic nanovaccines using metallo-organic nanoparticles (mof) coated with fused membrane (fm) of dendritic cells and cancer cells. presence of whole tumor antigens and immunological co-stimulatory molecules on its cytomembranes impart them apc like properties for cancer specific t cell immuno-activation. since, fusion of dc and cancer membrane is accompanied by dc maturation, mof@fm nanovaccines possessed lymph node homing molecules. therefore, enhanced retention and homing of mof@fm in lymph nodes and spleen as compared to only cancer membrane fused (mof@cm) and dendritic cell membrane fused (mof@dm) nanovaccines was observed. pre-immunization of mice with mof@fm has significantly prevented the development of t tumors by promoting differentiation of cd + t cell precursors into cytotoxic t cells with enhanced generation of ifn-ɣ and il- immuno-stimulatory cytokines (refer fig. ) [ ] . tumor heterogeneity with differential expression of antigens makes it challenging to develop a prophylactic treatment strategy. therefore, most of the nanovaccines being developed for cancer are therapeutic. the detailed description of the same is given in the next section. unlike conventional therapeutics, therapeutic nanovaccines offer the prospect of eliciting antigen-specific responses without any additional non-specific responses. therapeutic vaccines have been used against autoimmune disorders by antigen-specific targeting of t-regulatory cells [ ] . clinical studies revealed the therapeutic efficacy of vlp in allergen-induced asthma. bacteriophage derived vlps were packed with cpg and tlr and induced th response in immune system. a total of asthmatic patients were treated with injections of vlps as a part of double-blind randomized trial and it was observed that the asthma was well-controlled in patients treated with vlps as compared to the placebo group [ ] . unlike cancer, nanovaccines showed promising results in pathogenic infections because of the presence of extremely potent foreign non-self antigens. therefore, vaccine-based therapy is still limited in cancer treatment but can provide an alternative to, or complement conventional cancer treatments. till now, cancer vaccines have failed to make a mark clinically as compared to other immunotherapies like t cell therapy and checkpoint blockade. the only us fda approved therapeutic cancer vaccine, provenge (sipuleucel-t), has shown a moderate increase in clinical efficacy in prostate cancer [ ] . the major problem in successfully developing a cancer vaccine is the selection of tumor-specific antigens called "neoantigens". conventionally, cancer vaccines were developed by targeting tumor-associated antigens (taas), which are overexpressed specifically in a particular tumor type across patients. however, taas are also present in normal tissues; therefore vaccines specific for them can induce peripheral or central immunotolerance leading to autoimmunity or low vaccination efficiency [ ] . neoantigens are tumor-specific antigens derived from random somatic mutations but absent in normal cells. hence, vaccines targeting such neoantigens using nanoparticles may enhance the outcome of cancer immunotherapy. cancer nanovaccines can present unique and highly immunogenic tumor-specific antigens with increased antigen loading and efficient delivery, controlled antigen presentation, and retention in lymphoid organs [ ] . a recent study showed the targeting of bone marrow and splenic dcs with peptide ty using mesoporous silica nanoparticles conjugated with ova/cpg for immune activation (msn-ty/ova/cpg). therapeutic application of (msn-ty/ova/cpg) in naïve c bl/ j b -ova mice enhanced dc activation and tumor elimination by eliciting tumor-specific cd + t cell response, with prolonged survival and less systemic toxicity [ ] . to overcome inadequate loading efficiency, weak immune response, and complex preparation process, dong et al., prepared antigen nanoparticles (ovalbumin nps) containing cpg adjuvant (onps-cpg) with high antigen and adjuvant loading for cancer immunotherapy. the in vitro and in vivo results showed enhanced immune response including dc maturation, t cell activation and ifn-γ production. onps-cpg induced remarkable anti-tumor efficacy in mouse lymphoma model [ ] . biomimetic-approach for developing cancer nanovaccines: overcoming cancer heterogeneity by using patient-derived cell membranes for the development of smart nanovaccines can be a promising biomimetic approach for personalized therapy. various strategies have been explored in the form of developing artificial antigen-presenting cells (aapcs) based nanovaccines, use of different cell membrane fused nanovaccines and membrane coated viruses for enhanced cross-presentation and immune stimulation (refer fig. ) . recently, kuai et al., have shown the anti-tumor efficacy of high-density lipoprotein (hdl) nanodiscs loaded with neoantigenic peptide and cpg t cell receptor agonist in b f and mc- colon carcinoma model. nanodiscs have delivered the neoantigen to draining lymph nodes and enhanced antigen presentation by apcs. it causes enhanced stimulation of cytotoxic t cell response that potentially inhibited tumor growth. delivery of nanodiscs with checkpoint blockade (anti-pd and anti-ctla- ) therapy also eradicated established b f and mc- colon carcinomas. this strategy showed a powerful therapy to generate personalized cancer medicine [ ] . zhang et al., developed a magnetic artificial antigen-presenting cell (aapc) for enhancing cd + t cell-mediated tumorigenic response. they developed fe o magnetic nanoclusters coated with leucocyte membrane antigens decorated with mhc-i loaded peptide (siinfekl) and anti-cd co-stimulatory ligand for eg- tumors. these biomimetic aapcs stimulated ot- cd + t cells and visually guided them to tumor magnetically, causing reduced tumor burden [ ] . this offers a great platform for t cell-based immunotherapy. the fusion of biological membranes with nanovaccines has emerged as an interesting concept towards biomimetic nanovaccines. a recent study showed biomimetic cancer-derived magnetosome with enhanced retention in lymph nodes after subcutaneous administration. toll-like receptor (tlr) agonist (cpg) loaded fe o magnetic nanoclusters camouflaged with cancer membrane and was decorated with anti-cd for preferential recognition by cd + dc population for cross-presentation to cd + t cells via mhc-i. enhanced stimulation of tumor-specific cd + t cells due to prolonged retention of magnetically guided nanocluster in lymph nodes showed anti-tumor efficacy with improved survival in different mice models [ ] . similarly, guo et al., developed an erythrocyte membrane coated plga nanoparticle-containing hgp - antigenic peptide and monophosphoryl lipid-a (mpl-a) tlr- agonist for enhanced anti-tumor immunity. intradermal injection in c bl/ had shown reduced tumor burden along with metastasis and prolonged tumor occurring time after re-challenge in metastatic models [ ] . studies exploiting cancer cell membranes as an envelope to impart a wide repertoire of cancer-specific antigens can offer an advantage to develop various personalized vaccines towards multiple tumor types. another interesting strategy for developing personalized biomimetic cancer nanovaccines is the use of cancer cell membrane coated virus for increased adjuvanticity, infectivity and oncolytic activities to generate a strong anti-tumor immune response. a recent study showed the prophylactic and therapeutic potential of a . ± . nm biohybrid artificially enveloped virus extracrad (extra conditionally replicating adenovirus). oncolytic adenovirus serotype containing a cpg island with a base pair deletion was encapsulated into b .ova cancer cell line membrane. coating of the cancer membrane was found to enhance the in vitro cytotoxicity of extracrad as compared to the naked virus indicating the enhanced infectivity of viral vaccine as compared to the naked virus in multiple cell lines. intra-tumoral injection of extracrad in c bl/ mice containing b .ova, b f and ll/ induced models showed in vivo therapeutic vaccine efficacy in facilitating reduced tumor proliferation as compared to the naked virus, individual vaccine component controls and virus membrane mixture because of enhanced cross-presentation and t cell activation. the prophylactic potential was explored by vaccinating mice times with extracrad.cmt .ova and extracrad.b f vaccine before cmt .ova and b f cells re-challenge, respectively. prolonged overall survival and enhanced inhibition of tumor proliferation in homologous membrane enveloped viral vaccinated groups as compared to the naked virus and heterologous membrane enveloped group indicated the strong potential of viral-based vaccine imparting wide repertoire of cancer-specific antigens as preventive vaccines [ ] . another viral protein-coated dendrimer nanovaccine containing e and e peptide epitope of hpv showed enhanced cytotoxic t cell stimulation followed by hpv infected cells eradication. tumor elimination was observed in % of treated mice with no recurrence up to months post initial challenge [ ] . therefore, to address the problems associated with cancer vaccine development, special emphasis has been given on biomimetic personalized nanovaccines. the use of an apcs and nanoparticles coated with the patient's own cancer cell membranes can provide effective antigenic diversity to overcome tumor heterogeneity and tumor-specific immune response. this provides a key strategy to train one's own immune system with appropriate antigens. research using such a biomimetic personalized approach can significantly improve the outcome of current treatment modalities. nanovaccines have shown potential in augmenting the therapeutic and prophylactic efficacy of cancer vaccines. in the past decade, research on vaccines has taken a big leap forward, exploring their vast potential to combat numerous diseases. specifically, strong interactions of nanoparticles with the immune system and the associated benefits have attracted atten- tion with the hope of producing less toxic and effective nanovaccines. physicochemical variations in nanoparticles are one of the prime factors affecting their fate as "nanovaccines" inside the body in terms of their clearance, specific bioaccumulation, adjuvanticity, antigenicity and toxicity. potent antigenicity with prolonged retention and release can cause nanovaccines to elicit both cellmediated and antibody-mediated antibody along with memory effector response, thus alleviating the use of frequent booster doses. although nanovaccines are still in their infancy because of its limited clinical trials but new generation nanovaccines possess enormous potential both as prophylactic or therapeutic. hence, exploring new avenues to increase nanovaccine safety and efficacy should be the driving force for further research in this domain of bionanotechnology. nanomedicine toxicity, scaling-up processes and lack of regulatory guidelines can be considered as the limiting factors in the production of nanovaccines. the nano-scale size can work as a double edge sword as pre-clinical and clinical reports showed their dosedependent acute and chronic toxicities with preferential bioaccu-mulation depending on the route of administration. few classes of nanoparticles possess inherent toxicity after prolonged exposure like inorganic nanoparticles (e.g., metallic nanoparticles). moreover, iscoms are being used in a number of animal vaccines, but toxicity associated with saponin-based adjuvants prohibited their use in humans. therefore, concerns over nanoparticle usage for vaccines are still in existence. scaling up is another major problem that has been minimized up to an extent because of technological advancements, but scale-up in a sterile environment is still a significant challenge [ ] . though, the conventional treatments in this era of modern medicine is saving countless lives. the steady rise in antibiotic resistance and lack of curative cancer therapy because of continuous causative strain variations and cancer heterogeneity respectively drives the focus towards personalized therapeutics. hence, on similar lines, vaccines against bacterial infections and tumors could benefit significantly, but the current vaccine's antigenic breadth and lack of potency are the limiting factors. therefore, researchers have recently shifted towards biomimetic nanotechnology to develop biomimetic nanovaccines as personalized medicines. such nanovaccines can be inherently immuno-stimulatory and multiantigenic. coating of bacterial outer membrane vesicles (omvs) jid: actbio [m g; april , ; : ] onto nanoparticles for anti-virulence vaccination to deliver and neutralize bacterial toxins outplays the pathogen by utilizing its own survival mechanism. this can prevent bacterial colonization effectively and reduce direct selective pressure to develop antibiotic resistance. a large number of such nano-toxoid formulations can be developed by varying the outer membrane coating of these biomimetic nanovaccines [ ] . despite 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satchell, liam p.; fido, dean; latzman, robert d. title: functional fear predicts public health compliance in the covid- pandemic date: - - journal: int j ment health addict doi: . /s - - - sha: doc_id: cord_uid: ztj o ob in the current context of the global pandemic of coronavirus disease- (covid- ), health professionals are working with social scientists to inform government policy on how to slow the spread of the virus. an increasing amount of social scientific research has looked at the role of public message framing, for instance, but few studies have thus far examined the role of individual differences in emotional and personality-based variables in predicting virus-mitigating behaviors. in this study, we recruited a large international community sample (n = ) to complete measures of self-perceived risk of contracting covid- , fear of the virus, moral foundations, political orientation, and behavior change in response to the pandemic. consistently, the only predictor of positive behavior change (e.g., social distancing, improved hand hygiene) was fear of covid- , with no effect of politically relevant variables. we discuss these data in relation to the potentially functional nature of fear in global health crises. that the true rate to be no more than % (rajgor et al. ) . while the symptoms of covid- mimic the conditions caused by other coronaviruses (e.g., coughing, fever, and fatigue; wang et al. ), many infected individuals can appear asymptomatic (holshue et al. ) , and thus can unwittingly transmit the virus to others in their vicinity. this claim was supported through a comparison of confirmed case rates between south korea (who adopted widespread public testing for covid- in february ) and italy (who tested only those with symptoms), with backhaus ( ) reporting how, in south korea, substantially higher rates of younger people (below the age of ) tested positive for the virus in the absence of noticeable symptoms. as demonstrated by the differences in testing practices mentioned above, countries across the globe have taken different steps to contain and delay the spread of the virus within their borders, with differing degrees of success. china, for example, appeared to have halted the exponential increase of confirmed cases of the virus by limiting the freedom of citizens to move around their cities, provinces, and the country as a whole (graham-harrison and kuo ) . the italian government has implemented a similar strategy, placing much of the country into lockdown and preventing groups of people from congregating in public spaces. other countries have been slower to respond with such drastic action. the usa, for example, waited more than weeks after the first confirmed case within its borders to enact localized testing procedures (baird ) . elsewhere, the british government followed the instructions of its scientific advisors to delay the cancellation of mass gatherings and the closure of schools, instead favoring a policy that looked akin to pursuing herd immunity (whereby vulnerable groups are isolated, and healthier individuals are exposed to the virus to gain immunity at the population level; fine et al. ) . the one common piece of advice across different countries is that those who display symptoms of covid- should self-isolate from others for a period of - days, while those without symptoms (who, importantly, could still be infected) should practice "social distancing" (world health organization ) . this means that everybody should be limiting non-essential social interactions, not travel unless absolutely necessary, and work from home wherever possible to slow the interpersonal transmission of the virus. however, this is being practiced inconsistently in the absence of government enforcement. in this paper, we explore some of the potential psychological triggers for this inconsistency in social distancing behavior. in spite of us still only being in the early stage of the covid- pandemic, a rapidly growing body of research into social responses to the virus is emerging. this research examines how to encourage prosocial and virus-mitigating responses (e.g., social distancing, or the non-hoarding of food and household supplies, and good hand hygiene). for example, everett et al. ( ) reported how communicating advice using deontological moral advice (i.e., in ways that invoke a sense of civic duty) has a modest effect on increasing a propensity to engage in behaviors that enhance a delay in the transmission of the virus (e.g., hand washing, avoiding large gatherings, and sharing government advice on preventing the spread of . this sense of duty taps into themes of interpersonal empathy, with oosterhoff and palmer ( ) findings that american adolescents who demonstrated higher levels of subjective self-interest were more likely to hoard supplies and less likely to refrain from nonessential social interactions. pfattheicher et al. ( ) reported how trait empathy for vulnerable members of the population encourages the reduction of physical social interactions, while inducing empathy maintains this behavior. further, a rapid review of the evidence into compliance with quarantine advice during pandemics recently reported that appealing to altruistic motivations to comply with distancing instructions appears to maintain motivation to maintain social distance from others over an extended period of time (brooks et al. ) . while this raft of empirical work appears to be focused around persuading the community to follow social distancing advice, there may also be intrinsic or individual difference reasons for following such isolating practices. moral foundations theory (for a review, see haidt and joseph ) asserts that individuals make social and political judgments based on their endorsement of propositions rooted in a finite set of intuitive moral domains. in the main, political liberals value making decisions on the basis of care/harm (i.e., protection of the vulnerable) and fairness/reciprocity (i.e., proportionality) principles, while political conservatives also value authority/respect, ingroup/loyalty, and purity/sanctity. there is a case to be made that these moral foundations may play a role in decisions to engage in advice about delaying the spread of covid- . for example, framing of vulnerability among certain demographic groups (e.g., the elderly, pregnant, and chronically ill) has the potential to trigger the care/harm foundation. further, the advice coming from governmental or scientifically authoritative sources may trigger instincts related to the authority foundation, while the infectious nature of the virus, by definition, is likely to trigger a behavioral response associated with pathogen avoidance via the disgust foundation. for these reasons, it is unlikely that "political orientation" as a composite or self-identified variable would predict behavioral responses to covid- . however, specific moral foundations that are associated with positions across the ideological spectrum may differentially predict actions that mitigate the risk of the virus being spread. according to ahorsu et al. ( ) , one unique feature of pandemic viral infections is the fear that they can instill across large swathes of the population. fear is a negative emotion symptomized by extreme levels of emotive avoidance in relation to specific stimuli (perin et al. ) . it is associated with clinical phobias and social anxiety disorder (krueger et al. ) , and as such the potential for widespread public fear caused by pandemic viral infections could lead to significant levels of mental distress at the population level. this was implicated in a tweet published by dr. david murphy (president of the british psychological society) that highlighted fear and anxiety (operationalized as managing health anxiety and coping with worry about relatives) as targets for psychological input within the covid- pandemic (murphy ) . in spite of the documented negative wellbeing implications of fear and anxiety, these negative emotions do also motivate a range of behaviors that reduce the engagement in risky behaviors. for example, early levels of anxiety in children are associated with lower levels of alcohol use (kaplow et al. ) and cigarette and marijuana use (colder et al. ) . further, pathological low levels of fear are widely associated with psychopathic personality traits (see patrick et al. ; patrick and drislane ) , and this has been related to various risk-taking for social and recreational purposes (satchell et al. ) . this is also consistent with emerging evidence that higher levels of so-called dark personality traits (e.g., psychopathy, meanness, and disinhibition) are associated with a lack of engagement with health-promoting behaviors in the covid- pandemic (blagov ) . as such, the potential utility of a fear or anxiety response in the current context should be explored. specifically in relation to covid- , while perceiving the virus to be severe has been linked with worse mental health outcomes (li et al. ) , feeling personally at risk of infection predicted a greater propensity to engage in hand washing and social distancing behaviors in the early stages of the pandemic (wise et al. ) . even among the aforementioned li et al. ( ) research, subjectively judged self-control attenuated the link between perceived covid- severity and poorer mental health, suggesting that combining a sensible level of fear about the illness with messages related to personal agency could encourage safetypromoting behavior in the form of hand hygiene and social distancing. this claim is supported by zettler et al. ( ) , who reported that the hexaco personality domain of emotionality (characterized by exaggerated levels of anxiety, fear, and emotional reactivity) was associated with a greater level of acceptance of government-mandated personal restrictions. further, kuper-smith et al. ( ) found that community members in the usa, the uk, and germany consistently underestimated their likelihood of both becoming infected and transmitting covid- in comparison with estimates about the "average" member of the public (see also raude et al. ) . they also reported negative correlations between self-perceived likelihood of infecting others and engaging in hygiene-related behaviors (e.g., hand washing and social distancing), suggesting that increasing fears about contracting the virus might lead to less risky social behaviors. in this study, we explored multiple predictors of engaging in virus-mitigating behaviors within the context of covid- . specifically, we were interested in measuring concrete behaviors in response to the covid- pandemic as they occur, and predicting these using established psychological constructs. secondary to this, we explored whether fear of covid- and self-perceived likelihood of contracting the virus were associated with risk-mitigating behaviors. this is in response to an apparent mismatch between established psychopathological research into the potential utility and fear and anxiety in reducing risky behaviors, and the suggestion by ahorsu et al. ( ) that fear might be considered a unidirectional precursor to psychopathological responses within the current context. finally, we investigated the role of political ideology in changing behaviors in response to covid- . in doing so, we acknowledged the partisan nature of some social attitudes toward the virus itself (pennycook et al. ) and governmental responses to the pandemic (see pepinsky ) while contemplating the potential for intuitive moral foundations to overcome these identity-based political differences. to determine our target sample size, we conducted an a priori power analysis using g*power (version . . . ). due to the lack of previous research to inform our expected size of effects, we define our smallest effect size of interest by what the psychological literature typically observes. for example, funder and ozer ( ) reviewed various summative analyses of the psychology and reported r = . as the typical effect size. thus, we set a conservative type i and ii error rate both to . , and aimed to detect r = . , concluding a target sample size of n ≥ . a total of individuals clicked on the study link. of this, n = participants (m age = . years, sd = . , % female) met all four attention checks and were retained for analysis. in our sample, % reported "british" or "uk" nationality and % reported residence in the uk. the majority of those with complete responses had an undergraduate degree ( %) or had attained less than undergraduate degree ( %). most participants considered themselves "medium" risk for covid- ( %), and many considered themselves "low" risk ( %). on a scale of very liberal (− ) through "centrist" ( ) to very conservative ( ), participants were, on average, "somewhat liberal" (m politics = − . , sd = . , skew = . ). these participants were recruited via prolific, a crowdsourcing platform, whereby survey responders receive small monetary compensation for taking part in research. participants received the equivalent of £ . for their time. all data collection occurred between march and , . demographics and perceived risk of covid- participants were asked to report their age, gender, nationality, level and years of education, self-identified political orientation (rated on a -point scale from " -very liberal" to " -very conservative"), and their current country of residence. a self-report measure of perceived risk of covid- was also requested, with participants being asked to self-report whether they considered themselves "low-," "medium-," or "high-risk" (scored from to ). items (e.g., "it makes me uncomfortable to think about coronavirus- ") measuring one's fear of covid- (greater scores indicate greater fear). participants are asked to rate their agreement with each statement on a -point scale from " -strongly disagree" to " -strongly agree." yougov behavior change (ygbc; yougov blue ) shortly after the us public health campaign against covid- began, the polling group yougov asked a sample to self-report the degree to which seven behaviors have changed over the last week. we adapted this question in light of varied governmental responses to the pandemic, and asked participants to consider their behaviors in the month prior to any official "lockdown" in their country or state. however, we used the same behaviors as on the initial ygbc measure: hand washing, changed travel, working from home, stockpiling food, stockpiling medicine, child and elder care, and social distancing. participants reported the perceived change on a -point scale from " -it has not changed at all" to " -it has changed dramatically." promis emotional distress short forms (promis-sfs; cella et al. ) we used two of the promis-sf measures to examine recent (past days) emotional wellbeing. one measure used items to quantify diagnostic and statistical manual ( th edition; dsm- ; american psychiatric association ) depression symptoms (e.g., "i felt worthless"), and the other used items to measure dsm anxiety symptoms (e.g., "i felt worried"). participants rated each item on a -point scale from " -never" to " -always" with higher scores indicative of higher levels of negative affect, and a greater autonomic arousal and experience of threat, respectively. moral foundations questionnaire (mfq- ; graham et al. ) the mfq- consisted of statements spanning five moral foundations (care/harm, fairness/reciprocity, authority/ respect, ingroup/loyalty, purity/sanctity; four items per foundation) using two different response formats. the first section asked participants to rate the relevance of a particular domain when they make a moral decision ( items; e.g., "whether or not someone acted unfairly"; fairness foundation). the second section asked participants to rate their endorsement of a range of moral propositions ( items; e.g., "i am proud of my country's history"; loyalty foundation). two items on the mfq- were fillers: "whether or not someone was good at math" (section ) and "it is better to do good than to do bad" (section ). these are designed to catch careless responding, and were not included in calculating foundation scores. each statement was rated on a -point scale (scored functionally from to ). in section , anchor labels are " -not at all relevant" to " -extremely relevant," while in section they are " -strongly disagree" to " -strongly agree." responses are averaged for each moral foundation, with higher scores being indicative of greater endorsement of each respective moral domain. world health organization: quality of life-bref (whoqol-bref; world health organization ) the whoqol-bref measures how one feels about their quality of life and health through items (e.g., "how satisfied are you with your ability to perform your daily living activities?"). participants are asked to rate their agreement with each statement on a point scale from " -very dissatisfied" to " -very satisfied." greater scores were indicative of greater quality of life. participants initially provided their informed consent before entering their demographic information and perceived risk of contracting covid- . following this, all study questionnaires were presented in a randomized order by the survey software (qualtrics) to reduce the likelihood of order effects influencing the quality or validity of the data collected. on average, the study took . min to complete. this procedure followed british psychological society ethical standards, and was approved by an institutional ethical review panel prior to data collection. all analysis code and data (plus a redacted version of the survey file) can be found here: https://osf.io/cek q/?view_only= d a c c b c cd a df. the mean responses to domains were retained for analysis. we report pairwise correlations between all variables (with notable correlations highlighted when they meet a conservative α = . ). to analyze psychological predictors of engagement with who recommended behaviors, we first built a linear model (using base r) to explore the extent to which fcv- s scores predicted engagement with the change in behavior (ygbc) scores. then, we tested the additive effect of the two promis-sf scales and whoqol-bref when they were introduced into the base model. we compared the variance explained by these two models to investigate the unique variance explained by the fcv- s. next, we tested for the effect of the mfq and political orientation on behavior change (ygtc) scores. this base model was then compared with a second model including the fcv- s and promis-sf to answer whether political orientation has a greater effect on behavior change than anxiety or fear. mean scores, standard deviations, and internal consistency coefficients for all measures are reported in table . pairwise correlations between the predictor measures and ygbc and fcv- s scores can be found in table . there was a notable moderate positive correlation between increased change in behavior and fear of covid- , suggesting that those with higher fear scores were those who were engaging with more public health behaviors. the only other notable correlates of behavior change included a small positive correlation with promis anxiety and a small-to-moderate positive relationship with self-reported risk. that is, those who show more anxiety symptoms and believed themselves to be at risk of contracting the virus changed their behavior more. there were moderate-to-strong correlations between the fcv- s scale and promis anxiety and depression, suggesting this novel measure of fear is highly related to anxiety symptomatology. increased fcv- s scores also moderately correlated with increased selfreported risk of contracting the virus. physical and environmental quality of life decreased with care/harm r = . , p = . r = . , p < . * fairness/reciprocity r = . , p = . r = . , p = . authority/respect r = . , p = . r = . , p = . ingroup/loyalty r = . , p = . r = . , p = . purity/sanctity r = . , p = . increased fear of coronavirus. the moral foundations of care/harm and purity/sanctity were weakly-to-moderately positively related to fcv- s scores. a baseline model using fcv- s scores to predict behavior change explained a significant amount of variance (r adj = . , p < . ), with the fear score being a positive predictor (β = . , p < . ). an additional model, using the dsm's promis measures explained slightly more variance in behavior change (r adj = . , p < . ), but this what not significantly more variance when considering the standard of our conservative alpha correction (f , = . , p = . ). the additive model maintained fcv- s as a positive predictor (β = . , p < . ), alongside a negative effect of promis depression (β = − . , p = . ) and a non-significant predictor of promis anxiety (β = . , p = . ). a model predicting behavior change using the moral foundations questionnaire and participants' political orientation explained little variance and did not meet our conservative alpha, and no predictors in this model met our significance criterion. a model adding in the fcv- s and promis scales to the base model explained significantly more variance in behavior change than moral and political orientation (f , = . , p < . ). in this model (see table ), the only significant predictor was the positive effect of fcv- s. the current study explored psychological predictors of behavior change and fear in response to the covid- pandemic of . we found relationships between behavior change and the new fcv- s scale (ahorsu et al. ) , dsm-based anxiety and depression measures, and self-perceived risk of contracting the virus. critically, these relationships were generally positive, in that those participants who were more concerned about covid- (as measured by the fcv- s) were those who engaged more with public health-compliant behaviors (e.g., regular hand washing, and social distancing). it is of interest that the measures of fear and anxiety symptoms were stronger predictors than moral and political orientation, all of which explained small to no variance, potentially suggesting more emotional (rather than sociopolitical) influences on compliant behavior. there was also no notable decline in quality of life in relation to behavior change. however, fear of covid- was related to decreased physical and environmental wellbeing. overall, these results suggest that "fear" and anxiety at the current time have a functional role, and are related to increased compliance for improving public wellbeing. the data that we have presented above lead to a number of important implications, not only for the ways in which we understand behavioral responses to pandemics but also for how we conceptualize the utility of negative emotions, which may not necessarily always be reflective of psychopathology, and the political context within which such behaviors take place. we now consider these two broad implications in turn. functional "fear" in the development of the fcv- s the authors rightly state that "[w]ith high levels of fear, individuals may not think clearly and rationally when reacting to covid- " (ahorsu et al. , p. ) . this is true for the pure emotion of fear, which represents the reactive removal of oneself from a position of immediate risk. however, many of the items in the fcv- s scale are pertinent to anxiety, a preparatory reaction to ambiguous or distant stimuli. beyond the conceptual similarity in the wording of items, our analysis revealed a strong relationship between the fcv- s and dsm-based measures of anxiety. this is important as fear and anxiety are behaviorally and neuroendocrinologically distinct responses (mcnaughton and corr ), with anxiety potentially having a functional preparative role to encounter future negative stimuli consistent with the data we have presented here. indeed, as has been described in detail previously (for a review, see perkins and corr ), negative emotions, broadly, may have evolved to serve more adaptive and protective functions and may, in certain contexts, help to keep us safe. in the current context, this appears to be the case with negative emotions being protective (i.e., encouraging of public health-promoting behaviors) during the covid- pandemic. that is, the results of the current study suggest that negative emotions in response to the current pandemic predict adaptive public health-compliant behavior change (e.g., hand washing, social distancing). these findings, situated within the aforementioned larger literature pertaining to the oftentimes adaptive nature of the experience of negative emotions, raise serious concerns with efforts to identify "mental health issues" associated with strong emotional responses, which, for the majority of individuals, are both normative and protective. that is, for most individuals, the anxious responses being assessed by the fcv- may represent a normal and adaptive response to a real and present danger that one cannot fight or flee from, within the environment (i.e., the covid- pandemic). those working in the mental health field should be sensitive to the context in which behaviors emerge, especially when these behaviors exist in a culture of preparedness for coping with the new cultural and governmental demands that may be critical for personal and family survival. notwithstanding the above, it is important for mental health professionals to be attentive to the needs of individuals for whom highly emotional responses to the current pandemic, coupled with pre-existing risk factors (e.g., those with a history of mental illness), may result in pathological levels of negative emotions and related behaviors. indeed, increased and prolonged exposure to a community crisis, like the pandemic we are currently experiencing, has been found to result in increased maladaptive levels of anxiety leading to unnecessary behaviors associated with increased levels of impairments within individuals as well as overburdening of community resources (garfin et al. ) . overall, we believe that mental health professionals have an important role to play in supporting the wellbeing of the public in this current time. however, it is not clear how this work is supported by the classification of rational concerns about a pandemic and the labelling of functional "fear" as a psychological issue. politically driven responses to the pandemic in spite of recent reviews of the political psychological literature advocating for the presence of behavioral asymmetries between ideological groups (see, e.g., jost ), we found no such evidence in our data. this is perhaps surprising given recent coverage of the covid- pandemic, and reports about how attitudinal and behavioral responses to the virus were inherently partisan in nature (pepinsky ) . in this previously reported work, us-based democrats were more likely than republicans to engage in health-promoting behaviors (e.g., regular hand washing, social distancing, and self-quarantining), and to express attitudes that were critical of governmental responses to the pandemic (e.g., that inadequate testing was available, or that more money should be made available to tackle the virus). an obvious difference in our data is the political contextslightly less than % of our sample were british, rather than american. however, it could be that this work reflects the stage of the pandemic in which the partisan data were collected. at the time of writing, the usa is in an earlier phase of the covid- pandemic. in a manner consistent with a social intuitionist account of political and moral decision-making (haidt ) , it is therefore plausible that initial (and perhaps intuitive) judgments of the pandemic in the us context were guided by president trump's initial rejection of covid- as a serious and unique public health concern (trump ). these initial intuitions then led to asymmetrical responses between partisans in pepinsky's ( ) work: ambivalence and inaction among republicans, and concern and derision of the response among democrats. however, in the uk (where the majority of our participants were based), the pandemic is at a slightly more advanced stage, meaning that the seriousness of the situation is clearer. we argue that the lack of any significant political orientation or moral foundations effects on behavior change is a positive point from a social perspective, and suggests that, in times of (inter)national crisis, people can forgo their ideological commitments and behave consistently with governmental advice in pursuit of a common public health good. the results of this study are not without limitation. first, the data presented here are entirely self-reported and so may be subject to response biases. specifically, virus-mitigating behavior changes such as increased hand washing, working from home, and social distancing are all targets of governmental strategy to "flatten the curve" and reduce the spread of the virus (e.g., public health england ). as such, it may be expected that this constellation of behavioral changes will be reported to a greater degree as a function of a reluctance to deviate from this normative social shift. the collection of "other-reported" data on the participants would help to mitigate this limitation. second, while our regression models have utility for informing how public health behavioral compliance can be predicted from an individual differences perspective, it lacks the identification and understanding of any potential barriers to bringing about said change. it is here where experimental studies emerging about the response to covid- (e.g., everett et al. ) can be supplemented by correlational data such as ours in order to bring about a more comprehensive view of public health compliance and the effectiveness of government messaging. finally, we are mindful that data has been collected at a single time point within an unprecedented period of time. therefore, not only do results of this investigating warrant replication, but also future investigations should consider any potential variation in governmental guidance, policy, or social perception pertaining to covid- in their discussion and interpretation of results. this paper has presented cross-sectional data demonstrating, in real time, predictors of behavior change in response to a global viral pandemic. we have shown how "fear" may be a normal and, crucially, functional response within this context. that is, scores on the recently developed fcv- s (ahorsu et al. ) , which specifically measures "fear" toward the new virus, consistently predicted engaging in culturally and governmentally recommended public health behaviors (e.g., improved hand hygiene and social distancing). in light of this, we argue that researchers and mental health professionals would be mindful to consider the context within which negative emotional states are experienced before considering whether such emotional states are necessarily pathological. further, and in spite of the increasingly polarized nature of our political landscape, we found that politically relevant outcomes (i.e., self-identified orientation, and the endorsement of moral foundations; graham et al. ) were unrelated to behavior change. these nonsignificant effects highlight how universal polarization in social responses between political groups is not inevitable, and that there are some issues that unite us, rather than divide. just as other (inter)national crises have previously brought people together (e.g., willer ) , it seems that the covid- pandemic has the potential to do the same, and bring people of opposing political positions together in a sense of common humanity. this procedure followed british psychological society ethical standards, and was approved by an institutional ethical review panel prior to data collection. the authors declare that they have no conflicts of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. fear of covid- scale: development and initial validation diagnostic and statistical manual of mental disorders coronavirus: why it's so deadly in italy what went wrong with coronavirus testing in the adaptive and dark personality traits in the covid- 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psychologists can contribute to coping with the pandemic. here's my very rough st draft psychological correlates of news monitoring, social distancing, disinfecting, and hoarding behaviors among us adolescents during the covid- pandemic triarchic conceptualization of psychopathy: developmental origins of disinhibition, boldness, and meanness triarchic model of psychopathy: origins, operationalizations, and observed linkages with personality and general psychopathology fighting covid- misinformation on social media: experimental evidence for a scalable accuracy nudge intervention the public response to #covid in the united states is utterly experience of group conversations in rehabilitative medicine: methodological approach and pilot study anxiety as an adaptive emotion the emotional path to action: empathy promotes physical distancing during the covid- pandemic guidance on social distancing for everyone in the uk the many estimates of the covid- case fatality rate. the lancet. advance online publication are people excessively pessimistic about the risk of coronavirus infection? risk as reward: reinforcement sensitivity theory and psychopathic personality perspectives on everyday risk-taking so last year , americans died from the common flu. it averages between , and , per year. nothing is shut down, life & the economy go on. at this moment there are confirmed cases of coronavirus clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china the effects of government-issued terror warnings on presidential approval ratings changes in risk perception and protective behavior during the first week of the covid- pandemic in the united states world health organization quality of life (whoqol) -bref coronavirus disease (covid- ) advice for the public new coronavirus polling shows americans are responding to the threat unevenly individual differences in accepting personal restrictions to fight the covid- pandemic: results from a danish adult sample publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - trhh authors: frey, andreas; lunding, lars p.; ehlers, johanna c.; weckmann, markus; zissler, ulrich m.; wegmann, michael title: more than just a barrier: the immune functions of the airway epithelium in asthma pathogenesis date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: trhh allergic bronchial asthma is a chronic disease of the airways that is characterized by symptoms like respiratory distress, chest tightness, wheezing, productive cough, and acute episodes of broncho-obstruction. this symptom-complex arises on the basis of chronic allergic inflammation of the airway wall. consequently, the airway epithelium is central to the pathogenesis of this disease, because its multiple abilities directly have an impact on the inflammatory response and thus the formation of the disease. in turn, its structure and functions are markedly impaired by the inflammation. hence, the airway epithelium represents a sealed, self-cleaning barrier, that prohibits penetration of inhaled allergens, pathogens, and other noxious agents into the body. this barrier is covered with mucus that further contains antimicrobial peptides and antibodies that are either produced or specifically transported by the airway epithelium in order to trap these particles and to remove them from the body by a process called mucociliary clearance. once this first line of defense of the lung is overcome, airway epithelial cells are the first cells to get in contact with pathogens, to be damaged or infected. therefore, these cells release a plethora of chemokines and cytokines that not only induce an acute inflammatory reaction but also have an impact on the alignment of the following immune reaction. in case of asthma, all these functions are impaired by the already existing allergic immune response that per se weakens the barrier integrity and self-cleaning abilities of the airway epithelium making it more vulnerable to penetration of allergens as well as of infection by bacteria and viruses. recent studies indicate that the history of allergy- and pathogen-derived insults can leave some kind of memory in these cells that can be described as imprinting or trained immunity. thus, the airway epithelium is in the center of processes that lead to formation, progression and acute exacerbation of asthma. with more than million people affected bronchial asthma is one of the most common chronic inflammatory diseases worldwide ( ) . actually, out of deaths is associated with asthma and it causes annual direct medical (drugs, care, hospitalization) and indirect economic (productivity loss, early retirement) costs of about € billion for the eu ( ) and over billion for the united states ( , ) , making it a major burden for public healthcare systems ( ) . asthma is characterized by acute broncho-obstruction, in combination with additional symptoms such as cough, chest tightness, shortness of breath, and wheezing, which vary in extent and over time. these symptoms arise on the basis of chronic airway inflammation in response to a trigger, most commonly inhaled allergen(s), that causes airway hyperresponsiveness (ahr), airway remodeling and mucus hypersecretion ( ) . due to the complexity and variation of the symptoms along with its pathogenesis, asthma is nowadays described as a heterogeneous syndrome with distinct sub-or endotypes. however, the majority of asthma patients displays allergic inflammation of the airways, which can be classified by profiles of several characteristic mediators in "th high" or "th low" subtypes ( ) . thus, in sensitized individuals t helper (th ) cells orchestrate allergic inflammation by releasing a typical array of cytokines including interleukins (il)- , - , - , and - and granulocyte-macrophage colony stimulating factor (gm-csf). these mediators induce the production of allergen-specific immunoglobulin (ig) e, th cell development, goblet cell differentiation, submucosal gland activity, as well as recruitment, maturation, and activation of eosinophils and its precursors ( ) . activation of mast cells and eosinophils via ige-bound allergens results in their degranulation and, thus, in the release of a plethora of effector molecules and growth factors that on the one hand destroy airway tissue and on the other hand conduct its repair. chronic activation of these processes ultimately lead to signs of airway remodeling such as increased smooth muscle mass, subepithelial fibrosis, and epithelial desquamation, which in turn give rise to the pathological changes and clinical symptoms characteristic for asthma ( ) . allergic sensitization against aeroallergens represents the strongest factor predisposing for the development of asthma, indicating a hyperreaction of the immune system to be the central event within the pathogenesis of this disease. nevertheless, structural cells and particularly airway epithelial cells also appear to be of critical importance. this is not surprising since these cells represent the barrier that first encounters environmental stress factors like air pollutants, bacterial and viral pathogens, as well as allergens, and markedly contributes to their neutralization by a mechanism called mucociliary clearance (mcc). besides these barrier and cleaning functions airway epithelial cells also exert a number of immunological tasks interweaving the role of the epithelium with that of the above-mentioned cells of the immune system. here we aim to review these immune functions of the airway epithelium against the background of asthma pathogenesis. the main purpose of mucosae is to separate the body from its environment and therefore they are essential for the maintenance of the inner homeostasis. though this task is not commonly regarded as an "active" or "typical" immune function, it is absolutely central for the defense against allergens, pathogens and other harmful environmental factors. in order to fulfill this function the airway epithelium forms a continuous, selfcleaning barrier with a considerable resistance against biological, chemical or physical stressors ( ) . together with the physical barriers of the mcc and glycocalyx, this is achieved by three types of intercellular epithelial junctions that form the structural adhesion forces of the airway mucosa by linking the intracellular structures of the cytoskeleton of one epithelial cell to that of its neighbors. these junctions involve adherens junctions (ajs), hemidesmosomes, and tight junctions (tjs). ajs can appear as spots (adhesion plaques) or as bands encircling the cell (zonula adherens). in the junctional zone ajs interconnect the actin filaments of the adherent cells via homotypic transmembrane e-cadherin adhesions and anchor proteins like actinin, vinculin, and α-, β-, and p catenins, while adhesion plaques attach the cells to the extracellular matrix ( ) . similarly, hemidesmosomes are focal structures that form adhesive bonds between the cytoskeleton of epithelial cells and the lamina lucida, which is a part of the lamina propria. hemidesmosomes utilize integrin α β , plectin a and the tetraspanin cd connecting laminin and fibronectin of the extracellular matrix to the intermediate filaments of the cytoskeleton ( ) . in contrast, tjs form a multiprotein junctional complex called zonula occludens (zo) that in turn appears as the main regulator of the paracellular permeability. these complexes are formed by several transmembrane and cytoplasmic proteins that are attached to actin filaments of the cytoskeleton. the main components of tjs are claudins and occludins, proteins with four transmembrane domains, as well as so-called junctional adhesion molecules (jams) belonging to the immunoglobulin superfamily with only one transmembrane domain. these proteins are connected to actin filaments by cingulin and zo proteins , - , and - ( ) . in the airways of healthy individuals, the tjs of the zonula occludens and ajs of the zonula adherens constitute dense protein networks that interconnect the basolateral sides of epithelial cells in such a way that they prevent the paracellular passage of basically all molecules, including water, ions and proteins, as well as of pathogens or other inhaled particulate matter. several findings strongly indicate that in asthma patients the barrier function is impaired by epithelial disruption. for example, endobronchial biopsies revealed a fragile or even injured airway mucosa with partially or completely uncovered areas and detachment of columnar, ciliated cells ( ) . epithelial desquamation is further indicated by the presence of epithelial cells in bronchoalveolar lavage (bal) and of creola bodies (epithelial cell aggregates) in sputum of asthmatics ( ) . furthermore, bronchial biopsies of asthmatic subjects displayed patchy disruption of tjs ( ) and the expression of a number of proteins that are essential for the formation of tjs and ajs has been shown to be markedly reduced. among these proteins are α-catenin ( ), β-catenin ( ), occluding ( ) , zo- ( , ) , and e-cadherin ( , ) . the levels of the latter one in sputum also correlate with asthma severity ( ) . these data are further supported by in vitro studies where primary bronchial epithelial cells are kept in air liquid interface (ali) culture, a method that allows the cells to differentiate and form a pseudo-stratified epithelial monolayer largely resembling the physiological structure of the airway mucosa. once this structure has been established, in vitro barrier integrity can be assessed by measuring the transepithelial electrical resistance (teer), a characteristic that is indicative of the tightness of a cell layer ( ) . several studies showed that ali cultured airway epithelia from asthma patients display a decreased teer in comparison to epithelia derived from healthy controls ( , , ) . to date, three different factors are discussed to have a harmful impact on the barrier integrity of the airway epithelium in asthma pathogenesis: allergens themselves, viral infection, and (allergic) inflammation. according to the "protease hypothesis" allergens with an inherent protease activity are capable of cleaving the protein components of the aforementioned intercellular epithelial junctions so that the barrier function is disrupted and allergens can penetrate the airway mucosa on the paracellular route, which eventually could result in sensitization against them. accordingly, a considerable number of allergens has been tested in vitro for proteolytic potential and for an effect on epithelial barrier integrity. several studies provided evidence for a direct cleavage of e.g., occludin and zo- proteins by the major allergen from house dust mites (dermatophagoides), der p ( , ) . house dust mite extracts as well as der p have been shown to increase the permeability and to decrease teer of epithelial layers in vitro ( , , ) . comparable effects have been shown for extracts of the allergenic fungus alternaria alternata that reduced teer of human bronchial epithelial cells in vitro ( ) or the aspergillus fumigatus-derived alkaline protease (alp- ) ( ) . similarly, a variety of different pollen extracts has been investigated for their effect on the barrier integrity of epithelial cells in vitro. diffusates of italian cypress (cupressus sempervirens), orchard grass (dactylis glomerata), olive (olivia europaea), and scots pine (pinus sylvestris) have been shown to affect claudin- , e-cadherin, and occludin expression and thus to disrupt epithelial junctions in ali cultures of calu- cells, an effect which could be suppressed by protease inhibitors ( ) . japanese hop (humulus japonicus) extract also reduced expression of occludin in a comparable setting ( ) . another study provided evidence for proteolytic activity of giant ragweed (ambrosia trifida), kentucky bluegrass (poa pratensis), and white birch (betula pendula) as shown by reduced expression of claudin- , occludin, and zo- in calu- as well as in mdck cells ( ) . however, an inherent protease activity appears not to be the only way, by which allergens can impair the barrier integrity of the airway epithelium. cockroach, hdm, fungus, and mold extracts have also been shown to activate the protease-activated receptor (par-) and/or , which in turn leads to degradation of aj components ( , ( ) ( ) ( ) . the effect of viral infections on airway barrier function is even more pronounced than that of allergens. respiratory viruses cause junction dysfunction by different mechanisms: human rhinoviruses (hrv), respiratory syncytial virus (rsv), human metapneumovirus (hmpv), influenza and parainfluenza viruses bind to their entry receptor, which are typically protein or sugar structures expressed on the cellular surface for other purposes, leading to endocytosis of the virus. once the virus has been internalized, it uncoats and initiates the viral replication process, which has certain consequences for infected cells. on the one hand, the cell starts with the production of type i interferons (ifn) in order to slow down the internal virus replication and to activate the cellular immune response against the virus. in consequence, infected airway epithelial cells are killed by virus-specific, cytotoxic cd + t cells. on the other hand, the virus itself also kills epithelial cells, since it induces morphological alteration of the cells summarized as cytopathic effect (cpe). for hrv and influenza viruses, the cpe manifests in rounding and detachment of airway epithelial cells that are ultimately lysed by the virus in order to release freshly produced viruses. paramyxoviruses such as rsv and hmpv induce cell fusion so that four or more cells form typical syncytia ( , ) . additionally, at least hrv and rsv affect the barrier integrity of the airway epithelium by reducing the expression of epithelial junction proteins ( ) ( ) ( ) ( ) . it could be shown that hrv increases epithelial permeability by a reduction of occludin and zo- expression ( , ) . rsv also disrupts junctional complex structures by fostering the activity of protein kinase d (pkd) ( ). the antiviral immune response also includes the release of cytokines that directly affect epithelial barrier function as well. this is especially true for il- β, ifn-γ and tumor necrosis factor (tnf). these cytokines have been shown to support epithelial permeability and to decrease expression of claudins, jam, occludin, and zo- in several in vitro studies ( - ). in case of asthma, these effects are even more pronounced because of the allergic inflammatory response that already exists before the viral infection of the airway epithelium. hence, th type cytokines like il- and il- also increase barrier permeability by inhibiting the surface expression of β-catenin, e-cadherin, occludin, and zo- ( , ). in addition to cytokines, mast cell derived mediators also appear to have an effect on the barrier function of the airway mucosa. histamine for example has been shown to contribute to transient disruption of apical junctional complex integrity and thus to increase epithelial permeability in vitro ( ). allergens, viruses, and the inflammatory response to their exposure represent extrinsic factors that impair the barrier integrity of the airway epithelium. however, some studies suggest that epithelial cells of asthma patients inherently predispose for an increased permeability. as already mentioned above, airway epithelial cells that have been isolated from asthmatics and propagated in vitro to form an epithelial monolayer under ali culture conditions, display a decreased teer as compared to cells from healthy donors ( , ) . this observation indicates that the cellular properties leading to an increased barrier permeability are somehow imprinted within the cells. whether this is a matter of genetic predisposition encoded in epithelial stem cells or whether epithelial cells from asthma patients "remember" previous insults by epigenetic modifications that predispose for asthma development in later life remains elusive. besides the barrier function of the epithelium provided by the mere presence of the sealed cell layer itself, two additional barrier structures are "exported" onto the luminal surface by the airway epithelium, a static one dubbed glycocalyx or periciliary layer (pcl) and a mobile one termed mucus. the glycocalyx or pcl is a sponge/fleece-like, cell membraneanchored layer of glycolipids and glycoproteins -mainly mucins (see below) -that vertically stick out of the apical epithelial cell membrane. although mainly attributed to the gut epithelium where it can extend up to nm ( ) and to the vascular endothelium ( , ) a glycocalyx/pcl is also present throughout the airway epithelium even down to the alveoli ( ), again with heights up to nm in certain areas ( ) (figure ). this static glycoprotein and glycolipid coat stores water to control mucus hydration but also serves as a protective zone against the compression of the mucus lying above in order to allow persistent cilia beating for ongoing functionality of the mucociliary clearance (mcc; see below) ( , ). beyond that, the glycocalyx/pcl regulates receptor specificity by architectural means and prevents the progression of viruses through the occasionally patchy mucus layer. it has been shown that the height and density of the epithelial glycocalyx can determine whether a ligand-equipped nanoparticle may attach to its membrane receptor or not ( , ). in line with this, the inefficiency of adenovirus-mediated gene transfer into the airway epithelium was found to be caused by the membranetethered glycocalyx/pcl proteins that put a halt to the advance of the viral vectors ( , ). consequently, the susceptibility of the airway epithelium toward infection is at least to some extent controlled by the glycocalyx/pcl. very small viruses such as bocavirus (hbov ) and hrv, which are - nm in size ( , ) should readily advance through the pcl to the epithelial plasma membrane as has been observed with nanoparticles of the respective size ( ). consequently, those viruses should be able to luminally infect airway epithelial cells as long as their receptor is present on the apical side. little is known about receptor distribution in vivo but at least on cultured airway epithelial cells apical receptor expression and/or infectivity has been demonstrated for both viruses ( - ). larger particles of about nm and above, on the other hand, are efficiently blocked by the pcl ( ). hence, viruses such as rsv, hmpv, influenza and parainfluenza viruses, adenovirus or coronavirus, which are in this size range ( ) ( ) ( ) ( ) , should be hindered efficiently by the pcl to infect the host. yet, those viruses often are associated with respiratory infections and asthma exacerbations ( , ) . one possibility for them to infect the airway epithelium may be the presence of the viral receptor on structures that extend from the pcl such as the tips of the cilia. an example for this is chemokine receptor cx cr via which rsv can infect its host. in differentiated human airway epithelial cells this molecule is highly abundant on cilia ( , ) . another possibility is the preceding action of a door-opener such as hbov which may pave the way for further viral infections. hbov was shown to persist for several months in the human airway epithelium ( ) and causes pyroptotic cell death, epithelial cell hypertrophy, loss of cilia and disruption of the tight junction barrier ( , ). such a predamaged epithelial barrier may then readily fall victim to an influenza, parainfluenza or hpmv infection. with up to % of asthma exacerbations in small children found to be associated with hbov infection ( ) it may be worthwhile to further investigate possible coinfections with hbov in asthma exacerbation cases. in this context, it may also be of interest that the treatment of chronic inflammatory diseases of the airways such as asthma with corticosteroids (cs) seems to reduce the glycocalyx/pcl height on the alveolar epithelium ( ) thereby rendering the lung more susceptible to e.g., pneumocystis carinii infection. consequently, alleviating chronic inflammation in asthma with cs may make the patient more susceptible to certain infections, which in turn may enhance inflammation again, clearly a two-edged outcome of cs therapy in asthma. although the glycocalyx appears to be static on the architectural level, it may not be invariant in terms of its molecular composition. it was shown that lipopolysaccharide exposure could lead to heparan sulfate shedding from the airway epithelium thereby causing increased lung permeability ( ). allergen exposure of experimental animals resulted in different glycosylation patterns of the glycocalyx ( ), which may result in a deviant presentation of viral and bacterial receptors on the cell surface. in light of the above, the airway epithelial glycocalyx seems to play a so far underestimated but possibly important role in airway epithelial defense. whether or not the molecular composition figure | protection of epithelial surfaces by physical barriers. in the healthy state, ciliated cells (cc) form a tight epithelial layer where paracellular passage is prevented by sealing of lateral intercellular spaces with tight junctions (tj). the apical epithelial cell surface, including the cilia, is covered by a layer of membrane-anchored glycoproteins and glycolipids, the glycocalyx. the dense meshwork of glycostructures restricts access of luminal matter to the apical cell surface; depending on their size, larger pathogens can be cut off from their receptor if it is not present on cilia (inset). goblet cells (gc) secrete mucus, consisting of highly glycosylated mucins which absorb large quantities of water to form a viscous gel. the mucus -and any matter trapped within -is transported upward in the airway lumen by the coordinated beating of the cilia. bc, basal cells. in the asthmatic state, barrier functions can be compromised by partial disruption of tight junctions and gaps in the pcl/glycocalyx meshwork due to loss of cilia. mucus clearance is impeded by increased mucus viscosity and swelling of the gel matrix, and by disturbance of ciliar beating due to disorganization and dykinesia of cilia. of this static cell coat is different in asthma, remains to be investigated. while the role of the glycocalyx/pcl is still subject of debate, the importance of mucus in airway luminal defense is unchallenged. mucus is an unstirred discontinuous sheet of secreted mucous hydrogel which floats on top of the epithelium and is transported toward the oral cavity like the cargo on a conveyor due to the constant and coordinated beating of underlying ciliated cells ( ) . the mucus carried upward by this mucociliary clearance (mcc) mechanism can be swallowed or expectorated. as the mucus layer separates the airway lumen from the epithelium only objects that diffuse faster "vertically" toward the epithelial surface than the mucus is transported "horizontally" toward the oral cavity can reach the epithelial cell membranes. thus, only nanoscalar objects and smaller, like gases, water, salts and nutrients are able to reach the epithelial cells ( , ) . this way the mucus carpet provides a protective line of defense against pathogens, dust and other harmful objects that might be inhaled by an individual. in addition to that, the sticky texture of the mucus slows down airborne objects and further prevents their advance to the epithelial cell layer. in order to exert these functions properly, mucus requires a specific composition. mucus consists mainly of water, further components are salts, lipids and proteins. among those, antimicrobial proteins like lysozyme, immunoglobulins and antimicrobial peptides are major molecular scavengers distributed within the mucus layer ( ) ( ) ( ) . the characteristic viscous, elastic and sticky properties of the mucus are provided by a group of macromolecules named mucins ( , ) . to date, genes coding for mucins have been described ( ) . their protein products are secreted either to form mucus or remain immobile on the apical membranes of the airway epithelial cells where they become part of the glycocalyx/pcl. muc ac and muc b are the major secreted mucins in the airways. in addition, muc and muc are also part of airway mucus, albeit to a considerably smaller proportion, and thus belong to the family of secreted mucins ( ) . in contrast, muc , muc and muc are tethered to the cells of the airway epithelium (figure ) thereby contributing to the static luminal epithelial barrier, which resides underneath the mobile mucus layer ( , ) . the viscous and elastic properties of the mucous gel are primarily given by the secreted polymeric mucins muc ac and muc b ( ) . these mucins are highly o-glycosylated proteins enriched with amino acids like proline, serine or threonine ( ) . although both have a similar structure, muc b and muc ac differ in charge due to differential glycosylations ( ) . their production depends on cell type and site of production. in the upper airways, muc ac is produced by epithelial goblet cells while muc b is secreted from mucous cells in submucosal glands from secretory cells in the tracheal and bronchial epithelium. in the distal airways, muc b is also produced by secretory cells of the epithelium and seems to be the major mucin of this airway region ( , , ) . before secretion, polymeric proteins are stored in secretory granules in a compacted, dehydrated state. upon release, they switch to a hydrated form, which is necessary for the mucous gellike properties ( , ) . whether the two different mucins have different functions restricted to their site of production or whether the two mucins mingle to create a novel type of barrier structure is not clear yet. at least some studies analyzing airways of piglets have shown that muc b strands are becoming coated with muc ac to some extent after release at the epithelial surface ( , ) . a possible role of muc and muc has not been identified yet. howsoever, under healthy conditions, the viscous mucus traps noxious substances, which are then removed from the airway via cilial beating by the mcc ( ) . in asthma, the mcc is impaired leading to mucus plug formation which in turn results in the characteristic obstruction observed in asthmatics. this is already a feature of mild stable asthma and the dysfunction worsens during aggravation of asthma and in asthma exacerbations ( ) ( ) ( ) . one reason is an increased mucin content of the mucus thereby disturbing its regular composition. normally, the airways' mucus consists of ∼ % water and only ∼ % solid factors mainly mucins. in obstructive diseases, the amounts of mucins rise up to - % ( , ). due to its hygroscopic nature, this leads to acquisition of water from the underlying pcl/glycocalyx and shrinking of this static layer. the now protruding cilia either project into the mucus or get bend ( ). both effects impede passing on of the mucus to the next cell. loss and/or disorientation of cilia as it is typical for asthmatics will further disturb the "bucket chain"-like transport process ( ) . on the cargo side enhanced intermolecular crosslinking of mucus constituents by oxidative processes may further complicate forwarding. it will also increase mucus viscosity eventually leading to plug formation. oxidative intramolecular crosslinking of biomolecules is predominantly caused by cysteines whose thiol side chains can form disulfide bridges. all mucins are rich in cysteines, especially in their less glycosylated carboxy-and amino terminal regions. in the "normal" mucous gel of healthy individuals these cysteines are believed to be only moderately crosslinked, forming a lightly entangled network. in asthma, however, the degree of crosslinking and the density of the mucin network increases considerably ( , ) (figure ). increased oxidative stress appears to play an important role in this respect with eosinophils being the main suspects for oxidant production. the abnormally high levels of eosinophil peroxidase detected in the sputum of asthma patients may form an oxidative milieu. this would also bring the widely observed correlation between airway eosinophilia and airway obstruction into a causative relationship ( ) . lastly, the muc ac of asthmatics tends to tether to the epithelium, which also complicates mucus forwarding ( ) . in asthmatics not only the amount but also the composition of the mucus changes, especially the ratio of muc ac to muc b as well as the posttranslational modification of muc b. mucus from healthy individuals contains predominantly muc b, which is essential for the mcc and protection against pathogens ( ) ( ) ( ) . in asthma, the proportion of muc b relative to muc ac often decreased ( , ) along with the expression of a lowcharge form of muc b. consequently, there was a changed ratio between the two differently glycosylated forms of muc b ( , ) . the importance of muc b is indicated by muc bdeficient mice, which showed an accumulation of undesired substances e.g., bacteria, resulting in severe inflammation and airway obstruction ( ) . the ratio between muc b and muc ac changes dramatically in asthma because muc ac expression and protein production are substantially upregulated in asthmatic patients ( , , ) . especially patients with an eosinophilic type asthmatic phenotype showed a shifted ratio toward higher muc ac concentrations ( , ) . this is in line with the assumption that muc ac seems to be important for the defense against enteric nematodal and influenza infections ( , ) . the increased expression of muc ac seems to depend on substantially increased levels of il- . the il- signaling pathway activates the signal transducer and activator of transcription (stat ), which induces the expression of muc ac via various regulators ( ) and appears to be involved in ahr development ( , ) . several studies using in vitro systems with human epithelial cells or murine models validated this mechanism ( ) ( ) ( ) ( ) . furthermore, egfr, which is also overexpressed in asthma, also induces the expression of muc ac ( ) ( ) ( ) ( ) . this excessive production of mucins in the asthmatic airway epithelium leads to an increased volume of intracellular stored mucins, a mucus metaplasia ( ) . thus, a higher number of goblet cells compared to the healthy situation appears in case of asthma ( ) . it is not exactly understood, whether this switch from a muco-ciliary phenotype to a mucous metaplastic phenotype develops from goblet cell hyperplasia, metaplasia or both as reviewed before ( ) . in animal models, goblet cell metaplasia/hyperplasia arises from an increased expression of primarily il- , but also of il- and il- ( ) ( ) ( ) ( ) . these cytokines are highly upregulated in asthmatic individuals ( ) ( ) ( ) ( ) . one important factor for the development of the goblet cell metaplasia is notch regulated by il- ( ) . studies analyzing the function of sam-pointed domain containing ets transcription factor (spdef) highlighted its essential role in the development of goblet cell differentiation, hyperplasia and mucous metaplasia ( ) ( ) ( ) . therefore, spdef seems to inhibit the expression of forkhead box protein a (foxa ) which is an important negative regulator of genes associated with mucous metaplasia and goblet cell hyperplasia ( , , , , ) . thus, the physical barriers provided by the airway epithelial layer seem to be deeply disturbed in asthmatic individuals. although mucin is one of the most important barrier molecules in the airways its unbalanced overproduction is clearly detrimental to the desired outcome. mucus plugging impedes egress of the active luminal defense molecules necessary to eliminate invaders. although strong walls (tight junctionally sealed epithelial cell layer) surrounded by a glacis (pericilial layer/glycocalyx) and a moat filled with flowing liquid (mcc) are crucial to prevent invaders from entering a castle, active defenses are necessary to end the siege. this is of particular importance when the besieger can replicate and thus may increase continuously by number, as is the case when pathogenic bacteria colonize the luminal side of the airway epithelium. in order to get rid of a potential invader the airway epithelium possesses a battery of defense molecules, with which a potential microbial enemy can be attacked, destroyed or removed out of the airway lumen. prominent innate molecular scavengers are lysozyme, transferrin and antimicrobial peptides. lysozyme is produced in large amounts ( mg/day) by serous cells of the upper human airway epithelium ( ) and is able to destroy the polysaccharide capsules of many bacterial species. it has been shown that the production of lysozyme by serous cells residing in the serous glands of the upper airways is crucial for defending against bacterial airway invaders ( ) . once the polysaccharide capsule is destroyed or damaged, so called defensins or antimicrobial peptides may exert the lethal hit to the invader. defensins can form holes or pores into a bacterial cell membrane thereby killing a pathogen that aims to enter the body ( , , ) . in addition, lactoferrin is produced and secreted by serous cells ( ) , and transferrin is expressed by alveolar type i cells ( ) . these ferrins are iron-binding proteins, which deplete their environment from iron ions that are essential for the growth of a self-replicating organism ( , ) . consequently, the pathogen is starved out. besides this innate "rapid response team, " the polarized epithelium of the human airways is also able to transport and apically release immunoglobulins that carry a j-chain (joining chain) by using its poly ig receptor (pigr) ( ) ( ) ( ) that is expressed by all non-stratified epithelial cells (figure ) . only igm and multimeric iga are equipped with j-chains ( , ) . these two immunoglobulin classes not only circulate in the bloodstream but are also produced directly underneath the airway epithelium by b cells, given those lymphocytes express the j-chain ( , ) . functionally, igm can substitute for multimeric iga. for that reason iga-deficient individuals do not show a strong phenotype concerning susceptibility to infection. nevertheless, secreted iga (siga) outperforms igm in terms of mucosal protection as it usually displays a higher affinity toward its antigen and, more importantly, is able to crosslink with mucins upon target binding ( , ) . this way an incoming viral or bacterial pathogen becomes trapped in mucus and is removed from the airway surface via the mcc. the protective function of secreted iga has been demonstrated with various model systems, both for the gastrointestinal mucosa as well as for the airways, using passively administered monoclonal iga ( ) ( ) ( ) ( ) , injected hybridoma cells whose target specific, dimeric igas are then transported across the mucosae ("backpack tumor model") ( ) ( ) ( ) and by neutralization of preexisting mucosal iga immunity with mucosally administered anti-iga immunoglobulins ( ) . although adaptive multivalent target binding via its hypervariable regions is probably the main mode of protection in those models, siga is also able to bind in an innate manner to luminal pathogens via its carbohydrate components by presenting decoy structures that mimic target cell surface receptors ( ) . if both modes of repelling fail and a pathogen has nonetheless invaded an epithelial cell, dimeric iga may still be able to protect the infected cell, this time from inside. this is possible whenever the respective pathogen does not directly infect the cytosol of its target cell or inject its nucleic acids directly into the cytosol but rather uses an initial endocytosis step for infection. depending on the infected organelle, vesicles, which concurrently translocate iga toward the apical site, may fuse with the infected organelle, bind to the invader and carry it away into the lumen. in addition to this removal activity, mucus crosslinking and the tricking of pathogens by offering decoy receptors, siga also scavenges il- and thereby inhibits il- -driven neutrophil chemotaxis ( ) . in addition to these molecular interactions with a pathogenic target, iga also binds to numerous cell types that patrol at the airway epithelium. the most important cellular partner seems to be the eosinophil as this cell possesses a total of five different receptors for iga: fcαri (cd ), transferrin receptor (tfr) (cd ), pigr, asialoglycoprotein receptor (asgpr) and a receptor for secretory component (scr) with the integrin mac- (cd b/cd ) serving as a coreceptor for fcαri ( , ) . depending on the receptor addressed and the form of iga offered, i.e., soluble versus target-bound and cross-linked, eosinophils are either calmed down or activated ( ) ( ) ( ) . yet, eosinophils are not only manipulated by iga, they also influence iga production themselves ( , ) . thus, immunoglobulin a and eosinophils share a really intimate relationship. equipped with less receptors but still responsive to iga are neutrophils, dendritic cells, macrophages, basophils and even epithelial lining cells that express the transferrin receptor such as alveolar-type cells ( ) . an additional, so far unidentified receptor is present on m cells (microfold cells) ( , ) . m cells are a specialized epithelial lining cell type that is responsible for antigen sampling at mucosal surfaces and predominantly occurs in the epithelium above organized mucosa-associated lymphoid tissue ( , ) . this receptor senses the distance between two heavy chain domains in iga. thus, it is not able to bind iga , an iga subclass present only in primates. iga is different from iga in that it contains a mucin-like, highly glycosylated extension of its hinge region. it is believed that this hinge region also serves as a ligand for yet other iga receptors ( ) . if this holds true, subclass switching may be another adjusting wheel for iga function. the class switch from iga to iga depends on the presence of the cytokines april and baff which were shown to be produced by the epithelial layer itself, at least in case of the gut, upon bacterial stimulation ( ) . this way the microbiome as sparring partner of the epithelium comes into play as the true master of this adjusting wheel. in contrast to the gut where iga prevails, iga is the predominant iga subclass in the airways ( ) . this, however, does not imply that iga is of less importance for airway defense. iga simply may be the first class formed after pathogen challenge. those initial secretory iga (siga) responses are believed to be not very mature. upon pathogen challenge the human body apparently rapidly switches its current igm repertoire to iga even if most of such "first line of defense" iga are of low affinity ( ) . this can be regarded as just a "better than nothing" attempt; yet it creates a window of opportunity for the body to develop more powerful siga via affinity maturation. such optimized immunological scavengers are then able to block and eventually clear a microbial infection. thus, the iga system in which the transporting epithelium plays a key role is a complex defense machinery that combines innate with adaptive immune responses. it is therefore not surprising that the role of secretory iga attracted attention in asthma research in recent years. the role of siga in chronic inflammatory lung diseases is still ambiguous. some studies show that siga is necessary to maintain immune homeostasis, other reports claim that siga may play a detrimental role in asthma. a harmful effect of iga in asthma may be explained by its ability to activate eosinophils and neutrophils because both cell types play a central role in the pathogenesis and persistence of asthma ( ) . when iga is able to activate those cell types, this would readily lead to the hypothesis that in case of allergic asthma, allergen-specific iga is responsible for this activation upon allergen exposure. this assumption is supported by the finding that increased levels of both, allergen-specific ige and iga were observed in the airway mucosa of patients with atopic asthma and/or rhinitis ( ) ( ) ( ) ( ) ( ) , and it was shown that allergen-specific iga levels were positively correlated to eosinophil activation marker release after segmental lung challenge of asthmatic patients ( ) . yet, coincidence and correlation do not necessarily imply a causative relationship. in the abovementioned study, where a positive correlation of allergen-specific iga and eosinophil activation was observed, the non-allergic control patients also displayed allergen-specific iga in their airways; but in contrast, they did not have any allergen-specific ige as was the case for asthmatics. either so the allergen-specific ige was responsible for eosinophil activation in asthmatics or the eosinophils of asthmatics underwent some kind of imprinting or immune training that rendered them more sensitive to allergen-specific iga. with the expression of five different iga receptors on the eosinophil described so far ( ) , locked-in differences in iga receptor expression in eosinophils of asthmatics versus healthy individuals are not impossible. on the other hand, a coincidence of allergen-specific iga and ige does not necessarily imply a pathological role of iga either. it may still be the case that iga are beneficial to chronic airway inflammation, and the concomitant production of allergenspecific iga can also be interpreted as a rescue attempt of the body to counteract the allergen-specific ige. in fact, more evidence points toward a beneficial role of iga in asthma and other chronic airway inflammations. it was shown for instance that upon aging iga knockout mice tend to develop chronic airway inflammation that resembles chronic obstructive pulmonary disease (copd) in humans ( ) . a copd-like phenotype also develops in pigr knockout mice upon exogenous bacterial challenge ( ) , and it has been shown in the past that copd patients have an impaired pigr expression ( ) and reduced siga levels on the airway epithelium ( ) . recently a similar phenomenon was reported for asthma ( ) and rhinosinusitis ( ) . in the study of ladjemi et al., asthmatics show a reduced immunostaining of pigr in airway epithelia, with il- and il- being the suppressors of pigr formation in the airway epithelium. notably, there were no significant differences in the pigr gene expression rate among asthmatics and healthy individuals ( ) . thus, a posttranslational event such as proteolytic degradation of pigr in the epithelium may be responsible for the observed differences. a beneficial effect of allergen-specific iga in the airway lumen was highlighted by schwarze et al. ( ) . they showed that local application of a human monoclonal iga antibody directed against the ragweed allergen amb a attenuated the proinflammatory response to allergen inhalation in mice sensitized to amb a i, whereas a control iga against ovalbumin did not. notably, the instilled anti-ragweed iga induced the formation of amb a i-specific igg a in the animals upon allergen challenge which indicates a shift toward th . thus, iga residing in the airways may have an anti-allergic/anti-asthmatic immunomodulatory activity. this effect may be explained by the iga feedback loop, via which a secretory iga response is adjusted to current needs. in order to provide an optimal defense against luminal noxa luminal iga are continuously sampled at the epithelium and transported to the basolateral side, where it is inspected by immune cells whether it is loaded with antigen or not. if this is the case, an immune response is mounted or boosted ( ) . although this type of transcytotic event has been attributed primarily to m cells, the set-up of the ragweed-study rather precludes that route in this specific case in as much as a human iga against amb a i was used and this type of iga does not bind to murine m cells ( ) . however, with a plethora of iga receptors known, other epithelial cell types may have taken over the task. the iga-binding transferrin receptor, for instance, is expressed by type ii pneumocytes and was shown to transport transferrin conjugates to the basolateral site ( ) . in addition, similar to the gut, airway dendritic cells, which also carry iga receptors, send protrusions to the epithelial layer via which luminal antigen can be sampled ( , ) . yet, sampling antigen-loaded iga from the airway lumen and driving the airway immune response toward th requires the presence of iga in the lumen, which is reduced by the th micro-milieu in allergic asthma. this results in a vicious circle of a locked-in th environment where a lack of iga causes a further lack of iga. this is in line with clinical observations on asthmatic patients that suggests a critical role for iga in asthma pathogenesis. patients with selective iga deficiency tend to bronchial hyperresponsiveness ( ) and children that show a delay in maturation of iga production display atopic manifestations more often ( ) . moreover, immunotherapy against the respective aeroallergen result in higher specific mucosal iga levels along with lower skin prick test sensitivity ( ) or lower airway hyperreactivity ( ) . nevertheless, most of the above suggests a prominent role of siga or, more precisely, the lack thereof in the pathogenesis and chronicity of atopic asthma. whether a lack of siga also plays a prominent role in asthma exacerbations remains to be elucidated. in addition to all the homeostatic defense functions like the maintenance of barrier integrity, transcytosis, and the mucosal clearance the airway epithelium also plays a major role against inhaled materials by producing several defense proteins such as mucins, defensins, antimicrobial peptides, cytokines, and chemokines ( ) . thus, it contributes to local acute inflammatory reactions by regulating early inflammatory events via transcription and secretion of antimicrobial and pro-inflammatory proteins and by activating of mucin production ( , ) . consequently, it is also a critical player during sensitization processes, asthma pathogenesis and acute exacerbations of the established disease (figure ). inhaled pathogens that are not cleared by mcc are recognized by airway epithelial cells ( ) . equipped with a large number of prrs such as cytoplasmic nod like receptors (nlr) and transmembrane toll like receptors (tlr) that can respond to figure | inflammatory response of the airway epithelium during stable allergic asthma and exacerbation. during stable allergic asthma airway epithelial cells (aecs) release il- , il- and tslp supporting differentiation of t helper (th) cells that are activated by dendritic cells (dcs). th cells in turn secrete il- and gm-csf that together with aec-derived eotaxins, rantes and mcps regulate the production, maturation, recruitment and activation of eosinophils. local degranulation of eosinophils in the lung eventually leads to damage of the airway epithelium. in parallel, the th -type cytokines il- and il- induce goblet cell (gc) metaplasia in airway epithelium. during viral induced asthma exacerbations, several other additional factors lead to an aggravation of this inflammatory response. viral infection can be detected by the airway epithelium via pattern recognition receptors (prr). subsequently, aecs secrete on the one hand tarc, the main chemokine for the recruitment of th cells that amplifies the proinflammatory effects of th cells via release of il- , and on the other hand il- , which leads to the recruitment of neutrophils. local degranulation of neutrophils in the lung eventually leads to additional damage of the airway epithelium. viral infection of aecs also directly leads to damage of the airway epithelium. in summary, these conditions result in a markedly increased damage of the airway epithelium compared to the stable disease, which further impairs barrier integrity and leads to release of matrikines further amplifying the ongoing inflammation. damps and pamps aecs represent the first line of cells, which can respond to pathogens and other danger signals like cell stress and cell death in the lung ( ) . damps are molecules that are release from injured cells. their presence is a clear sign for the loss of homeostatic integrity of specific cell compartments or even whole cells. hence, they originate from the cytoplasm (s proteins, heat shock proteins, defensins, galectins, uric acid), the nucleus (high-mobility-group-protein (hmgb )), the endoplasmic reticulum (calreticulin), mitochondria (atp, mitochondrial dna, n-formylated peptides) or from the extracellular matrix (fibronectin, hyaluronan, versican) ( ) . airway epithelial cells are both, responder to and producer of damps ( , ) . the dna binding protein hmgb for example, set free during necrosis of one cell can be detected from nearby cells by binding to their receptor age (rage), which leads to activation of nuclear factor kappa b (nfκb) and thereby to the production of pro-inflammatory mediators and consequently the recruitment of immune cells. in turn, pamps are preserved molecules and structures from pathogens and toxins. they can originate from such different sources as bacteria, mycobacteria, viruses, fungi and parasites ( ) . pamps and damps activate signaling pathways resulting in the transcription and production of cytokines and chemokines. in brief, signal transduction through myd and myd independent mechanisms leads to the activation of nfκb, mitogen-activated protein (map) kinases, and interferon regulatory factor (irf) ( ) . based on the nature of the triggering pamps and damps and a possible preexisting inflammatory environment in the lung its signals can result in protective effects or pathological effects for the host organism ( ) . repeated cell stress and exposure to pathogens trigger chronic activation of prr pathways in airway epithelial cells that are highly active and play an important role in chronic airways diseases ( ) . activation of prrs by damps leads to a massive secretion of proinflammatory mediators like il- , cxcl , tnf that consequently entail infiltration of activated immune cells. some of these cells like dendritic cells (dc), lymphocytes and mast cells are also involved in the pathogenesis of asthma ( , , ) . after contact for example with hdm extracts, representing a major source of asthma associated allergens, tlr dependent activation of nfκb and protease induced injuries in airway epithelial cells lead to secretion of chemokines and cytokines like thymic stromal lymphopoietin (tslp), gm-csf, il- , and il- ( ) ( ) ( ) ( ) ( ) . this results in the activation and infiltration of dcs, innate lymphoid cells type (ilc ) and th cells ( ) ( ) ( ) . during infection with bacterial pathogens airway epithelial cells can sense bacterial cell wall components via tlr (recognizing e.g., lta), tlr (recognizing e.g., lps), nucleotidebinding oligomerization domain-containing protein (nod ) and nod (recognizing peptidoglycans) leading to activation of nfκb and subsequent immune responses and consequently to regulation of bacterial clearance ( , ) . nucleic acid patterns arising during viral infection can be sensed via tlr , tlr / , retinoic acid inducible gene i (rig- ), and melanoma differentiation-associated protein (mda- ) ( ) ( ) ( ) ( ) ( ) . in response to tlr activation airway epithelial cells can also produce antimicrobial peptides such as human β-defensin (hbd- ) after tlr activation ( , ) . the signals of different prrs like tlrs, nlrs, and rage cooperate to regulate cellular immune responses to cell stress, infection and inflammation, which can amplify or dampen their effects ( ) . airway epithelial cells are very potent producers of cytokines and chemokines. the presence of aggressors like toxins and pathogens leads to production and fast and early secretion of il- β, il- , tnf, cxcl , ccl , and ccl ( , ( ) ( ) ( ) . thereby, airway epithelial cells regulate and orchestrate local immunity by interacting with the recruitment of dcs, t-cells, and b-cells (ccl ), eosinophils (ccl ), and neutrophils (cxcl ). during viral infections they constitutively produce ifnβ to reduce viral replication and to support epithelial apoptosis ( ) . thereby, airway epithelial cells represent the frontline of antiviral defense mechanisms. as mentioned earlier, increased concentrations of proinflammatory cytokines like il- β, il- , il- , and tnf can directly lead to damage of the barrier function of the airway epithelium ( - , , ) . in allergic asthma airway epithelial cells are one of the main producers of proinflammatory cytokines and chemokines like il- , il- , tslp, ccl (rantes), ccl (mcp- ), ccl (tarc), ccl , and several eotaxins. all of these cytokines strongly direct or support the development of a th polarized inflammation ( ) . the chemokines ccl and ccl play a prominent role in the recruitment of th cells by binding to the ccr receptor, since activation of it is a key event for th cell specific chemoattraction ( , ) . as a highly potent producer of tslp the airway epithelium can create a local micro-milieu that supports and maintains a th polarized inflammation ( , ) . in response to different epithelial injuries, airway epithelial cells secrete so-called alarmins like tslp, il- , and il- that direct t helper cell differentiation toward an th phenotype ( ) . additionally, secreted gm-csf from airway epithelial cells leads to maturation and survival of eosinophils ( ) ( ) ( ) . both effects are supporting allergic inflammation in asthma. taken together the airway epithelium plays a major role for the recognition of pamps and damps in the lung. binding of these molecules to their respective receptors enables the airway epithelium to regulate pathways important for barrier function, mcc and local immune responses. functional disorders of the airway epithelium in the ability to answer the presence of pamps and damps favor the development of chronic airways diseases. viral infections and exposure to bacteria in early life modulate the acquisition of th and th immunity during further development and influence the responses to following exposures. these effects could play an even greater role in patients with asthma since they show disrupted mcc that could amplify the disease morbidity ( , ) . a cytokine induced th polarization of the epithelium in combination with a barrier dysfunction induced by the same cytokines augments barrier impairment, further infiltration of proinflammatory cells, and enhanced penetration of inhaled allergens, which can be described as a self-reinforcing mechanism that predisposes for the development and perpetuation of allergic asthma ( ) ( ) ( ) . consequently, it has been suggested that an abnormal programming of the airway epithelium in general paired with an impaired capability to produce anti-inflammatory mediators such as il- or α melanocyte stimulating hormone (α-msh) may be the origin of chronic inflammatory airway diseases ( , ) . viral infections of the airways is of critical importance for the pathogenesis of allergic bronchial asthma: on the one hand recurrent respiratory viral infections during early childhood represent one of the strongest factors increasing the risk for the development of asthma in later life ( ) ( ) ( ) ( ) ( ) . on the other hand such infections are by far the most common cause for acute exacerbation of already established asthma leading to acute aggravation of disease symptoms and necessitating increased medication, gp visits, and can lead to hospitalization and critical care measures under certain conditions ( ) . indeed, the airway epithelium is in the center of action during such an exacerbation, since it is not only the barrier that first comes into contact with viral pathogens, but its cells are also the target for their infection and the site of their replication. thus, viral infection of the airway epithelium does not only impair the barrier integrity as already mentioned before, but also triggers the release of chemokines, cytokines, alarmins, and matrikines of the epithelial layer that affect the pre-existing inflammatory response in the asthmatic airway, which largely contributes to the formation of an acute exacerbation. the viruses that have been implicated in asthma pathogenesis and especially the formation of acute exacerbation are hrv, rsv, influenza and parainfluenza viruses, human metapneumovirus, corona and adenoviruses, however, hrv infections appear to be the most common cause ( , ) . hrv is a non-enveloped, icosahedral virus, which belongs to the family of picornaviridae (genus enterovirus) and is subdivided into three clades (a, b, and c). the single-stranded positive rna genome of hrv is constituted of ca. nucleotides ( , ) . clades a and b, which comprise the most common serotypes, are further subdivided into a major and minor group. the major group utilizes the intracellular adhesion molecule- (icam- ) to bind to and transfect the host cell ( ) ( ) ( ) . the minor group hrv bind to the low density lipoprotein (ldl) receptor ( ) ( ) ( ) ( ) and are considered to be more infectious. clade c consists of serotypes ( ) , which all bind to the cadherin-related family member (cdhr ). all species of hrv have been shown to infect and replicate in airway epithelial cells ( ) . viral engagement with the specific receptor leads to transfection of the host cell (e.g., bronchial epithelial cells) and subsequently to a multitude of cellular responses. it is this cellular response that is believed to facilitate acute asthma exacerbation. the cellular response to the virus is initiated by the detection of single-stranded rna via tlrs - , - , and - , mad , and rig-i ( , ) . activation of these receptors ultimately leads to the secretion of cytokines such as il- , il- , il- , il- , chemokines like cxcl , ccl (eotaxin), ccl and anti-viral interferons of type i and iii ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the interferons have not only innate but also adaptive immune-system functionality to keep the viral infection locally at bay by mobilizing the adaptive immune response for effective viral clearance ( ) . furthermore, proinflammatory cytokines like il- β and il- are not specific for special types of immune response and thus, not only support the immune response against the invading virus but also promote the allergic immune response already established in the airways. consequently, augmentation of the allergic immune response results in acute amplification of tissue damage, mucus production, and mediator release, and therefore in acute symptom aggravation. especially, the interferon response is thought to be an early post-infection event. in asthmatics epithelial interferon responses are believed to be hampered and as a consequence antiviral responses lack sufficient clearance ( ) . not only interferons seem to be differently expressed in epithelial cells from asthmatics but also tslp, which promotes th responses ( ) . another critical cytokine elevated in humans after hrv infections is il- , which also augments th cell development ( ) . it is of note that viral infections not only initiate an immune response but also drive remodeling of the epithelial barrier and the subepithelial extracellular matrix (ecm). hence, hrv induces perlecan, collagen v, tenascin c and matrix-associated (ma-) vegf expression in an either tlr- or tlr- /- associated manner in vitro ( , ) . elevated expression of ma-vegf and tenascin c was replicated in a mouse model of hrv infection, in which also collagen i and fibronectin was found to be increased ( , ) . in addition, our group found human nasal epithelial cells infected with rv- in vitro to significantly downregulated genes associated with ecm receptor interaction and focal adhesion ( ) . after infection and viral replication, the release of a vast array of mediators is among the earliest responses. in tissue culture a pneumocyte cell line expresses large amounts of il- and ccl readily after h post-infection ( ) . also, the interferon response is thought to be an early post-infection event. in asthmatics epithelial interferon responses have been suggested to be hampered and as a consequence antiviral responses lack sufficient clearance ( ) . but not only interferons seem to be differently expressed in epithelial cells from asthmatics but also tslp, which promotes th responses ( ) . another critical cytokine elevated in humans after hrv infections is il- . in line with that, jackson et al. impressively showed, that the release of il- by bronchial epithelial cells induces il- , il- , il- , and gata expression in th cells. an effect, which could be entirely blocked by an antibody against the il- receptor ( ) . recently, active fragments from epithelial deposited ecm molecules have gained some recognition in asthma and asthma exacerbation. matrikines are a class of molecules derived from ecm proteins (e.g., via proteolysis) with different properties from the parent molecule ( ) ( ) ( ) . in , burgess et al. reported diminished levels of the collagen iv isoform alpha (col a ) in airways from asthmatic subjects. the non-collagenous domain of col a is referred to as tumstatin and a biologically active matrikine. treatment of mice with experimental allergic asthma with human recombinant tumstatin let to a significant reduction of hallmark disease features (e.g., airway hyperresponsiveness, ma-vegf, eosinophil influx, il ) ( ) . in another study, van der velden et al. identified an anti-angiogenic effect of tumstatin in a sheep model of asthma ( ) . further investigations revealed a novel active region in tumstatin (cp ), which significantly reduced neutrophil influx, mucus production in a mouse model of viral asthma exacerbation and reduced migrational speed and production of reactive oxygen species of neutrophils in vitro ( , ) . while the matrikine tumstatin conveys protection from experimental features of asthma and asthma exacerbation, a collagen i derived matrikine (pgp, acetylated-(ac) pgp) has been shown to be a more potent inducer of neutrophil chemotaxis then il- and is found to be increased in severe asthmatics, a group of patients prone to develop exacerbations ( , ) . albeit ecm derived matrikines follow a different kinetical pathway (deposited first, released during inflammation) as compared to cytokines and chemokines (de novo production after viral infection), they can serve as protective or aggravating factors in asthma exacerbations. in addition, the notion of epigenetic modification due to hrv infection in epithelial cells in asthma has gained attraction. mcerlean et al. infected nasal epithelial cells from asthmatics and found evidence of reproducible changes to the methylome ( ) . we confirmed these results, which may unriddle how asthmatic airway epithelial cells may be able to respond differently to the same stimuli as compared to healthy epithelial cells ( ) . first studies to investigate this effect in vivo are underway ( , ) . infection associated stimuli appear not to be the only factors that imprint mucosal immune reactions of the airway epithelium. in addition, the interaction between epithelial cells and leukocytes can lead to sustained alteration of respiratory epithelial cell biology. even though these cells are definitely not able to constitute an immunological memory, it becomes more and more obvious that especially epithelial cells somehow memorize their exposure to certain environmental factors and the following insults and thereby develop some kind of trained immunity. we are just at the beginning to understand these processes and questions of which parts of the epithelium are trained and of how long the training effects sustain in the mucosa remain to be answered. thus, respiratory epithelial cells are constantly exposed to many types of challenges, including pathogens, allergens and environmental pollutants. consequently, they are able to respond quickly and effectively to cellular damage such as the local cytokine production, lateral transport by ion exchanges, wide arrays of mucus compositions, secretion of antimicrobial peptides, and epithelial shedding. to date, it appears possible that different inflammatory environments as originated by for example typical th -or th -directed immune responses have a different impact on the biology of the respiratory epithelium and lead to some kind of e -or e -polarization of the respective epithelial cells ( ) (figure ) . there is in vivo evidence for the inhibitory role of ifnγ on asthma pathogenesis at the epithelial level indicating that type- responses counteract allergy ( , ) . the direct implication of airway epithelial cells was demonstrated by selective transgenic expression of the ifn-γ receptor on the airway epithelium and showed that ifn-γ inhibits mucus secretion, release of chitinases and eosinophilia independent of the activation of th cells ( ) . in turn, gata- inhibition causes an increase of t-bet und ifn-γ expression levels, leading to a dampened allergic phenotype ( ) . in addition, an increase in dna-methylation of ifn-γ was observed during allergic sensitization ( ) , while perinatal prevention of allergy mediated by acinetobacter does not show the anticipated drop in h acetylation in the ifn-γ promoter ( ) . the immunological consequence of epithelial differentiation becomes increasingly interesting, as sensitization, but also recovery processes and airway remodeling could open new options for intervention and prevention of lung damage. increasing evidence of mechanisms involving epithelial cytokine production such as ccl- , and the epithelium-derived alarmins tslp and il- are substantiating the current focus on the cross talk between airway epithelium and immune cells in allergy research. the role of tissue cells in the early phase of disease is largely unknown, but could provide important information about the pathologic development and could help to identify the causal relationships. however, bronchial epithelial cells are pre-committed to a type- (e ) or type- (e ) like phenotype. e epithelial cell activation by allergens takes place and their pro-inflammatory cytokines and chemokines induce inflammation and contribute to an epithelial type- response, so called "e response" with epithelial alarmins tslp, il- , ccl- , il- , and il- . local type- responses involve multiple cytokines such as il- , il- , il- , il- , il- , il- , and increase of eosinophils. a series of chemokines are produced and migration of inflammatory cells to the allergic tissues takes place. the activation of e.g., smooth muscle cells by adam lead to remodeling. bronchial hyperreactivity takes place leading to an enhanced susceptibility to bronchoconstriction. e epithelial cells respond to an infection releasing cxcl , cxcl , il and ccl , thus stimulating the local synthesis of ifn-γ, il- , il- , il- , il- , and tnf-α that present a wide range of antiviral activities, inducing up-regulation of mhc-i molecules and antiviral resistance in uninfected cells. neutrophils respond to the infection signals il- and ifn-γ by releasing pro-inflammatory cytokines, leading to the containment of the infection, rise of body temperature and to the recruitment of further phagocytic cells. however, it is unknown whether epithelial cells are influenced by il- prior or with entry into terminal differentiation. this early influence could imprint the offspring cells that populate the epithelial surface and therefore have major consequences for the physiology of the airways. il- was shown to have a major effect on the epithelium, as mice overexpressing this cytokine under the club cell-secretory protein (cc ) promoter show increased cellular infiltration, epithelial hypertrophy, mucus cell hyperplasia, secretion of gastric mucins and surfactant proteins (sp) a and b ( ) . while this model effectively demonstrated all hallmarks of experimental allergic asthma, it did not demonstrate whether il- itself is inducing differentiation of basal cells or whether secondary effects trigger epithelial differentiation. however, the differentiation effects could also be observed in human primary epithelial cells of the nose, where il- modulates the differentiation toward less ciliated and more secretory cells ( ) . to date, it is controversially discussed, whether il- and il- can also affect fully differentiated epithelial cells in air liquid-interphase cultures or whether this is only possible in immature submerged cultures ( , ) . however, during the epithelial differentiation process induced by air-liquid exposure, the addition of il- enhances expression of certain antimicrobial peptides ( ) and eicosanoids ( ) . furthermore, it was demonstrated that il- and il- , through inhibition of tlr expression and signaling (irf ), impair immune responses to hrv infection ( ) . this is in line with the finding that chronic house dust mite exposure in the airways not only causes a strong th -directed inflammation but also diminishes anti-rhinovirus responses and local ifn expression, particularly of epithelial ifn-λ ( ) . in line with this, transgenic il- expression in the lungs reduces cytotoxic t cell responses against influenza viruses ( ) . on the level of secreted factors, it was shown that cytokines such as wnt a (wingless-type mmtv integration site family, member a) or il- are expressed as response to il- stimulation only, while proteins with known pathological roles such as the il- induced protein ccl- or periostin were shown to be upregulated by il- and down-regulated in ifn-γ environment. these results were consistent when comparing upper and lower airway secretions, thus confirming nasal lining fluids as a proxy for the lower respiratory tract, particularly for epithelial type- biomarker like ccl- and il ( , ) . the e -related transcription factor network contained the e hub-transcription factors gata , nfe , meis , hey , and ahr: gata is well known as the master transcription factor of type- response in immune cells, however it was also shown to be expressed in airway epithelial cells. nfe was demonstrated to have a cytoprotective activity against epithelial cell injury by cigarette smoke, which could hint on a protective role in an il- dominated micromilieu ( ) . for meis , it has been demonstrated that its inactivation produces an increase in airway smooth muscle mass and a corresponding decrease in cartilage and suggesting an important role in allergic airway diseases. a loss of hox gene function, however, does not preclude airway repair, but regenerated epithelium displays goblet cell metaplasia and less scgb a -positive cells, demonstrating the essential role of hoxa for correct differentiation. this goblet cell metaplasia is further associated with increased notch signaling activity. consistent with these findings, expression levels of activated notch and the effector gene hey are in turn enhanced in patients with allergic disease ( , , ) . taken together, e polarization has a marked impact on the barrier and especially immune functions of the airway epithelium and at least supports impairment of the mcc and antiviral responses, both factors that are critical in asthma pathogenesis. in summary, the airway epithelium exerts a broad variety of immune functions that range from passive barrier over mcc, active production and transport of pathogen-neutralizing molecules to pathogen recognition and targeting as well as cytokine and chemokine release. the airway epithelium is usually the first tissue that is exposed to inhaled allergens, pathogens or pollutants. since it is able to react on this contact by inducing local inflammatory reactions, it is clearly a central part of the local immune response and bridges innate and adaptive immune functions against all types of harmful intruders entering the respiratory system. therefore, the airway epithelium is a key factor in asthma pathogenesis and plays a critical role in the development as well as in the progression and exacerbation of the disease: hence, a disturbed cellular barrier enables allergens to enter the body and to induce a sensitization reaction, which is widely regarded as the starting point of an asthma career. down the line, the protective mucus and pcl layers provided by the epithelium as physical barriers are compromised and the release of pathogen deterring molecules such as secretory iimmunoglobulins becomes impaired. as a consequence, this frontline toward invading pathogens can be breached and airway epithelial cells are infected and even destroyed by respiratory pathogens. 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regulates alveolarization and goblet cell hyperplasia distal airways are protected from goblet cell metaplasia by diminished expression of il- signalling components cftr and lysozyme secretion in human airway epithelial cells lysozyme secretion by submucosal glands protects the airway from bacterial infection antimicrobial peptides in lung inflammation defensins in lung host defense the serous cell alveolar type i cells protect rat lung epithelium from oxidative injury protein transport across the lung epithelial barrier transepithelial transport of immunoglobulins the j chain is essential for polymeric ig receptor-mediated epithelial transport of iga the role of the polymeric immunoglobulin receptor and secretory immunoglobulins during mucosal infection and immunity immunoglobulin m and secretory immunoglobulin a: presence of a common polypeptide chain different from light chains role of j chain in secretory immunoglobulin formation mucosal b cells: phenotypic characteristics, transcriptional regulation, and homing properties putting j chain back on the map: how might its expression define plasma cell development? siga binding to mucosal surfaces is mediated by mucin-mucin interactions secretory component: a new role in secretory iga-mediated immune exclusion in vivo intranasal monoclonal iga antibody to respiratory syncytial virus protects rhesus monkeys against upper and lower respiratory tract infection urease-specific monoclonal antibodies prevent helicobacter felis infection in mice intranasal monoclonal immunoglobulin a against respiratory syncytial virus protects against upper and lower respiratory tract infections in mice immunoglobulin a monoclonal antibodies protect against sendai virus new model for analysis of mucosal immunity: intestinal secretion of specific monoclonal immunoglobulin a from hybridoma tumors protects against vibrio cholerae infection monoclonal secretory immunoglobulin a protects mice against oral challenge with the invasive pathogen salmonella typhimurium analysis of the roles of antilipopolysaccharide and anti-cholera toxin immunoglobulin a (iga) antibodies in protection against vibrio cholerae and cholera toxin by use of monoclonal iga antibodies in vivo secretory iga n-and o-glycans provide a link between the innate and adaptive immune systems il-. released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component iga receptors in health and disease mac- (cd b/cd ) as accessory molecule for fc alpha r (cd ) binding of iga secretory iga induces antigenindependent eosinophil survival and cytokine production without inducing effector functions ragweed-specific antibodies in bronchoalveolar lavage fluids and serum before and after segmental lung challenge: ige and iga associated with eosinophil degranulation iga-induced eosinophil degranulation eosinophils promote generation and maintenance of immunoglobulin-a-expressing plasma cells and contribute to gut immune homeostasis il- β in eosinophil-mediated small intestinal homeostasis and iga production secretory iga accumulated in the airspaces of idiopathic pulmonary fibrosis and promoted vegf, tgf-β and il- production by a cells binding and transepithelial transport of immunoglobulins by intestinal m cells: demonstration using monoclonal iga antibodies against enteric viral proteins selective adherence of iga to murine peyer's patch m cells: evidence for a novel iga receptor antigen sampling across epithelial barriers and induction of mucosal immune responses molecular insights into the mechanisms of m-cell differentiation and transcytosis in the mucosa-associated lymphoid tissues intestinal bacteria trigger t cell-independent immunoglobulin a( ) class switching by inducing epithelial-cell secretion of the cytokine april cells containing iga subclasses in bronchi of subjects with and without chronic obstructive lung disease rat salivary gland reveals a more restricted iga repertoire than ileum eosinophilic and neutrophilic inflammation in asthma: insights from clinical studies antigen-specific ige and iga antibodies in bronchoalveolar lavage fluid are associated with stronger antigen-induced late phase reactions dissociation of allergen-specific ige and iga responses in sera and tears of pollen-allergic patients: a study performed with purified recombinant pollen allergens elevation of specific immunoglobulin a antibodies to both allergen and bacterial antigen in induced sputum from asthmatics specific iga and igg antibodies to house dust mite dermatophagoides farinae in nasal secretions ragweed-specific iga in nasal lavage fluid of ragweed-sensitive allergic rhinitis patients: increase during the pollen season airway bacteria drive a progressive copd-like phenotype in mice with polymeric immunoglobulin receptor deficiency bacterial-derived neutrophilic inflammation drives lung remodeling in a mouse model of chronic obstructive pulmonary disease bronchial secretory immunoglobulin a deficiency correlates with airway inflammation and progression of chronic obstructive pulmonary disease secretory iga deficiency in individual small airways is associated with persistent inflammation and remodeling bronchial epithelial iga secretion is impaired in asthma. role of il- /il- downregulation of polymeric immunoglobulin receptor and secretory iga antibodies in eosinophilic upper airway diseases antigen-specific immunoglobulin-a prevents increased airway responsiveness and lung eosinophilia after airway challenge in sensitized mice delivery of antigen to nasal-associated lymphoid tissue microfold cells through secretory iga targeting local dendritic cells confers protective immunity transcytosis of gcsf-transferrin across rat alveolar epithelial cell monolayers dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria accelerated antigen sampling and transport by airway mucosal dendritic cells following inhalation of a bacterial stimulus bronchial hyper-responsiveness in selective iga deficiency correlation between serum immunoglobulin a concentrations and allergic manifestations in infants sublingual immunotherapy alters salivary iga and systemic immune mediators in timothy allergic children production of salivary immunoglobulin a and suppression of dermatophagoides pteronyssinus-induced airway inflammation by local nasal immunotherapy chronic inflammatory airway diseases: the central role of the epithelium revisited innate immunity in the lung: how epithelial cells fight against respiratory pathogens innate immunity in the respiratory epithelium airway epithelial regulation of pulmonary immune homeostasis and inflammation the airway epithelium in asthma damps activating innate and adaptive immune responses in copd s a , s a and s a activate airway epithelial cells to produce muc ac via extracellular signal-regulated kinase and nuclear factor-κb pathways dangerassociated molecular patterns and danger signals in idiopathic pulmonary fibrosis toll-like receptors and their crosstalk with other innate receptors in infection and immunity type i interferon response to extracellular bacteria in the airway epithelium the danger within: endogenous danger signals, atopy and asthma innate and adaptive immune responses in asthma house dust mite allergen induces asthma via toll-like receptor triggering of airway structural cells transcription of interleukin- and extracellular release of the protein is regulated by allergen proteases in airway epithelial cells tlr signaling in stromal cells is critical for the initiation of allergic th responses to inhaled antigen interleukin- α controls allergic sensitization to inhaled house dust mite via the epithelial release of gm-csf and il- proteases induce production of thymic stromal lymphopoietin by airway epithelial cells through proteaseactivated receptor- allergens and the airway epithelium response: gateway to allergic sensitization commensal bacteria-derived signals regulate basophil hematopoiesis and allergic inflammation innate lymphoid cells: critical regulators of allergic inflammation and tissue repair in the lung klebsiella pneumoniae increases the levels of toll-like receptors and in human airway epithelial cells various human epithelial cells express functional toll-like receptors, nod and nod to produce antimicrobial peptides, but not proinflammatory cytokines role of double-stranded rna pattern recognition receptors in rhinovirus-induced airway epithelial cell responses dynamic cross talk model of the epithelial innate immune response to double-stranded rna stimulation: coordinated dynamics emerging from cell-level noise respiratory syncytial virus induces tlr protein and protein kinase r, leading to increased double-stranded rna responsiveness in airway epithelial cells cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells airway epithelial versus immune cell stat function for innate defense against respiratory viral infection activation of toll-like receptor on human tracheobronchial epithelial cells induces the antimicrobial peptide human beta defensin- rage and tlrs: relatives, friends or neighbours? bronchial epithelial cells: the key effector cells in the pathogenesis of chronic obstructive pulmonary disease? preferential recruitment of ccr -expressing th cells to inflamed joints via ccl in rheumatoid arthritis and its animal model the nucleotide-binding domain, leucine-rich repeat protein inflammasome/il- receptor i axis mediates innate, but not adaptive, immune responses after exposure to particulate matter under µm critical role of constitutive type i interferon response in bronchial epithelial cell to influenza infection chronic il and il- exposure leads to an altered differentiation of ciliated cells in a well-differentiated paediatric bronchial epithelial cell model cytokines induce tight junction disassembly in airway cells via an egfr-dependent mapk/erk / -pathway sensing the outside world: tslp regulates barrier immunity blockade of ccr in a humanized model of asthma reveals a critical role for dc-derived ccl and ccl in attracting th cells and inducing airway inflammation tlr -and th cytokinedependent production of thymic stromal lymphopoietin in human airway epithelial cells bronchial epithelium as a target for innovative treatments in asthma effect of interleukin- and granulocyte-macrophage colony stimulating factor on in vitro eosinophil function: comparison with airway eosinophils group innate lymphoid cells facilitate sensitization to local, but not systemic. th -inducing allergen exposures lung natural helper cells are a critical source of th cell-type cytokines in protease allergen-induced airway inflammation the lung tissue microbiome in chronic obstructive pulmonary disease microbes and mucosal immune responses in asthma epithelial barrier function: at the front line of asthma immunology and allergic airway inflammation role of viral respiratory infections in asthma and asthma exacerbations gabriela transregio study group. exposure to environmental microorganisms and childhood asthma requires il- rα and sigirr/il- r to diminish allergic airway inflammation in mice the alpha-melanocyte-stimulating hormone acts as a local immune homeostasis factor in experimental allergic asthma respiratory syncytial virus in early life and risk of wheeze and allergy by age years respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age rhinovirus illnesses during infancy predict subsequent childhood wheezing early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma wheezing rhinovirus illnesses in early life predict asthma development in high-risk children costs of asthma in the united states: - asthma exacerbations: origin, effect, and prevention the role of rhinovirus in asthma exacerbations sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution cdna cloning reveals that the major group rhinovirus receptor on hela cells is intercellular adhesion molecule the major human rhinovirus receptor is icam- evidence for the direct involvement of the rhinovirus canyon in receptor binding type rhinovirus infection of cultured human tracheal epithelial cells: role of ldl receptor similar frequency of rhinovirus-infectible cells in upper and lower airway epithelium structure of a human rhinovirus complexed with its receptor molecule the minor receptor group of human rhinovirus (hrv) includes hrv and hrv , but the presence of a lysine in the vp hi loop is not sufficient for receptor binding cadherin-related family member , a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication rhinoviruses infect the lower airways co-ordinated role of tlr , rig-i and mda in the innate response to rhinovirus in bronchial epithelium rhinovirus replication causes rantes production in primary bronchial epithelial cells interleukin- : stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness rhinovirus infection of primary cultures of human tracheal epithelium: role of icam- and il- beta role of p mitogen-activated protein kinase in rhinovirus-induced cytokine production by bronchial epithelial cells human rhinovirus recognition in non-immune cells is mediated by toll-like receptors and mda- , which trigger a synergetic pro-inflammatory immune response poly(inosinic-cytidylic) acid-triggered exacerbation of experimental asthma depends on il- a produced by nk cells airway epithelial orchestration of innate immune function in response to virus infection. a focus on asthma double-stranded rna induces disproportionate expression of thymic stromal lymphopoietin versus interferon-beta in bronchial epithelial cells from donors with asthma il- -dependent type inflammation during rhinovirusinduced asthma exacerbations in vivo rhinovirus infection induces extracellular matrix protein deposition in asthmatic and nonasthmatic airway smooth muscle cells airway epithelial cells generate pro-inflammatory tenascin-c and small extracellular vesicles in response to tlr stimuli and rhinovirus infection rhinovirus infections change dna methylation and mrna expression in children with asthma gene expression patterns induced at different stages of rhinovirus infection in human alveolar epithelial cells matrikines and the lungs an extracellular matrix fragment drives epithelial remodeling and airway hyperresponsiveness bioactive extracellular matrix fragments in lung health and disease reduction of tumstatin in asthmatic airways contributes to angiogenesis, inflammation, and hyperresponsiveness the effects of tumstatin on vascularity, airway inflammation and lung function in an experimental sheep model of chronic asthma lamstatin-a novel inhibitor of lymphangiogenesis derived from collagen iv tumstatin fragment selectively inhibits neutrophil infiltration in experimental asthma exacerbation novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation human rhinovirus infection causes different dna methylation changes in nasal epithelial cells from healthy and asthmatic subjects atopic asthma after rhinovirus-induced wheezing is associated with dna methylation change in the smad gene promoter dna methylation in nasal epithelium, atopy, and atopic asthma in children: a genome-wide study interleukin- and interferon-γ orchestrate an epithelial polarization in the airways helper cells and interferon gamma regulate allergic airway inflammation and mucus production allergen-specific th cells counteract efferent th cell-dependent bronchial hyperresponsiveness and eosinophilic inflammation partly via ifn-gamma ifn. -γ acts on the airway epithelium to inhibit local and systemic pathology in allergic airway disease effective prevention and therapy of experimental allergic asthma using a gata- -specific dnazyme dna methylation of th /th cytokine genes affects sensitization and progress of experimental asthma epigenetic regulation in murine offspring as a novel mechanism for transmaternal asthma protection induced by microbes interleukin- alters epithelial cell differentiation and surfactant homeostasis in the postnatal mouse lung epithelial differentiation is a determinant in the production of eotaxin- and - by bronchial epithelial cells in response to il- and il- differentiationdependent responsiveness of bronchial epithelial cells to il- / stimulation il- and il- exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides eicosanoid biosynthesis during mucociliary and mucous metaplastic differentiation of bronchial epithelial cells th cytokines impair innate immune responses to rhinovirus in respiratory epithelial cells no exacerbation but impaired anti-viral mechanisms in a rhinovirus-chronic allergic asthma mouse model local il- expression in the lung reduces pulmonary influenza-virus-specific secondary cytotoxic t cell responses biomatrix for upper and lower airway biomarkers in patients with allergic asthma early il- producing b-cells and coinciding th/tr shifts during three year grass-pollen ait the cytoprotective role of dj- and p nfe against human primary alveolar type ii cell injury and emphysema the loss of hoxa function promotes notch-dependent goblet cell metaplasia in lung airways current and future biomarkers in allergic asthma all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. this work was supported by the german center for lung reearch (dzl). key: cord- -i fjwzm authors: woodland, david l.; winslow, gary m. title: immunity to emerging pathogens date: - - journal: immunol rev doi: . /j. - x. . .x sha: doc_id: cord_uid: i fjwzm nan emerging infectious diseases constitute one of the most significant health and security challenges facing the world today. pathogens that are recently adapted to humans, such as human immunodeficiency virus (hiv), have become rampant worldwide, and pathogens that were thought to be under some control are creating new concerns, such as drug-resistant tuberculosis. furthermore, the threat of 'new' viruses, such as severe acute respiratory syndrome (sars) virus, and agents of bioterror, such as franciscella, present novel health challenges. many factors influence the emergence and spread of these pathogens including climate change, man's encroachment into new environments, patterns of travel and migration, and deliberate dissemination. thus, emerging infectious diseases will pose a major threat to human health well into the future. the challenge for mankind is to develop effective strategies to control these emerging diseases, including establishing surveillance and response networks, improving diagnostics, promoting public health initiatives, establishing vector control strategies, and developing effective vaccines and therapeutics. the battle against infectious disease has been ongoing since humans developed the capacity for medical intervention. the advent of vaccines and antibiotics was a huge step forward, and, based on early successes, it was expected that in time mankind could eventually conquer infectious diseases. however, in the first article in this volume ( ), prof. frank snowdon (yale university) offers a historical perspective of the fight against infectious diseases that highlights our hubris. today, we realize that the task is more difficult than was imagined. despite the many challenges that lie ahead, it is nonetheless essential that humans continue the fight to control major infectious disease killers in this century. indeed, several national and international organizations are dedicated to this task, including the world health organization (who), the centers for disease control (cdc), the national institutes of health (nih), the bill & melinda gates foundation, and others. it is an article of faith for research scientists that new advances will require a deep fundamental understanding of how the pathogen interacts with the host. the articles in this volume highlight recent and important advances in our quest for fundamental knowledge of the immunology of emerging infectious diseases. emerging pathogens have been defined as infections that have newly appeared in a population or have existed previously but are expanding in incidence or geographic range. both the cdc (http://www.cdc.gov/ncidod/diseases/eid/disease_sites.htm) and the nih (http://www .niaid.nih.gov/healthscience/ healthtopics/emerging/) have classified a number of microbes as emerging pathogens. these lists are overlapping and contain obvious candidates; however, each list also contains pathogens that are unique. because our focus for this volume of immunological reviews was immunity, we chose to include reviews of emerging pathogens where studies have led to novel insights into immune processes. we excluded some pathogens that have been highly studied, either because of their medical importance (e.g. hiv) or because they have been utilized in welldeveloped experimental models (e.g. listeria, borrelia). we also excluded pathogens for which relatively little is understood regarding the immune response (e.g. histoplasma, cryptosporidium, hanta virus, sars). the emerging pathogens that we chose come from a wide spectrum of infectious agents from several different phyla (i.e. viruses, bacteria, protozoa). these include pathogens that cause high disease incidence (mycobacteria, plasmodium, influenza), low disease incidence (franciscella, monkeypox, ebola), and that differ widely in host niche (i.e. intracellular pathogens such as yersinia, franciscella, and salmonella, versus extracellular pathogens such as group b streptococcus). the pathogens we have included also mediate a very wide range of infections, from acute (influenza) to chronic (hepatitis), to latent (tuberculosis). furthermore, our list includes pathogens that although highly virulent, are not considered to be major killers. for example, despite the fact that % of bubonic infections are lethal and only colony-forming units of franciscella are sufficient to kill an adult human, these pathogens cause few deaths worldwide. finally, several of the emerging pathogens included are important because they are major biothreats (franciscella, yersinia, vaccinia). our choice of pathogens and authors has therefore resulted in reviews that cover many different types of infectious disease and a breadth of immune responses. an important theme from this compendium is that immunity is pathogen-specific. each pathogen therefore informs us about a particular feature of the immune response and provides an opportunity to study particular aspects of immunity. thus, these articles explore the host response to infection, ranging from innate immune responses, to adaptive immunity, as well as the development of vaccines. innate immunity is an important component of anti-microbial immunity and associated pathology. an excellent example of an innate response to a pathogen is the development of an antiviral state. we now know that infected cells undergo multiple changes in response to a viral infection to block viral replication. we have also learned that viruses, in turn, go to considerable lengths to avoid or block these changes to ensure their survival. for example, type i interferons play a central role in establishing the anti-viral state. the underlying mechanisms of type i interferon function are discussed in detail by drs kate ryman and william klimstra (louisiana state university health sciences center) ( ) with regard to alphavirus infections. these authors focus on differences between particular viruses, as they relate to viral replication and host responses in dendritic cells and macrophages. these concepts are further illustrated by dr paul rota and colleagues (centers for disease control) ( ) with regard to paramyxoviruses and dr jacqueline katz and colleagues (centers for disease control) ( ) with respect to influenza. in these cases, the authors focus on viral interference with type i interferon signaling and induction pathways and on the regulation of inflammatory responses. the article by dr william golde and colleagues (plum island animal disease center) ( ) discusses the role of dendritic and natural killer cells in the control of foot and mouth disease virus in cloven-hoofed animals. these innate immune responses are essential to control the infection, and consequently, this highly successful virus has evolved mechanisms that interrupt these immune mediators to ensure rapid replication and spread. understanding the mechanisms at play will be important for the development of therapeutics against this economically important disease. the relationship between innate immune responses and viral success in the host population is also discussed by drs jessica weaver and stuart isaacs (university of pennsylvania school of medicine) ( ) with respect to monkeypox virus. the key question is what changes need to occur for the virus to establish uncontrolled infection in the human population. as the authors note, it is changes in genes encoding innate immune response proteins that appear to be critical, again highlighting the central role played by innate immunity in the host response to infection. toll-like receptors (tlrs) are well known to play major roles in the recognition of bacterial pathogens. although typically viewed as important components in protective immunity, innate tlr signals may be detrimental to the host. this is illustrated by dr philipp henneke and colleagues (university medical centre, freiburg) ( ), who discuss how excessive inflammatory signaling mediated by tlr during group b streptococcus infection causes excessive inflammatory signaling, which may have neurological sequelae in newborns. another example of a deleterious innate immune response is described in the review by dr henry tabel and colleagues (university of saskatchewan) ( ), who discuss dysregulation of macrophage function by cd t cells and regulatory t cells during trypanosome infections. dendritic cell and macrophage maturation via tlr-dependent and -independent mechanism in intestinal lymphoid-associated salmonella infections are discussed in the review by dr mary jo wick and colleagues (goteborg university) ( ) . adaptive immune responses are characterized by their ability to enhance the specificity, magnitude, and quality of the response as the infection progresses. such responses include the development of pathogen-specific antibodies and t cells. the interdependence of t cell and antibody-mediated immunity and the underlying relationship to innate immunity is outlined in the article on hantaviruses by dr rainer ulrich and colleagues (friedrich-loeffler institute) ( ) . these authors consider all aspects of the immune response, including immune evasion by the pathogen, immunopathology, and the role of the immune response in pathogen dissemination. the article by dr ralph baric and colleagues (university of north carolina) ( ) builds on some of these concepts to illustrate the interplay between the antibody response and the evolution of norovirus infections. in this case, the persistence of this virus is linked to its ability to evade antibody responses, while retaining the ability to bind to viral receptors in the host. the blockade of pathogen binding to the host receptor is another important component of antibodymediated pathogen neutralization. this is illustrated for west nile virus in an article by dr michael diamond and colleagues (washington university school of medicine) ( ) . here, beautiful structural and molecular studies identify the immunologic basis of antibody protection against west nile virus. in a related study, drs john fraser and thomas proft (university of auckland) ( ) describe the structural characteristics of a large family of staphylococcal and related bacterial superantigens and superantigen-like proteins that play important roles in human diseases. until relatively recently, antibodies have not been considered to play important roles in intracellular bacterial infections, but recent work suggest that antibodies are important components of host defense against such emerging pathogens. this is highlighted by dr dennis metzger and colleagues (albany medical college) ( ) and dr stephen smiley (trudeau institute) ( ) in their reviews that document roles for antibodies in studies of franciscella and yersinia infections, respectively. several of the reviews in this volume discuss the important role of t cells in viral, bacterial, and parasite emerging infections. for example, cd t cells play a major role in protective immunity during plasmodium liver stage infection, as described by dr fidel zavala and colleagues (johns hopkins university) ( ) in their review of malaria immunity. dr gary winslow and colleagues (wadsworth center) ( ) describe the genesis of the protective cd tcell response following mycobacterium tuberculosis infection; cd t-cell responses are delayed after m. tuberculosis infection, relative to many other respiratory pathogens, and this likely allows the pathogen time to gain a foothold before the development of adaptive immunity. while t cells can play a key role in protective immunity, they can also mediate considerable pathogenic effects. dr alan rothman and colleagues (university of massachusetts medical school) ( ) discuss the impact of t-cell responses on dengue virus infections. in this case, immune responses to secondary virus infection can result in enhanced disease and dengue hemorrhagic fever. this effect appears to be mediated by a skewed t-cell cytokine response that was primed by the initial, and largely asymptomatic, primary infection. an important goal of immunological studies is the development of effective and durable vaccines. while vaccines have considerable benefits over drug treatments, the major limitation in developing novel vaccination approaches is our lack of understanding of immunity to many emerging pathogens. furthermore, many emerging infections are prevalent in third-world countries, where it can be problematic to test new vaccines. one area where vaccines are being developed, without the constraints associated with clinical studies, is animal vaccines. for example, highly pathogenic influenza is a major economic concern for poultry production throughout the world. it is also classified as an emerging disease in poultry that has the potential to infect the human population. protection against disease in chickens is dependent on the development of neutralizing antibody. while homotypic vaccines are generally very effective, it is difficult to develop vaccines with broad heterosubtypic protection against drift variants. drs david swayne and darrel kapczynski (us department of agriculture) ( ) discuss the challenges in developing and delivering avian vaccines against avian influenza. the lessons learned from these studies have considerable implications for vaccines against human influenza, especially pandemic influenza. vaccination is a major goal of much of the research described in this volume, including the work described by drs golde, diamond, metzger, smiley, rothman, and swayne ( , , , , , ) . together these articles on various aspects of the immune response highlight what we feel are important areas of research in immunity to emerging infections. one 'emerging' theme that these reviews illustrate is not only the wide variety of lifestyles encompassed by emerging pathogens, but the wide variety of immunological defenses and pathological responses employed by the host to combat such a range of pathogens. research into this panoply of pathogens will continue to inform us of the plasticity of the immune response and the variety of different mechanisms that are utilized for host defense. emerging and reemerging diseases: a historical perspective host responses to alphavirus infection inhibition of interferon induction and signaling by paramyxoviruses pathogenesis of emerging avian influenza viruses in mammals and the host innate immune response immune evasion during foot-and-mouth disease virus infection of swine monkeypox virus and insights into its immunomodulatory proteins induction and termination of inflammatory signaling in group b streptococcal sepsis t cells and immunopathogenesis of experimental african trypanosomiasis early cellular responses to salmonella infection: dendritic cells, monocytes and more hantavirusinduced immunity in rodent reservoirs and humans norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations the structural immunology of antibody protection against west nile virus the bacterial superantigen and superantigen-like proteins humoral and cell-mediated immunity to the intracellular pathogen francisella tularensis immune defense against pneumonic plague protective cd t cells against plasmodium liver stages: immunobiology of an 'unnatural' immune response early t-cell responses in tuberculosis immunity understanding the contribution of cellular immunity to dengue disease pathogenesis strategies and challenges for eliciting immunity against avian influenza virus in birds key: cord- -ltkpt u authors: lee, kyu-myoung; jung, kyujin title: factors influencing the response to infectious diseases: focusing on the case of sars and mers in south korea date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: ltkpt u following the the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) outbreak in south korea, this research aims to explore and examine the factors influencing the response to infectious diseases, which encompasses both communicable and non-communicable diseases. through a qualitative research method, this research categorizes the factors as inputs, processes and outputs and applies them into the sars and mers outbreak in south korea. as the results conducted meta-analyses to comprehensively analyze the correlations of factors influencing disaster response from a korean context, the findings show that the legislative factor had direct and indirect influence on the overall process of infectious disease response and that leadership of the central government, establishment of an intergovernmental response system, the need for communication, information sharing and disclosure and onsite response were identified as key factors influencing effective infectious disease response. recently, a wide array of disasters, including earthquakes, forest fires, various infectious disease outbreaks and marine accidents have occurred in korea. accordingly, the importance of disaster response has drawn more attention than it ever has in korea, which was once considered a safe zone from various disasters. however, following the recent and sudden increase in the frequency of disasters, sufficient learning and legislature, as well as policy-making processes in korea have exposed limitations in the disaster management system, relative to other countries and regions where disasters have occurred more frequently. the recent efforts to change the disaster response system after certain disasters has exposed many limitations. under such circumstances, it is difficult to respond appropriately to disasters and there are limitations to avoiding or reducing social and economic losses caused by disasters. after the sinking of mv sewol, also called the sewol ferry disaster, in april , the korea coast guard was dissolved and the ministry of public safety and security (mpss) took on the role of a control tower. every episode of failed disaster response was always blamed on the lack of a control tower. therefore, the introduction of mpss was expected to be a panacea for effective disaster response. however, the initial response still failed during subsequent disasters, including in the middle east respiratory syndrome (mers) outbreak, the geyongju earthquake and various marine accidents, which confirmed that the disaster response was still not being effectively executed. when president the present study conducted an in-depth review of the concept of infectious disease and infectious disease response, which are the subjects of its theoretical review. in the past, the terms "contagious" or "communicable disease" were generally used. however, because the term "communicable diseases" implied diseases that were transmitted from one person to another, which further implied difficulties in controlling them, the term was changed to "infectious diseases," which encompasses both communicable and non-communicable diseases. for effective prevention and management of infectious diseases, the existing "parasitic disease prevention act" and "communicable disease prevention act" were merged. according to the "infectious disease control and prevention act," infectious diseases include class - of infectious diseases, designated infectious diseases, who-monitored infectious diseases, bioterror infectious diseases, sexually transmitted diseases, zoonotic infectious diseases and healthcare-associated infections. korea has experienced outbreaks of diseases that were traditionally regarded as "diseases that occur in developing countries," such as hepatitis a, tuberculosis, chicken pox and malaria, while cholera patients were identified for the first time in years in . moreover, despite continued outbreaks of emerging infectious diseases since the sars outbreak, there have been no noticeable changes in prevention and response measures. with the subsequent occurrence of the mers and zika virus in korea and the re-emergence of cholera, an infectious disease that had not been experienced for a while, public anxiety about health safety is growing. according to the infectious diseases surveillance yearbook published by the korea center for disease control and prevention (kcdc), the level of imported infectious diseases has been increasing every year, with - newly reported cases every year since . experts have warned that this is only the beginning of the war against infectious diseases, due to the following reasons. with the changing global environment and increased human migration, prevention of emergence or re-emergence of infectious diseases is fundamentally impossible. moreover, infectious diseases tend to evolve along with advances in medical technology and emerging infectious diseases are difficult to handle since they spread quickly and have no readily available treatment. in particular, knowledge about the characteristics, route of infection and control measures of emerging infectious diseases are lacking or uncertain and it is difficult to predict when, where and how these diseases may emerge. moreover, globalization has brought with it an increased level of international trade and travel. therefore, korea, which has a high foreign trade dependency, is constantly exposed to the risk of imported infectious diseases. despite the growing anxiety and concerns about infectious diseases, studies on infectious disease response and control are lacking. after the mers outbreak, numerous studies on the response to this outbreak were conducted . however, most of the studies focused mostly on medicine and communication, with relatively fewer studies focusing on administrative fields [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . studies in the medical field must precede the response to infectious diseases, so that information and knowledge about the infectious disease can be applied in response measures. however, if the national infectious disease response system is not ready when an actual infectious disease outbreak occurs, then medical determination and response, as well as crisis management and communication cannot be executed properly. this is because medical response, crisis management and communication are sub-elements in such a national-scale system. therefore, it is important to conduct studies on infectious diseases and responses in every specialty. however, there is also need for comprehensive discussions that include the establishment of laws; regulations; resources; information on infectious disease response from administrative and policy perspectives; information sharing system; and the establishment of an international cooperation system and national response system involving the central government, the regional government, private organizations and the public for effective response when an actual infectious disease outbreak occurs. in addition to infectious diseases being difficult to handle, the mers outbreak in also revealed that even if prevention and response measures are in place, a failed initial response can lead to an unanticipated increase in the rate and scope of infection transmission. moreover, disaster responses do not always pan out as planned and uncertainties and complexities that emerge after the disaster must be handled. therefore, it is necessary to identify government-level responses and make effort to improve the response system. however, previous studies on responses to infectious diseases are still lacking despite the importance of this issue. accordingly, the present study was conducted with the consciousness of the need to analyze response systems based on past response experiences in order to effectively respond to future infectious diseases, which are a threat. the present study analyzed two cases of infectious disease outbreaks based on the factors that influence response to disaster, as identified through existing studies and theories and aimed to derive factors that have a strong influence on the effectiveness of actual disaster response. in the following section, the factors that influence disaster response will be categorized from a system theory perspective to form a categorization framework. factors that influence disaster response have been identified through numerous studies over several years. most of the studies on disaster response analyzed actual cases by applying analytical tools based on major variables presented in existing studies and theories [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] or they analyzed the factors that influence disaster response by administering questionnaire surveys to members of agencies associated with disaster response [ , , ] . therefore, instead of comprehensively examining the factors influencing disaster response, these studies handled the subject at a macroscopic level, focusing on the major variables. the present study aimed to organize factors and variables that influence the entire process of disaster response from a comprehensive and systematic perspective and categorize these factors and variables based on a system theory perspective in order to present an analytical framework. this concept and context are similar to those of perry [ ] , who claimed that influencing factors of disaster response that are derived without classifying according to disaster types do not need different analytical frameworks since they can be described and analyzed according to the factors presented and they only differ in intensity according to the type of disaster. the present study reviewed existing studies, focusing on those with "disaster response" and "effective disaster response" as the outcome variables. the factors that influence disaster response can be broadly categorized into financial resources, human resources, physical resources, information, education and training, leadership, intergovernmental relationships, onsite response, information sharing, environmental context, characteristics of disaster and the legal/institutional environment. resources that influence disaster response can be categorized into financial, human and physical resources. financial resources include the government's budget for disaster response, funding to support processes involved in disaster response and support from the government or community [ ] [ ] [ ] [ ] . therefore, financial resources can be regarded as the disaster-related budget, the disaster management fund and financial support measures for processes entailed in korea's disaster response system. human resources included disaster response-related organizations and agencies, education and training of relevant organizations and the general public and utilization of specialists. existing studies have pointed out that the establishment of disaster response-related organizations or crisis management centers and the securement of specialists have a very significant influence on disaster response [ , , , , , ] . this is because identification of disaster response-related organizations and agencies must come first to allow effective communication about disaster response and secure accountability in disaster response [ ] . moreover, education and training for disaster response organizations and their members has been mentioned as an element that allows effective disaster response [ , ] . lastly, physical resources refer to securement of disaster management-related resources and establishment of disaster management facilities. as indicated in the study by lindell et al. [ ] , securement of disaster response and management resources within the organization allows timely and accurate disaster response, which was expressed as disaster response equipment in the study by jung [ ] . such physical resources can be viewed as disaster management resources and facilities and whether the expansion of negative-pressure units and emergency isolation units have occurred also influences the response to infectious diseases. other influencing factors besides resource-related factors include education and training. knowledge can be explained as a collection of disaster-related information and information sharing in advance, where information about different types of disasters must be collected before the occurrence of a disaster. factors associated with disaster information have a positive effect on disaster response, as indicated in the study by kim [ ] , which reported that when a disaster information system is established the recognition of the importance of information quality and higher information quality had a positive effect on achieving disaster management duties. next, leadership, intergovernmental relationships and communication, onsite response and information sharing have been identified as factors that influence disaster response. first, leadership in the context of this paper refers to leadership in the central government, which can be divided into leadership from the president and leadership from central organizations and agencies. the president's level of interest in disaster management and response and the governance style in running an organization, were analyzed as factors that have significant influence on disaster response [ ] . moreover, onsite leadership by the heads of organizations and agencies must be demonstrated in a timely manner, especially in initial disaster response, in order to prevent the disaster from spreading [ , , , , ] . second, intergovernmental relationships and communication are also factors that influence effective disaster response [ , , , , ] . in addition, network was also pointed out as a factor that influences disaster response. existing studies tended to use the concepts of network and inter-organizational cooperation without clearly differentiating them but both network and inter-organizational cooperation were analyzed as factors that positively influence disaster response [ , , ] . one of the reasons inter-organizational communication and coordination, network and cooperation have been identified as important influencing factors is that appropriate allocation and utilization of disaster response-related resources are essential for effective disaster response. accordingly, the present study analyzed intergovernmental relationships in order to comprehensively examine intergovernmental and inter-organizational relationships, communication and cooperation. information sharing has also been identified as an important factor in disaster response. information sharing between organizations and with the general public after a disaster was found to have a positive influence on actual disaster response. in particular, a study by hyun [ ] found that information disclosure-related legislation, the organization's budget, personal awareness and attitude and information quality influenced the effectiveness of disaster management. among various factors influencing disaster management and response, factors associated with disaster-related information disclosure and sharing were tested for their influence on the effectiveness of disaster management. the results showed that greater information quality in information disclosure and sharing and greater personal awareness and attitude positively influenced the effectiveness of disaster management. effective disaster response may comprise sub-variables from its outcome aspect. in a study by denise [ ] , the effectiveness of disaster response was determined by measuring life loss, property damage, satisfaction of stakeholders, society's resilience, operational efficiency and budget maximization. moreover, a study by byun ( ) examined fire service organizations and thus effectiveness was determined by evaluating fire containment and rescue, while efficiency was represented by reduction in damage and cost-effectiveness. other factors influencing disaster response include environmental factors [ , , , ] , disaster characteristics [ , , ] and legislative factors [ , , , ]. sars stands for severe acute respiratory syndrome. it has a latency period of days, after which the victim experiences high fever (≥ °c), coughing and respiratory distress. it is transmitted by respiratory routes to medical staff and family members who come in close contact with the patient. complete cure is possible if treatment is administered early, where approximately % of infected patients recover easily within one week. however, sars may rapidly become severe for elderly or frail patients or for patients with chronic illness, yielding a mortality rate of approximately . %. sars became known worldwide on february , when the chinese health authority announced that patients with sars had been identified in china between november , and february , five of whom had died. who, which had strengthened its surveillance activities in the asian region after identifying the likelihood of the emergence of influenza, issued a worldwide warning on march . according to the official statistics released by who in november , between november and july , a total of suspected sars cases from a total of countries were identified and a total of sars related deaths were reported. consequently, sars emerged as the first new disease in the st century that was highly contagious and severe and its transmission through international air travel received special attention. since february , when sars became known for the first time, korea continued to monitor sars outbreak trends through who data and recognized the need for national quarantine measures. accordingly, guidelines for strengthening nationwide sars quarantine measures were passed down on february and on march the korean government issued a sars alert, in keeping with the announcement of the global sars alert by who and established a quarantine system. with nih playing a central role, all healthcare institutions, including national quarantine stations and health centers, maintained a -hour emergency operation-ready status as part of the emergency sars quarantine measures. moreover, a quarantine system was established by designating hospitals as isolation treatment hospitals according to regions. in addition, the policy of measuring people's body temperature as they entered the country through airports and sea ports was implemented in order to detect sars inflow from overseas and to prevent the disease from spreading. furthermore, follow-up investigations were conducted on people who entered korea from sars-risk regions and a system through which patients suspected of being infected could be transported immediately for isolation and treatment was put in place. moreover, additional isolation measures were implemented for people who came in contact with infected patients to prevent the disease from spreading further, in addition to preventing the import of sars. recommendations were made to refrain from traveling to high-risk regions to further prevent the import of sars into korea and to encourage precautions during travel. during the response process to the outbreak, a meeting for city and provincial quarantine officials and experts was held on april . on april , a discussion was held on measures of blocking the importation of sars and preventing its spread. the decision to implement government-wide response measures by setting up a central sars measures headquarter, led by the minister of health and welfare and satellite stations at city and province levels was made. subsequently on april , a government-wide comprehensive sars situation room was introduced and prime minister kun goh released a general public statement, urging active cooperation from the general public regarding the response measures taken by the government. eventually, who declared on june that korea had won the war against sars, effectively subduing sars in less than days after the global sars alert was issued [ ] . mers stands for middle east respiratory syndrome. the outbreak of mers coronavirus started on april following its introduction into korea by a -year-old male, who worked in floriculture and was returning home to korea after a visit to the middle east. the patient was treated at a clinic for a fever he developed seven days after arriving in korea but his condition did not improve. after his visit to the clinic, he received inpatient treatment for three days at pyeongtaek st. mary's hospital and was subsequently discharged. because of continued symptoms of high fever and respiratory distress, he visited another clinic and was eventually admitted to a single-bed unit at the samsung medical center in seoul on may . the staff at samsung medical center had learned that the patient had visited the middle east and based on a suspicion of mers, the doctor in charge requested kcdc to perform further testing on the patient the following day ( may ) . the diagnostic test performed by nih detected middle east respiratory syndrome coronavirus: (mers-cov) genes in the patient. following the announcement of these findings on may , identification of the first mers patient in korea was officially reported [ ] . as shown in figure , the first mers patient in korea visited multiple clinics and large hospitals for treatment over a -day period since the symptoms first appeared, during which time he came in contact with family members, other patients and medical staff, resulting in many cases of secondary infection. [ ] . as shown in figure , the first mers patient in korea visited multiple clinics and large hospitals for treatment over a -day period since the symptoms first appeared, during which time he came in contact with family members, other patients and medical staff, resulting in many cases of secondary infection. as shown in the graph above, the highest number of confirmed mers cases outside of the middle east region was found in korea. the response to mers completely exposed contradictions in the national quarantine system, as well the healthcare system. kcdc and local government entities all proved inadequate in their ability to respond to the public health crisis caused by this infectious disease. there was confusion due to lack of clarity in the delegation of roles between the central and regional governments and the cooperation system between health authorities and medical institutions did not operate smoothly either. most medical institutions, including general hospitals, small-to-medium-sized hospitals and clinics, were not prepared to deal with healthcare-associated infections and as a result the infection continued to spread among patients and medical staff. in addition, problems in the transport and referral system for confirmed or suspected patients were discovered, while compensation for medical institutions and research and development of emerging infectious diseases became points of contention. moreover, medical staff who participated in the isolation and treatment of mers patients complained about job-related burden and stress. the mers outbreak exposed fundamental problems in the public healthcare system and vulnerabilities in the national quarantine system but the solutions to these problems have not been clearly identified to date. the mers outbreak caused restrictions in korean citizens' day to day lives and significantly impacted the national economy. the socioeconomic impact of mers has still not as shown in the graph above, the highest number of confirmed mers cases outside of the middle east region was found in korea. the response to mers completely exposed contradictions in the national quarantine system, as well the healthcare system. kcdc and local government entities all proved inadequate in their ability to respond to the public health crisis caused by this infectious disease. there was confusion due to lack of clarity in the delegation of roles between the central and regional governments and the cooperation system between health authorities and medical institutions did not operate smoothly either. most medical institutions, including general hospitals, small-to-medium-sized hospitals and clinics, were not prepared to deal with healthcare-associated infections and as a result the infection continued to spread among patients and medical staff. in addition, problems in the transport and referral system for confirmed or suspected patients were discovered, while compensation for medical institutions and research and development of emerging infectious diseases became points of contention. moreover, medical staff who participated in the isolation and treatment of mers patients complained about job-related burden and stress. the mers outbreak exposed fundamental problems in the public healthcare system and vulnerabilities in the national quarantine system but the solutions to these problems have not been clearly identified to date. the mers outbreak caused restrictions in korean citizens' day to day lives and significantly impacted the national economy. the socioeconomic impact of mers has still not been accurately assessed. what is clear at this point is that the entire korean society has become more interested in infectious diseases and that infectious diseases have become an agenda directly linked to public safety. moreover, people recognized that in order to respond to emerging infectious diseases it is necessary to continually assess and monitor infectious diseases that occur worldwide and establish manuals based on up-to-date knowledge through research, specialists and timely crisis analysis during the response process. in addition, the need to establish an infectious disease response network and partnerships between medical institutions and local government, as well as a central government, was also presented. the objective of this study was to inductively explore the factors that influence response based on studies related to disaster and infectious disease response. for this, a meta-analysis method called successive approximation was used [ ] . prior to inductive exploration of the factors that influence disaster response, sample articles were used to categorize these factors. the study also aimed to perform successive meta-analyses to present a model based on detailed explanation and revision of the previously established factors influencing disaster response. accordingly, the present study used a rough model based on the categorization of the factors influencing disaster response presented in existing studies to perform meta-analyses on sars and mers cases. in summary, after establishing the initial model, several rounds of meta-analyses were performed to refine the model. accordingly, the incomplete preliminary theoretical framework, which can be viewed as the initial model, represented simplification and categorization of major factors through existing disaster response-related studies. the study aimed to conduct successive analyses based on the incomplete framework to present a refined model by revising the factors and the relationships between them. accordingly, precedent studies previously examined in chapter were used to derive the factors influencing disaster response from a system perspective. on a review of numerous studies, it was discovered that the duties assigned to various organizations and agencies and the factors that actually influence disaster response show regularity [ , , ] . therefore, based on such regularity, the study aimed to categorize these factors according to timelines from a system theory perspective. first, the studies that presented communication, coordination, cooperation and network from the process level as the mediating variables for effective disaster response included those by [ , , , ] . other studies selected the process level variable as one variable among many independent variables in analyzing the influence on effective disaster response. it was commonly pointed out that resources related to disaster response influenced the outcome of disaster response through the interactions and coordination between organizations and agencies in the response process. kapucu [ ] analyzed the influence of the system, organizational environment, tools for organizational capability and cooperation and the decision-making process of actors in the entire process on effective disaster response. the system was a variable that included organizational structure, culture and goals, while the environment included time pressure, uncertainty and complexity of the situation. capability was a factor that involved decision-making support, communication tools, previous cooperation experience, flexibility in responding to disaster and immediate response capability, while actors included the number of stakeholders, experts, interdependence and trust. the study examined whether these independent variables influenced effective disaster response through cooperative decision-making processes, meaning open and honest exchange of opinions, shared model construction, negotiation and utilization of relevant knowledge and information. the proposed research model was used to conduct social network analysis through content analysis, in addition to in-depth case analysis on countries that were victims of terrorist attacks, including the us, indonesia, turkey, spain and the uk. moreover, a study by lindell et al. [ ] also revealed that various resources influenced disaster planning and such process had a significant influence on the efficiency of disaster response. on the basis of these studies, a framework consisting of independent variables having an influence on the effectiveness of disaster response through the disaster response processes was constructed. even in studies that do not present process variables as mediating factors, the majority of process variables were selected as independent variables for analysis and thus it is necessary to reorganize and categorize the variables that were presented in previous studies. as shown in figure the present study selected the factors influencing disaster response presented in existing studies and categorized them largely into environment, inputs and process based on system theory. this model was presented because disasters act as a single system that includes the aforementioned factors, regardless of their type (natural or social disaster) [ , [ ] [ ] [ ] . the strength of the influencing factors may vary depending on the type of disaster but because they were described and analyzed by the factors that are presented below, analyzing or describing social and natural disasters using different frameworks is unnecessary [ ] . based on the categorization of the influencing factors shown in the figure below, the study aimed to conduct future meta-analyses to explore detailed factors and identify the relationships between these factors. had a significant influence on the efficiency of disaster response. on the basis of these studies, a framework consisting of independent variables having an influence on the effectiveness of disaster response through the disaster response processes was constructed. even in studies that do not present process variables as mediating factors, the majority of process variables were selected as independent variables for analysis and thus it is necessary to reorganize and categorize the variables that were presented in previous studies. as shown in figure the present study selected the factors influencing disaster response presented in existing studies and categorized them largely into environment, inputs and process based on system theory. this model was presented because disasters act as a single system that includes the aforementioned factors, regardless of their type (natural or social disaster) [ ] , [ ] , [ ] [ ] . the strength of the influencing factors may vary depending on the type of disaster but because they were described and analyzed by the factors that are presented below, analyzing or describing social and natural disasters using different frameworks is unnecessary [ ] . based on the categorization of the influencing factors shown in the figure below, the study aimed to conduct future meta-analyses to explore detailed factors and identify the relationships between these factors. for inductive exploration of the factors influencing infectious disease response, meta-analyses were performed based on the aforementioned factor categorization framework and in-depth interviews were conducted for testing and supplementation. the present study used previous studies on disaster response to compile a list of the factors influencing disaster response and presented a theoretical framework. moreover, the factors influencing disaster response were explored through case review, while qualitative meta-analysis was performed to identify the correlations between the factors. qualitative meta-analysis was conducted to allow a comprehensive analysis of qualitative studies [ ] . this method of analysis is different from meta-analysis which integrates results from existing empirical studies using a quantitative method [ ] , [ ] . the approach in qualitative meta-analysis involves interpretive analysis, which strives to include major concepts that appeared in individual qualitative studies but at the same time, generate a higher-level concept that can connect these concepts to a higher dimensional theoretical structure to allow for comprehensive understanding of the phenomenon and possibility of new interpretation and theoretical creation [ ] . therefore, the major goal of qualitative meta-analysis is to contribute to knowledge. from this perspective, schreier et al. [ ] listed theory building, theory explication and theory development as the three overlapping goals of qualitative meta-analysis. in the present study, the factors influencing disaster response were reviewed from existing studies in the theory building process and organized from a system theory level. subsequently, meta-analysis was performed to explore factors through for inductive exploration of the factors influencing infectious disease response, meta-analyses were performed based on the aforementioned factor categorization framework and in-depth interviews were conducted for testing and supplementation. the present study used previous studies on disaster response to compile a list of the factors influencing disaster response and presented a theoretical framework. moreover, the factors influencing disaster response were explored through case review, while qualitative meta-analysis was performed to identify the correlations between the factors. qualitative meta-analysis was conducted to allow a comprehensive analysis of qualitative studies [ ] . this method of analysis is different from meta-analysis which integrates results from existing empirical studies using a quantitative method [ , ] . the approach in qualitative meta-analysis involves interpretive analysis, which strives to include major concepts that appeared in individual qualitative studies but at the same time, generate a higher-level concept that can connect these concepts to a higher dimensional theoretical structure to allow for comprehensive understanding of the phenomenon and possibility of new interpretation and theoretical creation [ ] . therefore, the major goal of qualitative meta-analysis is to contribute to knowledge. from this perspective, schreier et al. [ ] listed theory building, theory explication and theory development as the three overlapping goals of qualitative meta-analysis. in the present study, the factors influencing disaster response were reviewed from existing studies in the theory building process and organized from a system theory level. subsequently, meta-analysis was performed to explore factors through korean studies and articles and interviews. the protocol was constructed through data collection and analysis and effort was made to ensure reliability and validity of the study [ ] . using this approach, the study was conducted systematically, from the data collection stage to the final analysis. after comprehensively collecting data, including domestic research articles, media reports and audit reports from the board of audit and inspection (bai) of korea related to sars and mers cases, data to be analyzed were selected on the basis of the inclusion and exclusion criteria. the data collection method will be discussed in more detail in the data collection section. the collected data were codified and categorized on the basis of the meta-analysis framework consisting of the factors influencing disaster response extracted from existing studies and theories. as shown in figure , for the influencing factors identified from the data, the factor and source were recorded, and evidence of the correlation was identified. the evidence included statistical data, media reports, interviews with experts and claims made by authors. with this coding process, consistency of the results when the same analysis is performed by different researchers can be maintained and this can be used to ensure reliability. data for meta-analysis were collected from various sources, including listed academic journals, articles from daily and weekly periodicals and audit reports from bai. duplicate items were eliminated based on search results and data that met the objective of the present study were selected through in-depth reviews and discussions with fellow researchers. academic articles were limited to those published in journals listed in the national research foundation of korea, while duplicate articles and articles with low correlation to the research question were excluded. the period of data of academic articles was from (sars outbreak) to (at the time of the research). all searched articles were tallied and data were selected through validity testing and unanimity with fellow researchers. media reports were collected from daily and weekly periodicals to provide information that was not covered in academic articles. the search process used the naver news site and the official home pages of each newspaper. the search keywords were disaster case names: sars, mers and different variations of these terms in korean. additionally, data that mentioned a disaster name along with the term "response" were reviewed. to eliminate political bias, chosun ilbo, donga ilbo, kyunghyang shinmun and hankyoreh were selected from daily periodicals, while weekly chosun, weekly donga, weekly kyunghyang and hankyoreh were selected from weekly periodicals. the period included in data collection was set to one year to include the infection outbreak and response period between january and december for sars and between january and december for mers. lastly, the homepage of bai was searched for bai audit reports on sars and mers but since audit reports for sars did not exist only mers cases were analyzed. among the cases that appeared as search results for mers, the results that were unrelated to the selected cases were excluded. as a result, a total of cases of audit reports for various organizations were selected for the analysis (table ) . the results of factors coded and explored based on aforementioned media reports, bai audit reports, academic articles and in-depth interviews are provided here. to effectively demonstrate the results of exploring the factors influencing disaster response, analysis was performed by identifying how each factor, as an independent factor, influenced other factors; and by gathering evidence of the relationship between time, cause and outcomes. the present study underwent the process of identifying and testing correlations through a meta-analysis of the factors influencing infectious disease response. the analysis results on the factors influencing infectious disease response were as follows. legislation, sociocultural factors and disaster characteristics were identified as the environmental factors influencing disaster response. with respect to sars, although there was no legislative system for disaster response and management, some respiratory transmission diseases, including sars, were temporarily designated as "infectious diseases subject to quarantine and surveillance" for onsite response. enactment and amendment of laws have procedural and time requirements and thus quarantine or isolation was made possible by presenting them as subjects of quarantine and surveillance following the decree of the minister of health and welfare, which actually had a positive influence on onsite response. moreover, while legislation for mers was in place, it was incomplete and not detailed enough. this caused confusion in the response process because of the possibility of arbitrary decisions and because it contained inaccurate information about infectious diseases, it had a negative influence on onsite response. consequently, mers spread to other patients, leading to a failed initial response. moreover, unlike the sars outbreak, when international public health crisis was declared, there was no announcement of an international public health crisis with mers, which caused a lack of awareness on the importance of prevention and response. financial resources, human resources, physical resources, information and education and training were identified as the input factors influencing disaster response. human resources also acted as a mediating factor in the relationship between legislation and the effectiveness of response to infectious diseases. in the processes of responding to sars and mers, problems related to human resources, especially epidemiologists, were identified. this was also very apparent in the correlations. although epidemiological investigation in infectious disease response is very important for preventing the spread of infectious diseases and for timely response, an insufficient number of epidemiologists made it impossible to keep up with the rate at which the disease was spreading and since public health physicians were mostly responsible for epidemiological investigation, a lack of specialization was also a serious problem. moreover, budget, the proportion of public healthcare and infection control infrastructure, such as negative-pressure units, were also found to be insufficient during both sars and mers outbreaks. one of the factors that was identified as being important in the correlation analysis was education and training. since everyone may experience an actual disaster, simulated training according to given scenarios and education for response personnel are very important. leadership, intergovernmental relationships, information sharing and onsite response were identified as the process factors influencing response to infectious disease outbreak, while information sharing was found to influence stakeholder satisfaction. with respect to leadership, as mentioned earlier, the role of the prime minister and the president was an important factor in the implementation of timely and effective disaster response. during the sars outbreak, prime minister kun goh was at the forefront, urging the public and departments to cooperate. on the other hand, during the process of responding to mers, the control tower changed at least twice and the president made it clear through the spokesperson that the blue house was not the control tower. during this process, the intergovernmental relationship was not smooth either. moreover, poor information sharing and communication between departments and between the central and local government caused confusion and increased the level of distrust among the general public. among the process factors, intergovernmental relationships, information sharing and onsite response were independent variables that influenced the outcome and acted as mediating factors between legislation and outcome. lastly, although not presented in existing analytical frameworks, the factors identified through meta-analysis and interviews were interest and cooperation from the private sector (volunteerism). with respect to interest, an analysis of sars cases showed that the interest of local citizens, meaning regional self-centeredness, caused the designation of sars quarantine hospitals to be nullified, acting as a factor that interfered with infectious disease response. these factors were confirmed in the interview results. the interest of the agency in charge of the control tower emerged as a factor that interfered with the infectious disease response, albeit at a different level than the interest of local citizens. the agency in charge of determining the disclosure of information was the mohw and because the same agency was responsible for both promoting actual related projects and managing disaster, conflict of interest did not allow immediate response measures to be implemented. cooperation from the private sector (volunteerism) was a factor that did not appear in the meta-analysis but was identified in interviews with workers. its correlations were not analyzed in the meta-analysis data of the present study and existing studies did not discuss the role of volunteers in infectious disease response either. however, resource support for self-quarantine patients in actual infectious disease response was lacking but active participation by volunteers played a major role in helping to slow the spread of mers and to successfully implement self-quarantine. moreover, the hidden context of correlations identified through interviews, which was not identified in existing articles, was education and training. education and training was analyzed as a factor influencing infectious disease response, while the interview results revealed that education and training not only had a direct influence on response but also had an impact on the relationship between the people in charge of disaster response. timely response was made possible by relationships built between people in charge of disaster response through continued training, which may be attributed to the uniqueness of korean culture. as shown earlier, the factors influencing infectious disease response in korea were very diverse and they became more refined and detailed when compared to categorization of factors presented in the introduction of this paper. these were factors identified through meta-analyses and in-depth interviews and should be considered in the improvement of the infectious disease response system in korea. a comprehensive model that summarizes the aforementioned exploration of the influencing factors is shown in figure . factor influencing infectious disease response, while the interview results revealed that education and training not only had a direct influence on response but also had an impact on the relationship between the people in charge of disaster response. timely response was made possible by relationships built between people in charge of disaster response through continued training, which may be attributed to the uniqueness of korean culture. as shown earlier, the factors influencing infectious disease response in korea were very diverse and they became more refined and detailed when compared to categorization of factors presented in the introduction of this paper. these were factors identified through meta-analyses and in-depth interviews and should be considered in the improvement of the infectious disease response system in korea. a comprehensive model that summarizes the aforementioned exploration of the influencing factors is shown in figure . the present study conducted meta-analyses to comprehensively analyze the correlations of factors influencing disaster response from a korean context. for inductive exploration of the factors influencing infectious disease response in korea, the present study collected and selected reliable data from academic research on infectious disease response conducted in korea, newspaper articles and audit reports from bai. the reason for limiting the studies to those conducted within korea was based on the determination that it was necessary to review how well domestic studies and articles explained domestic cases. the objective was to use the findings to point out the limitations of infectious disease-related studies in korea and to present factors influencing infectious disease response within a korean context. the analysis results confirmed that, overall, the studies korea focused on factors from the process aspect when analyzing the factors influencing infectious disease response. a summary of other major findings are as follows: first, among environmental factors, the legislative factor had direct and indirect influence on the overall process of infectious disease response. other environmental factors were regarded as factors influencing disaster response based on their correlations but the legislative factor was considered especially important. disaster-related legislation enacted in various forms including basic laws, manuals and code of conduct should be systematic and exhibit high integrity to allow timely and accurate response in crisis situations. however, owing to insufficiencies in many aspects, it had a negative influence throughout the entire response process. the present study conducted meta-analyses to comprehensively analyze the correlations of factors influencing disaster response from a korean context. for inductive exploration of the factors influencing infectious disease response in korea, the present study collected and selected reliable data from academic research on infectious disease response conducted in korea, newspaper articles and audit reports from bai. the reason for limiting the studies to those conducted within korea was based on the determination that it was necessary to review how well domestic studies and articles explained domestic cases. the objective was to use the findings to point out the limitations of infectious disease-related studies in korea and to present factors influencing infectious disease response within a korean context. the analysis results confirmed that, overall, the studies korea focused on factors from the process aspect when analyzing the factors influencing infectious disease response. a summary of other major findings are as follows: first, among environmental factors, the legislative factor had direct and indirect influence on the overall process of infectious disease response. other environmental factors were regarded as factors influencing disaster response based on their correlations but the legislative factor was considered especially important. disaster-related legislation enacted in various forms including basic laws, manuals and code of conduct should be systematic and exhibit high integrity to allow timely and accurate response in crisis situations. however, owing to insufficiencies in many aspects, it had a negative influence throughout the entire response process. the legislative factor indirectly influenced disaster response, making it an important factor that influences the overall disaster response process. in other words, human resource was identified as the mediating factor in the relationship between the legislative factor, human resources and onsite response. on the other hand, intergovernmental relationships, information sharing and onsite response were identified as the mediating factors in the relationships between the legislative factor, intergovernmental relationships and the effectiveness of disaster response; the relationship between the legislative factor, information sharing and the effectiveness of disaster response and the relationship between the legislative factor, onsite response and the effectiveness of disaster response, respectively. along with the determination of mediating factors, the study also found that the establishment of legislation had an overall impact on infectious disease response. second, the results showed that most input factors, including physical resources, human resources and information were insufficient. within a korean context, it is believed that this problem stemmed from the lack of a disaster response system or many studies related to disaster response, as indicated by the fact that basic laws about disaster management were implemented in korea from . considering that the systematization of disaster response following the passing of basic related laws was relatively recent, more detailed issues, such as securement of resources, did not draw attention until a disaster actually occurred, leading to gradual improvement. therefore, factors related to these resources showed insufficiencies no matter which case was reviewed. however, considering the differences in the timeframes of the cases raises concern on whether the experience gained from the disaster response system is actually being used as an asset to improve the disaster response system in korea. third, major findings regarding process factors were as follows. leadership of the central government, establishment of an intergovernmental response system, the need for communication, information sharing and disclosure and onsite response were identified as key factors influencing effective infectious disease response. existing studies have found that information sharing occurred top-down, from the central government to local government [ ] . even so, information sharing was correlated with process factors. nondisclosure of hospital names by the government had an impact on the spread of infectious diseases and on failed initial response. further, the general public voluntarily shared information and made the effort to share accurate information, such as creating a mers map and sharing information on websites. in addition, the interests of local citizens and departments also acted as a factor that interfered with effective infectious disease response. by analyzing the factors influencing infectious disease response within a korean context, the present study presents the following theoretical and policy implications. theoretically, the study established a model of factors influencing infectious disease response by performing inductive exploration on the factors influencing infectious disease response in korea, which was utilized for comprehensive analysis. policy-wise, the study aimed to emphasize the need for improvement of infectious disease response-related legislation, strengthening the authority of kcdc, which currently serves as the control tower, qualitative and quantitative supplementation of disaster response-related human resources, legal grounds for the authority of response personnel and preparation of protective measures. lastly, since the importance of information collection and sharing and cooperation between agencies was demonstrated, it is necessary to establish a system for information sharing and disclosure, as well as a cooperation system involving the central government, the local government, health centers and medical institutions. author contributions: k.m. designed study and collected data; k.j. gave an important advice on analyzing data; and k.m. and k.j. wrote the paper. interorganizational coordination in dynamic context-networks in emergency response management interorganizational coordination in complex environments of disasters-the evolution of intergovernmental disaster response systems 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the cooperation and performance in disaster management a study on disaster management governance system building effectiveness in multi-state disaster management systems: the case of the caribbean disaster and emergency response agency the analysis about the potential effects of local overnment's disaster control type on the efficiency of its disaster control system collaborative networks for the infrequent emergency management: a comparative analysis on the case of controlling mers collaborative governance in theory and practice organized behavior in disaster facing the unexpected: disaster preparedness and response in the united states the network governance in response to acts of terrorism: comparative analyses; routledge: abingdon-on-thames a study on the establishment of integrated disaster management system by local governments a study of semantic network analysis of newspaper articles on mers situation: comparing conservative and progressive news media qualitative meta-synthesis on training and workplace experiences of individuals with disabilities: focusing on practical issues of applying qualitative meta-synthesis secondary and meta-analysis of research focus on qualitative methods qualitative metasynthesis: issues and techniques public health network structure and collaboration effectiveness during the mers outbreak in south korea: an institutional collective action framework the authors declare no conflict of interest. key: cord- -zieuc vk authors: brazee, patricia l.; sznajder, jacob i. title: targeting the linear ubiquitin assembly complex to modulate the host response and improve influenza a virus induced lung injury date: - - journal: arch bronconeumol doi: . /j.arbres. . . sha: doc_id: cord_uid: zieuc vk abstract influenza virus infection is characterized by symptoms ranging from mild congestion and body aches to severe pulmonary edema and respiratory failure. while the majority of those exposed have minor symptoms and recover with little morbidity, an estimated , people succumb to iav-related complications each year worldwide. in these severe cases, an exaggerated inflammatory response, known as “cytokine storm”, occurs which results in damage to the respiratory epithelial barrier and development of acute respiratory distress syndrome (ards). data from retrospective human studies as well as experimental animal models of influenza virus infection highlight the fine line between an excessive and an inadequate immune response, where the host response must balance viral clearance with exuberant inflammation. current pharmacological modulators of inflammation, including corticosteroids and statins, have not been successful in improving outcomes during influenza virus infection. we have reported that the amplitude of the inflammatory response is regulated by linear ubiquitin assembly complex (lubac) activity and that dampening of lubac activity is protective during severe influenza virus infection. therapeutic modulation of lubac activity may be crucial to improve outcomes during severe influenza virus infection, as it functions as a molecular rheostat of the host response. here we review the evidence for modulating inflammation to ameliorate influenza virus infection-induced lung injury, data on current anti-inflammatory strategies, and potential new avenues to target viral inflammation and improve outcomes. tratamiento dirigido al complejo de ensamblaje de cadenas lineales de ubiquitina para modular la respuesta del huésped y mejorar el daño pulmonar inducido por el virus de la gripe a la infección por el virus de la gripe se caracteriza por síntomas que van desde la congestión leve y los dolores corporales hasta el edema pulmonar grave y la insuficiencia respiratoria. aunque que la mayoría de las personas expuestas presentan síntomas leves y se recuperan con poca morbilidad, se estima que cada año personas en todo el mundo fallecen por las complicaciones relacionadas con esta infección. en estos casos graves, se produce una respuesta inflamatoria exagerada, conocida como "tormenta de citoquinas", que causa daños en la barrera epitelial respiratoria y el desarrollo del síndrome de distrés respiratorio agudo (sdra). los datos de estudios retrospectivos en humanos, así como de modelos animales experimentales de infección por el virus de la gripe, resaltan la delgada línea que existe entre una respuesta inmune excesiva y una inadecuada, cuando la respuesta del huésped debe mantener el equilibrio entre el aclaramiento viral y la introduction seasonal influenza a viral infection affects a significant proportion of the population worldwide, with an estimated , people succumbing to iav-related complications each year. while most patients infected with influenza a virus (iav) recover without major sequelae, severe viral pneumonia is one of the most common causes of acute respiratory distress syndrome (ards) [ ] [ ] [ ] [ ] . clinically, ards presents with bilateral pulmonary infiltrates, hypoxemia, pulmonary edema and widespread lung inflammation that lead to high mortality rates due respiratory and to multiple organ failure [ , [ ] [ ] [ ] . ards patients can be sub-grouped based on the severity of the inflammatory response, where patients with hyper-inflammation have worse clinical outcomes, spending more days on mechanical ventilation, experiencing increased incidence of organ failure and a higher mortality rate compared to hypo-inflammatory ards patients [ ] . impairment of gas exchange in iav-induced ards, in large part, is due to damage to the respiratory epithelial barrier and edema accumulation [ , [ ] [ ] [ ] [ ] [ ] . during iav infection, an exaggerated inflammatory response, known as "cytokine storm", can occur leading to the development of hyper-inflammatory ards, increasing iav-induced morbidity and mortality ( figure ) [ ] . post-mortem studies of lungs from iav-infected patients show extensive diffuse alveolar damage characterized by edema, cellular infiltration, thickening of alveolar walls, and necrosis [ ] . interestingly, a study of critically ill patients showed no differences in pulmonary viral load between those who died and those who recovered, while mortality directly correlated with exuberant inflammation, further supporting a maladaptive host response as the major driver of iav-induced lung injury [ ] [ ] [ ] [ ] [ ] . similar observations are being reported in patients with severe coronavirus disease (covid- ) , where severe lung damage is associated with increased pro-inflammatory cytokines and respiratory failure from ards is the leading cause of mortality [ , ] . with no virus-specific treatment options currently validated, therapies which target the inflammatory response are currently being considered for patients with severe covid- [ , ] . however, as it has been reported for severe iav infections, current antiinflammatory drugs have pleiotropic effects and may lack the specificity needed to carefully calibrate the host response. respiratory epithelial cells, as primary targets for iav infection and replication, initiate inflammatory signaling [ ] [ ] [ ] [ ] . in response to respiratory epithelial derived cytokines, innate immune cells, such as neutrophils, monocyte-derived inflammatory macrophages, and natural killer (nk) cells are recruited to the airspace [ ] . together with tissue resident alveolar macrophages, recruited innate immune cells are critical for control of viral replication through both lysis and clearance of virus-infected cells [ ] [ ] [ ] [ ] . however, in addition to controlling viral spread, innate immune cells contribute to the overproduction of pro-inflammatory cytokines that enhance iav-induced lung injury ( figure ). the pulmonary immune response must be carefully balanced, simultaneously promoting viral clearance and limiting excessive inflammation to maintain proper lung function. findings from animal models of iav infection have shown that modulation of the host immune response is associated with reduced lung injury and improved survival [ , , , ] . blockade of specific immune cell subsets has been shown to improve outcomes in mouse models of severe iav infection. for example, genetic deletion of the chemokine receptor ccr inhibited the recruitment of monocyte-derived inflammatory macrophages during iav infection and resulted in reduced lung injury with improved survival. however, loss of this myeloid cell population resulted in a delay in viral clearance [ , ] . moreover, adoptive transfer of nk cells from iav-infected lungs, as compared to nk cell from naïve lungs, resulted in increased mortality of influenzainfected mice [ ] , suggesting that inflammation-dependent activation, rather than recruitment, drives the observed pathology. taken together data from these animal models suggest that the determinant of influenza severity be orchestrated by respiratory epithelial cells. the linear ubiquitin assembly complex (lubac) is a multi-protein e ubiquitin ligase complex composed of two stabilizing proteins, the heme-oxidized iron responsive element binding protein ubiquitin ligase- l (hoil- l) and shank-associated rh domain-interacting protein (sharpin), and a catalytic component, hoil- -interacting protein (hoip) [ ] [ ] [ ] (figure ). the proteins within the heteromeric complex contain multiple domains for interactions within the complex, ubiquitin binding, as well as catalytic activity [ ] [ ] [ ] [ ] [ ] . lubac is an essential regulator of nf-κb activation and has been shown to act as a molecular rheostat, regulating the amplitude of the epithelial-driven inflammatory response dung iav infection [ , ] . the respiratory epithelium actively participates in the first line of defense against pathogens by orchestrating host innate immunity [ , [ ] [ ] [ ] . as iav replicates within the respiratory epithelium cells, the cytosolic pattern recognition receptor (prr), rig-i, is activated and initiates formation of a signaling platform to which lubac is recruited. lubac covalently attaches met- linked linear ubiquitin chains to the nf-κb essential modulator (nemo), a component of the inhibitor of nf-κb (iκb) kinase (ikk) complex along with ikkα and ikkβ [ , ] . due to the high affinity of nemo's ubiquitin binding domain for linear chains, linear ubiquitination of nemo facilitates the recruitment of additional ikk complexes, which results in the efficient trans-autophosphorylation and activation of proximal ikkβ followed by the phosphorylation and degradation of iκbα and robust nf-kb activation ( figure ) [ , , , , ] . recent reports show that destabilization of respiratory epithelial lubac, via loss of the non-catalytic component hoil- l, dampens the host response during severe influenza and promotes survival with reduced lung injury as well as reduced viral titers. however, when lubac activity is abolished through deletion of hoip, the alveolar epithelia driven inflammatory response is inhibited and mortality is increased. these findings highlight the fine line between an excessive and an inadequate immune response and suggest that therapeutic modulation of lubac activity may be crucial, as it functions as a rheostat regulating the amplitude of the host response to iav infection. lubac covalently attaches linear ubiquitin chains to nemo, which facilitates the recruitment of additional ikk complexes. stably docked ikk complexes result in the efficient transautophosphorylation and activation of proximal ikkα/β, followed by the phosphorylation and degradation of iκbα. nf-κb translocates to the nucleus to stimulate transcription of inflammatory genes. current anti-influenza strategies are limited to yearly vaccination or administration of antiviral drugs, however, short therapeutic windows, viral mutation, and resistance to current therapies limit their effectiveness. despite available vaccination and anti-viral drugs, the most recent pandemic in resulted in an estimated , - , deaths in its first year of circulation worldwide [ ] . the pandemic strain contained a novel assortment of viral genes not previously identified in animal or human populations. from its first detection in april , it was only months until resistance to anti-viral drugs was reported, and it took an additional months before the first vaccine offering protection from the pandemic strain was administered [ ] . in addition to emerging pandemic strains, between , and , people worldwide die from seasonal influenza-related respiratory illnesses each year [ ] . novel mutations and reassortments of the virus will inevitably lead to the next iav pandemic; therefore, the use and development of therapeutics that target conserved host pathways, rather than the virus itself, hold promise to curtail the impact of viral infection. moreover, a heterogeneous response to iav with the same virulence exists within the population, suggesting that host factors play a crucial role regulating the host response and determining the severity of lung injury [ , , ] . additionally, experimental evidence from human studies and animal models of severe iav show that viral titers do not always correlate with severity of disease, but rather ards induced "cytokine storm" is the major driver of morbidity and mortality [ , , , ] . severe iav infection is associated with inflammatory cytokines in humans and mice. due to their pleiotropic and redundant effects, targeting of individual cytokines may not be a suitable approach to reduce pathology during iav infection. instead, dampening of the immune response may be more effective, as was the case with lubac destabilization noted above [ ] . fda-approved anti-inflammatory drugs, including corticosteroids and statins, have been proposed for the treatment of "cytokine storm" associated with severe iav infection [ ] . moreover, current data regarding their efficacy is limited to mouse models and retrospective patient observations [ , ] . corticosteroids have been shown to be effective in limiting the inflammation in in some lung pathologies [ ] [ ] [ ] . however, observational studies of the impact of corticosteroid treatment of iav-infected patients suggest against their use; with administration associated with higher incidence of hospital-acquired pneumonia, longer duration of mechanical ventilation, and increased mortality [ ] . similarly, clinical evidence does not support corticosteroid treatment for covid- lung injury [ ] . statins are another class of drugs recognized for their ability to dampen inflammation [ ] . while experimental evidence from mouse models using statins during iav infection have been inconclusive regarding their benefit [ , , ] , retrospective analysis of patient data suggests an association between statin treatment and lower iav mortality rates [ ] . these examples highlight the need for new avenues of drug discovery and validations, as no currently available immune modulators have convincingly demonstrated their ability to improve outcomes during influenza infection. lubac represents a potential new target for limiting the pathological inflammation that occurs during iav infection. several chemical inhibitors as well as peptides that bind hoip have been used to inhibit lubac activity in cell culture [ ] [ ] [ ] [ ] and in vitro assays [ , ] and support the specific targetablility of lubac (figure ) . currently, lubac inhibitors fall into two categories: those that target the catalytic activity of hoip (ie. bay , gliotoxin, hoipin) or those that disrupt the interaction between lubac components to destabilize the complex (ie. stapled peptides). bay , a small molecule commonly used as an inhibitor of nf-κb activation. it has been observed that treatment of raw . macrophages with bay prevented il- stimulated formation of linear ubiquitin chains. further investigation revealed that bay irreversibly inhibits lubac through a chemical reaction with cysteine residues in the active site of hoip, the catalytic unit of lubac [ ] . while bay represents a potent inhibitor of lubac activity, it targets multiple components of the ubiquitin system, including inhibition of e ubiquitin conjugating enzymes, and possible proteasome inhibition [ ] . as such, use of bay is not suitable for the study of lubac-dependent physiological functions or therapeutic targeting of lubac activity in disease. gliotoxin, a fungal metabolite, was identified in a highthroughput screening for lubac inhibitors using a time-resolved fret-based screening system. while gliotoxin is known to have multiple cellular targets, it is able to inhibit lubac activity and downstream activation of nf-κb at x lower concentrations [ ] . gliotoxin's strong, irreversible binding to the catalytic site of hoip makes it a selective inhibitor of lubac activity [ ] . interestingly, the potency of gliotoxin has been shown to vary between cell types, with myeloid and lymphoid cells being more sensitive to gliotoxin-mediated nf-κb inhibition than epithelial cells [ , ] . however, this irreversible inhibition may quench the inflammatory response and increase susceptibility to secondary infections. hoipins are synthetic small molecules that reversibly inhibit lubac though targeting of hoip activity, displaying both lubac specificity as well as low cytotoxicity. several derivatives have been made with varying degrees of efficacy (hoipin- - ), with hoipin- showing significantly enhanced ability to prevent lubac-mediated nf-κb activation in response to tnf-α without cytotoxicity compared to the other derivatives in vitro [ ] . conversely, stapled α-helical peptides developed based on specific lubac structures disrupt interactions necessary for stable complex formation [ , ] . stapled peptides are a class of synthetic macrocycles where the secondary α-helix structure is stabilized by the introduction of a hydrophobic bridge or "staple" that rigidifies specific areas to inhibit protein:protein interactions [ ] . stapled peptides based on the hoip ubiquitin binding domain have been shown to successfully inhibit lubac activity in vitro by disrupting its interaction with hoil- l and destabilizing the overall complex [ , ] . while several inhibitors of lubac have been developed and shown promise in vitro, no data is available detailing their efficacy in vivo. further investigation in to these compounds which target lubac stability to modulate the degree of lubac activity is warranted as they may be therapeutically beneficial for the treatment of hyper-inflammatory response during viral infection, where a graded host response is necessary. within the past years, iav has been the causative agent of at least five pandemics ( , , , , ) [ , ] . in addition to iav, novel viral threats, such as the coronavirus outbreaks of , and , quickly spread worldwide before virus-specific vaccines or pharmacological options could be developed. thus, therapies that target conserved host pathways may provide a novel universal treatment strategies, regardless of viral sequence. findings from animal models of iav infection have shown that inhibition of the exuberant host immune response is associated with reduced lung injury and improved survival [ , , , ] . however, current fda-approved anti-inflammatory drugs, such as corticosteroids and statins, have failed to show benefit during severe iav infection [ , ] . beyond viral infections, the amplitude of the inflammatory response has been shown to be a critical determinant in outcome during bacterial sepsis in community acquired pneumonia, a common complication post iav infection [ , ] . analysis of a cohort of patients showed that reductions in the inflammatory response during bacterial pnumonia, both due to host inability to mount a response or the administration of anti-inflammatory steroids, lead to increased mortality [ , ] . these clinical observations highlight the need to balance the inflammatory response during viral infection, not only to improve lung injury during the primary viral infection, but also to prevent poor outcomes to secondary infections. in addition to the seasonal threat of influenza, we must also be cautious in the regulation of inflammation in treatment of the ongoing covid- pandemic. while a subgroup of patients with severe covid- develop 'cytokine storm' [ , , ] , it must be remembered that current anti-inflammatory drugs have pleiotropic effects and lack the specificity needed to carefully calibrate the host response. as such, newly developed pharmacologics such as those that target lubac, a molecular rheostat of inflammatory signaling, have the potential to fine tune inflammation and moderate the host response. further investigation of compounds which modulate lubac activity is warranted, as they may be therapeutically beneficial for the treatment of hyperinflammatory response during viral infections, where a milder host response should improve outcomes. nonventilatory strategies for patients with 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genomic landscape of the individual host response and outcomes in sepsis: a prospective cohort study key: cord- -c fry authors: dong, fen; tacchi, luca; xu, zhen; lapatra, scott e.; salinas, irene title: vaccination route determines the kinetics and magnitude of nasal innate immune responses in rainbow trout (oncorhynchus mykiss) date: - - journal: biology (basel) doi: . /biology sha: doc_id: cord_uid: c fry simple summary: many pathogens exploit the olfactory route to reach critical organs in the body such as the brain or lungs. thus, effective onset of an early innate immune response in the nasal epithelium is key to stopping pathogen progression. the stimulation of nasal immunity by vaccines may depend on the type of vaccine and vaccination route. the goal of this study was to evaluate the ability of a live attenuated viral vaccine to stimulate innate immunity in the olfactory organ of rainbow trout, a teleost fish of commercial aquaculture value. the kinetics and magnitude of the innate immune response depended on the route of vaccination, with the strongest and fastest responses recorded in intranasally vaccinated fish. injection vaccination had an intermediate effect, whereas immersion vaccination resulted in delayed and weak nasal innate immunity. injection vaccination, even with the vehicle control, induced mortality in fingerlings, whereas nasal and immersion vaccines were safe. challenge experiments with the live virus revealed that nasal and injected vaccines conferred very high and comparable levels of protection, but immersion vaccination only induced transient protection. in conclusion, the route of vaccination determines the type, magnitude and velocity of the innate immune response in the nasal epithelium of animals. abstract: many pathogens infect animal hosts via the nasal route. thus, understanding how vaccination stimulates early nasal immune responses is critical for animal and human health. vaccination is the most effective method to prevent disease outbreaks in farmed fish. nasal vaccination induces strong innate and adaptive immune responses in rainbow trout and was shown to be highly effective against infectious hematopoietic necrosis (ihn). however, direct comparisons between intranasal, injection and immersion vaccination routes have not been conducted in any fish species. moreover, whether injection or immersion routes induce nasal innate immune responses is unknown. the goal of this study is to compare the effects of three different vaccine delivery routes, including intranasal (in), intramuscular (i.m.) injection and immersion (imm) routes on the trout nasal innate immune response. expression analyses of immune-related genes in trout nasopharynx-associated lymphoid tissue (nalt), detected significant changes in immune expression in all genes analyzed in response to the three vaccination routes. however, nasal vaccination induced the strongest and fastest changes in innate immune gene expression compared to the other two routes. challenge experiments days post-vaccination (dpv) show the highest survival rates in the in- and imm-vaccinated groups. however, survival rates in the imm group were significantly lower than the in- and i.m.-vaccinated groups dpv. our results confirm that nasal vaccination of rainbow trout with live attenuated ihnv is highly effective and that the protection conferred by immersion vaccination is transient. these results also demonstrate for the first time that immersion vaccines stimulate nalt immune responses in salmonids. vaccination has become the most effective method of preventing infectious diseases in farmed fish [ ] . the ideal vaccine must provide long-term protection at both mucosal barriers and systemic tissues. the most common vaccination strategies in farmed fish are injection (intramuscular or intraperitoneal), immersion and oral vaccination [ ] [ ] [ ] . the majority of fish vaccines are delivered by injection, as it is considered the most effective vaccination route [ ] . however, the stimulation of mucosal immune responses by injection vaccines may be delayed compared to mucosal vaccines [ ] . nasal immunity is key to stopping the progression of neurotropic and respiratory pathogens to other body tissues such as the lower respiratory tract or the central nervous system. recent studies have identified nasal vaccination as an effective method to control infectious diseases in fish [ ] [ ] [ ] . nasal vaccines offer many advantages over other types of vaccines such as: (i) a needle-free delivery system; (ii) the induction of strong local and systemic immune responses; (iii) the need for low amounts of antigen. in support, nasal vaccination elicits both local and systemic innate and adaptive immune responses in rainbow trout without the need for an adjuvant [ , ] . combined, all these aspects make nasal vaccination a very attractive mucosal vaccination route for the control of aquatic infectious diseases in farmed fish. immersion vaccination is one of the most desirable ways to deliver vaccines in fish farms due to the ability to mass vaccinate large numbers of fingerlings without handling them one by one. during immersion vaccination, every mucosal surface of the fish, including the olfactory organ, is exposed to the diluted vaccine for a short period of time. however, several studies have evaluated how immersion vaccination induces immune responses in the skin, gut and gills of different fish species [ ] [ ] [ ] , the contribution of the fish nasopharynx-associated lymphoid tissue (nalt) during the immune response of immersion vaccines is yet to be determined. teleost nalt is formed by myeloid and lymphoid cells located at the tips and neuroepithelial regions of the olfactory lamellae that response to nasal antigens [ , ] . previous studies using a bath infection model with the parasite ichthyophthirius multifiliis (ich) have revealed that this parasite infects the trout olfactory organ and that trout nalt mounts innate and adaptive immune responses against this protozoan parasite [ ] . interestingly, the same study detected the highest parasite loads in the olfactory organ compared to common target tissues such as the skin and gills days after infection. these findings suggest that the olfactory organ may be a key site for antigen uptake during immersion vaccination and that nalt likely mounts immune responses to immersion vaccines. infectious hematopoietic necrosis virus (ihnv) is classified in the family rhabdoviridae and causes economically important disease known as infectious hematopoietic necrosis (ihn). ihn is a problem in salmonid farms worldwide, especially for atlantic salmon (salmo salar) and rainbow trout (oncorhynchus mykiss) which suffer significant morbidities and mortalities [ ] [ ] [ ] . we have previously reported that nasal vaccination using a live attenuated ihnv vaccine is highly protective both and days post-vaccination (dpv) [ ] . interestingly, ihnv immersion vaccination only elicits moderate levels of protection in rainbow trout [ , ] , but a comparison of all three routes of vaccination with the same vaccine formulation has not been conducted to date in any salmonid species. in this study, we compared three vaccination routes (intranasal (in), intramuscular injection (i.m.) and immersion (imm) on trout nasal innate immune responses using a live attenuated ihnv vaccine and found strong and quick immune responses in the olfactory organ in-vaccinated group. injection and immersion vaccines also triggered innate immune responses in rainbow trout nalt, albeit of a different magnitude and kinetics than those elicited by the in route. challenge experiments confirmed that immersion vaccination is not as effective against ihn compared to the nasal and injection routes. our findings open up new questions regarding how different teleost mucosa-associated lymphoid tissues (malt) and systemic lymphoid tissues communicate in response to pathogens and vaccines. the specific-pathogen-free (spf) rainbow trout ( . g mean weight, clear springs broodstock operations) were obtained from clear springs foods inc. (buhl, id, usa). fish were maintained in -l tanks that received single-pass ultraviolet-light-treated spf spring water at a constant temperature of . • c and a dissolved oxygen content of . ppm. spf status of brood stock was confirmed routinely in the entire facility. fish were fed twice daily with a commercial rainbow trout diet (clear springs foods inc.). a vaccine trial was conducted with a live attenuated ihnv as described in our previous study [ ] . the ihnv vaccine was experimentally generated at clear springs foods by serial passage in vitro as previously described [ ] . three vaccine routes were tested: intranasal vaccine method (in), intramuscular (i.m.) injection and immersion (imm) delivery. groups of spf rainbow trout were immunized by either pipetting µl of a live attenuated ihnv suspension that contained × plaque-forming units (pfu)/ml into the right nare (in), or by injecting µl of live attenuated ihnv suspension ( × pfu/ml) into the dorsal musculature just anterior to the dorsal fin (i.m.) or by immersion (imm) in pfu/ml for h in tank water. for in and i.m. groups, vaccine was diluted in pbs. a mock immunized group received µl of pbs both in and i.m. and were also immersed in pbs for h. duplicate -fish groups from each treatment were challenged with ihnv at and dpv. briefly, fish were anaesthetized in ms- ( mg/l, syndel, usa) and injected intraperitoneally (i.p.) with ( dpv) and ( dpv) pfu of live ihnv (isolate - [ ] ) ( figure ). an additional -fish group from each treatment was mock-challenged by injecting pbs at each challenge interval. after virulent ihnv challenge, each -fish group was held in separate l aquaria that received flow through • c ultraviolet light-treated spring water. all groups were monitored for mortalities for days post-challenge. dead fish were monitored every day at a.m. by two laboratory technicians and any dead animals removed with a clean net. ten percent of the deceased animals were checked for presence of ihnv by plaque assay on epithelioma papulosum cyprini cells (epc) from common carp (cyprinus carpio) [ ] . at each time point, fish (n = ) were anesthetized in ms- and bled from the caudal vein to avoid blood contamination in the olfactory tissue. trout olfactory organs were dissected at , and dpv (in, i.m. or imm) and placed in sterile . ml eppendorf tubes containing ml of trizol (invitrogen) and stored at − • c until use. total rna was extracted from both olfactory rosettes of each fish by homogenization using sterile tungsten carbide beads ( mm, qiagen) and shaking ( times for min) in a tissuelyser ii (qiagen). rna was extracted following a standard phenol-chloroform extraction protocol. the rna pellet was washed in % ethanol, air dried and resuspended in rnase-free water. rna concentrations were determined by spectrophotometry (nanodrop nd ) and the integrity of the rna was determined by electrophoresis (agilent bioanalyser, ). rna samples were stored at − °c until use. cdna was synthesized using µg of total rna per sample as previously described [ ] . the qpcrs reactions ( -µl reaction volume) consisted of µl of a diluted cdna template ( ng of total rna equivalents), . µl of power sybr green pcr master mix ( ×, applied biosystems) and nm forward and reverse primers. reactions were run in triplicate. the amplification profile consisted of an initial denaturation step at °c for min, and then cycles of • c for s and • c for min followed by melting (dissociation stage) from to • c in an abi prism (applied biosystems) sequence detection system. a negative control (no template) reaction was also performed for each primer pair. a sample from the serial dilution was run on a % agarose gel and stained with red gel stain and viewed under ultraviolet light to confirm a band of the correct size was amplified. in order to determine the efficiency of the amplification for each primer pair, reactions were carried out using serial tenfold dilutions of pooled cdna on the same plate as the experimental samples. the efficiency was calculated as e = (− /s) , where s is the slope generated from the serial dilutions, when log dilution is plotted against ∆ct (threshold cycle number). expression levels were normalized to those of the trout elongation factor a (ef- a) which was used as a single house-keeping gene. the relative expression level of the genes was determined using the pfaffl method [ ] . the primers used for qpcr are shown in table . all primer sets were designed to span an exon-exon boundary. absence of amplification of genomic dna contamination for each primer set was checked by standard pcr using total rainbow trout dna samples from a pool of lymphoid tissues of vaccinated fish as template. kaplan-meier survival curves were plotted to state the mortality of vaccination trials and challenge experiments. data were analyzed in prism version . (graphpad). data are expressed as mean ± standard error (s.e). unpaired student's t-tests were used for analysis of differences between groups. fisher's exact test of conditional independence for a × contingency table was used for follow-up tests using prism version . . p-values less than . were considered statistically significant. in this study, we vaccinated rainbow trout with live attenuated ihnv or pbs by different delivery routes (figure a -d) and then investigated changes in expression of immune-related genes at , and dpv in trout nalt by qpcr. these genes were selected based on a previous transcriptomics study performed in trout nalt dpv [ ] . at one dpv, il b and tgfb expression were greatly up-regulated (~ -and~ . -fold, respectively) in the in group, but not in the i.m. or imm groups. moreover, the pro-inflammatory cytokines tnfa, il and il were also significantly up-regulated dpv in the in group (~ . -,~ . -and~ . -fold) but not in the other two vaccinated groups (figure a ). in fact, il expression was down-regulated . -fold in nalt of i.m. vaccinated fish dpv. the il a expression was up-regulated~ . -and~ . -fold in both in and i.m. vaccinated groups dpv respectively, but not for the imm group. the expression of ck a, a chief nasal chemokine in trout, was only significantly up-regulated in in vaccinated fish dpv. the expression levels of il r, a marker for memory t cells following acute infections [ , ] , were significantly higher (~ . -fold) in in vaccinated fish, with no change in expression detected in the i.m. and imm groups. interestingly, except for the db and db expression in the imm-vaccinated group, all four beta defensin genes (omdb - ) examined were up-regulated in trout nalt dpv in all vaccinated groups compared to mock-vaccinated controls. four dpv, ck a as the most up-regulated gene (~ -fold) in the in group, followed by il , il r and tgfb (~ -,~ . -and~ -fold, respectively) ( figure b) . interestingly, whereas il b and il expression was still significantly higher compared to controls, il and tnfa expression levels were already similar to those of controls in the in group. at this timepoint, expression levels of omdb- , omdb- and omdb- had also returned to basal levels in all vaccinated groups. however, omdb- expression was significantly elevated in all vaccinated groups compared to controls (~ . - . -fold), indicating a unique behavior of this beta defensin in trout nalt. with regards to the i.m. and imm groups at dpv, transcription levels of tnfa, il b and il were significantly higher in the imm group but not the i.m. group, indicating that pro-inflammatory responses occur in trout nalt following imm vaccination albeit with a delay ( days vs. day) compared to the in route. (unpaired student's t-test) * p < . , ** p < . , *** p < . . one of the most remarkable findings of this study was the difference in the transcription kinetics of the innate immune responses that occur in nalt depending on the route of vaccination. as shown in figure c , i.m. injection resulted in significant up-regulation of tnfa (~ . -fold), il (~ -fold), il b (~ . -fold), and il (~ . -fold) expression dpv, while the expression of these genes was already down-regulated in the in group at this timepoint compared to dpv and showed very modest or no change in expression in the imm group (figure c ). ck a expression remained significantly up-regulated in both the in and i.m. groups (~ . -and~ . -fold, respectively) but no changes in expression was recorded in the imm group ( figure ). regarding beta defensins, no changes in expression were observed in any of the vaccinated groups compared to controls dpv except for a significant down-regulation in omdb- expression (~ . -fold) in the in vaccinated group (figure c,d) . taken together, these data show that in vaccination triggers quick (day ) and potent pro-inflammatory and anti-inflammatory immune responses in trout nalt and that these responses are rapidly dampened by day . imm vaccination also results in innate immune responses in trout nalt but these have lower magnitude and a delayed onset (day ) compared to those elicited by in vaccination. finally, i.m. injection vaccination also induces innate immune responses in trout nalt, but those occurred even later (day ) compared to the other two vaccination routes and with a magnitude more similar to the imm group than to the in group. percent cumulative mortalities for all vaccination trials are shown in table . fisher's exact tests show the survival in different vaccinated groups and challenged to pathogen (table ) . at dpv, survival rates in the in and imm groups ranged between % and % (p-value = in vs. i.m., table ), whereas the unvaccinated group had a mean survival rate of % (figure a) . i.m. vaccinated and challenged fish had a mean survival rate of . % (figure a) . i.m. vaccination without challenge resulted in a survival rate of % (percent cumulative mortality = %) (figure b and table , fisher's exact test p-value = . ), indicating losses due to the injection procedure and not the pathogen challenge in some fingerlings. table . statistical analyses of the survival between the different vaccinated groups and challenged to pathogen. significance symbols indicate the p-value: * p < . , ** p < . , *** p < . . at dpv, the mean survival rate of the unvaccinated and challenged group was %. the mean survival rate of the challenged imm group was % compared to the in and i.m. groups, which showed % protection (figure a,b) . fisher's exact tests show sufficient evidence for differences between the imm vaccinated group and the in-or i.m.-vaccinated group (p-value = . and . , respectively, table ) and no differences between the in and i.m. groups (p-value = , table ). vaccination has become the most effective method of preventing infectious diseases in farmed fish. the majority of the fish vaccines are delivered by injection, as it is still considered the most effective vaccination route [ ] . injection vaccination, however, is labor-intensive and can result in side-effects that impact fish welfare [ ] . injected vaccines do not directly stimulate mucosal surfaces, the first tissues to be infected by pathogens. mucosal vaccines for fish, such as immersion or oral vaccination may be the simplest and most cost-effective vaccination methods, especially is small fish, but they usually result in sub-optimal and short-term protection [ , ] . to date, very few viral vaccines have been developed for immersion because of their low efficacy [ ] . nasal vaccination, although labor-intensive, has been shown to be effective against viral and bacterial diseases, to be safe, and to stimulate both mucosal and systemic immunity in fish [ , ] . importantly, nasal vaccination provides additional welfare benefits for the fish as well as the handling staff, since needle use is avoided. vaccines based on live attenuated viruses have been amongst the most successful, cost-effective immune interventions in medical history [ , ] . live attenuated viral vaccines for fish typically elicit a strong and sustained immune response to the target disease [ ] . attenuated live vaccines are safe under most circumstances, although there are some risks such as the presence of residual virulence in vaccinates or virulence in immunocompromised hosts [ ] . in our results, we found no mortality in the in-vaccinated group after challenge with virulent ihnv, demonstrating once again that nasal delivery in rainbow trout is safe. furthermore, challenge experiments showed that the nasal route is very effective both at and dpv. some of these benefits are illustrated in the data from the present study, where we recorded % mortality in the unchallenged i.m.-vaccinated group but no mortalities in the unchallenged in or imm groups. although immersion vaccination is less labor-intensive and mimics natural exposure to infection [ ] , our data confirm that immersion vaccination with a live viral vaccine only affords short-lived immunity in rainbow trout, even in a prolonged exposure set-up like the one used in this study. teleost nalt shares the main features of other teleost mucosa-associated lymphoid tissues (malt) and mounts strong immune responses following infection or vaccination [ , ] . importantly, previous studies in trout demonstrated that intranasal antigen delivery results in very rapid and potent innate immune responses [ , ] and modest circulating specific antibody titers [ ] . here, we evaluated the expression of immune-related genes including cytokines, il r, the chemokine ck a and four β-defensins and found unique kinetics in the local nalt immune responses depending on the route of immunization. neuronal tissues such as the olfactory epithelium are particularly susceptible to pathological inflammation and, therefore, tight regulation of pro-inflammatory immune responses is critical [ ] . our gene expression studies clearly show a rapid pro-inflammatory state in trout nalt following nasal ihnv vaccination. the pro-inflammatory response was characterized by the elevated expression of classical cytokines including il b, il , il and tnfa. interestingly, our time series study revealed that the pro-inflammatory signature was no longer detected by dpv. moreover, elevated expression of anti-inflammatory cytokines il and tgfb was also recorded early on in response to intranasal ihnv vaccination, suggesting a tissue repair response. combined, these results highlight the ability of nalt to mount inflammatory responses against novirhabdoviruses while limiting the magnitude and duration of this response in order to protect tissue integrity. il- and its receptor il- r play critical roles in b and t cell growth, maturation and activation [ , ] . additionally, il- is involved in cd + and cd + memory t cell responses [ ] . at mucosal barriers, il- promotes il- a responses against respiratory bacterial infection [ ] and aids in the elimination of activated lymphocytes in the inflamed mucosa [ ] . our data show that in and i.m. vaccination induce il r expression in nalt, however, immersion vaccination results in transient down-regulation or no changes in nalt il r expression. this important finding may reflect the need to regulate inflammatory lymphocytes in the olfactory tissue in response to high antigenic doses such as those applied in nasal vaccination with this vaccine. moreover, the differential regulation of il r expression noted in each experimental group may shape the local and systemic b and t cell responses elicited by each of the vaccination routes. further studies are needed to support or reject this hypothesis. the β-defensin family of genes plays a significant role in antibacterial and antiviral immunity in fish [ , [ ] [ ] [ ] . previous studies showed up-regulation of β-defensins and other antimicrobial peptide (amp) genes in the kidney of brown trout infected with the novirhabodvirus viral hemorrhagic septicemia virus (vhsv) [ ] . however, the expression of antimicrobial peptides in the trout olfactory organ had not been investigated to date. our results highlight the key role of β-defensins as early antimicrobial effectors, in this case, in response to a viral antigen. importantly, induction of β-defensin gene expression in nalt was highest when the vaccine was delivered intranasally, compared to the other two routes. we observed that one of the four β-defensin genes studied, omdb- , showed a unique expression pattern characterized by a more sustained up-regulation compared to the other three β-defensin genes. similarly, in vhsv-infected brown trout, omdb- expression changes in the kidney differed from those of other amp genes [ ] . future studies should determine the unique function of each amp in the context of nasal immunity. chemokines play pivotal roles in coordinating leukocyte migration in immunity and inflammation [ , ] . in rainbow trout, the ccl -like chemokine ck is strongly expressed both at the mrna and protein level in mucosal tissues such as gill, gut and skin, suggesting its role as a mucosal chemokine [ ] . local nasal production of ck a plays a central role in antiviral immune protection both locally and systemically in trout [ ] . specifically, ck a is chemotactic in vitro and in vivo and recruits cd α + lymphocytes to the nasal mucosa [ ] . in line with these findings, our results show that the ck a mrna levels increase significantly in nalt after immunization with live attenuated ihnv vaccine both by nasal delivery and injection. previous studies revealed increased ck expression in the liver of trout infected by i.p. injection with vhsv [ ] and in the fin bases of trout following bath infection with vhsv [ ] . remarkably, we found that the immersion vaccinated group showed no change in ck a expression in nalt, suggesting that this route does not effectively stimulate this chemokine in the olfactory organ. therefore, it is unlikely that recruitment of immune cells such as cd α + lymphocytes into the olfactory organ occurs in response to this vaccination mode, perhaps explaining the lower effectiveness of this route compared to the other two. although we did not evaluate ck a responses at other malt in our immersion experiment, such studies will reveal if our observation in trout nalt is unique to nalt or universal across all malt following immersion vaccination. one of the caveats of the present study is that we did not measure transcriptional type i ifn responses or ifn-stimulated genes (isgs), known to play an important role in teleost antiviral immunity [ ] [ ] [ ] . we previously reported strong modulation of antiviral immunity genes in rainbow trout nalt days post nasal vaccination with live attenuated ihnv [ ] . given the rapid onset of type i ifn responses against novirhabdoviruses previously found in other studies in teleosts [ , ] , we predict that, similar to the genes investigated here, the vaccination route and antigen dose will be major determining factors of the type i ifn response in trout nalt. the teleost olfactory organ, although not a respiratory surface, shares many anatomical, cellular and molecular features with the mammalian olfactory system [ ] . thus, teleost fish models have been proposed as comparative models for human nasal infections and immunity [ ] . several viral pathogens infect the human host via the nasal epithelium including influenza virus [ ] and sars-cov- [ ] . understanding innate immunity in the nasal mucosa is therefore critical for the effective design of immunoprophylactic strategies against respiratory and neurotropic viruses. interestingly, currently, none of the covid- vaccines currently being tested in clinical trials are being delivered intranasally. our results indicate the immediate and potent immune responses in the trout nasal mucosa are best achieved when vaccines are delivered intranasally, and it is likely that this is the case in humans too. in conclusion, the present study shows that trout nalt mounts innate immune responses after vaccination with live attenuated ihnv vaccine regardless of the route of vaccination. importantly, the route of vaccination and antigen dose determine the magnitude, type and kinetics of the nalt innate immune responses. specifically, the responses induced by direct delivery of the vaccine into the nasal cavity of trout are not mimicked by neither injection nor immersion vaccination. finally, future studies should further investigate whether the limited stimulation of mucosal immune responses in nalt by immersion vaccination may explain the suboptimal protection conferred by this route in this or other vaccine models. this study shows that nasal vaccination with a live attenuated ihnv vaccine induces fast and potent innate immune responses in trout nalt. intramuscular vaccination with live attenuated ihnv vaccine induces a slightly delayed and less potent innate immune responses in trout nalt. immersion vaccination with live attenuated ihnv vaccine causes delayed and mild innate immune responses in trout nalt. finally, whilst nasal and injection vaccination are highly protective against ihn, immersion vaccination provides only transient protection. advances in fish vaccine delivery status and future perspectives of vaccines for industrialised fin-fish farming a review of fish vaccine development strategies: conventional methods and modern biotechnological approaches th -like immune response in fish mucosal tissues after administration of live attenuated vibrio anguillarum via different vaccination routes nasal immunity is an ancient arm of the mucosal immune system of vertebrates evaluation of dual nasal delivery of infectious hematopoietic necrosis virus and enteric red mouth vaccines in rainbow trout (oncorhynchus mykiss nasal vaccination of young rainbow trout (oncorhynchus mykiss) against infectious hematopoietic necrosis and enteric red mouth disease efficacy of a bivalent vaccine against eel diseases caused by vibrio vulnificus after its administration by four different routes polymeric immunoglobulin receptor and mucosal igm responses elicited by immersion and injection vaccination with inactivated vibrio anguillarum in flounder (paralichthys olivaceus) efficacy of a polyvalent immersion vaccine against flavobacterium psychrophilum and evaluation of immune response to vaccination in rainbow trout fry (onchorynchus mykiss l.) tissue microenvironments in the nasal epithelium of rainbow trout (oncorhynchus mykiss) define two distinct cd α + cell populations and establish regional immunity mucosal immunoglobulins protect the olfactory organ of teleost fish against parasitic infection epidemiological characteristics of 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immune responses in a trka-dependent manner the many roles of il- in b cell development; mediator of survival, proliferation and differentiation new insights into il- signaling pathways during early and late t cell development overexpression of interleukin (il)- leads to il- -independent generation of memory phenotype cd + t cells interleukin- protects against bacterial respiratory infection by promoting il- a-producing innate t-cell response mucosal il- -mediated immune responses in chronic colitis-il- transgenic mouse model a β-defensin gene of trachinotus ovatus might be involved in the antimicrobial and antiviral immune response b-defensin in nile tilapia (oreochromis niloticus): sequence, tissue expression, and anti-bacterial activity of synthetic peptides identification and characterization of a β-defensin gene involved in the immune defense response of channel catfish, ictalurus punctatus immune response modulation upon sequential heterogeneous co-infection with tetracapsuloides bryosalmonae and vhsv in brown trout (salmo trutta) chemokines: chemistry, biochemistry and biological function chemokines and their receptors: drug targets in immunity and inflammation ck , a rainbow trout chemokine with lymphocyte chemo-attractant capacity associated to mucosal tissues early immune responses in rainbow trout liver upon viral hemorrhagic septicemia virus (vhsv) infection specific regulation of the chemokine response to viral hemorrhagic septicemia virus at the entry site the peculiar characteristics of fish type i interferons the antiviral innate immune response in fish: evolution and conservation of the ifn system insights into the antiviral immunity against grass carp (ctenopharyngodon idella) reovirus (gcrv) in grass carp an oral dna vaccine against infectious haematopoietic necrosis virus (ihnv) encapsulated in alginate microspheres induces dose-dependent immune responses and significant protection in rainbow trout (oncorrhynchus mykiss) infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective molecular and neuronal homology between the olfactory systems of zebrafish and mouse comparative models for human nasal infections and immunity influenza a viruses are transmitted via the air from the nasal respiratory epithelium of ferrets more than a respiratory virus: its potential role in neuropathogenesis we thank the technical staff at clear springs foods research. division for their help with fish maintenance. the authors declare no conflict of interest. l.t. is employed by leica biosystems amsterdam, a manufacturer of products for cancer diagnostic. this work was not financed by leica biosystems amsterdam. the authors declare no additional conflicts of interest. key: cord- -sp tai authors: jiang, xinpeng; hou, xingyu; tang, lijie; jiang, yanping; ma, guangpeng; li, yijing title: a phase trial of the oral lactobacillus casei vaccine polarizes th cell immunity against transmissible gastroenteritis coronavirus infection date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: sp tai transmissible gastroenteritis coronavirus (tgev) is a member of the genus coronavirus, family coronaviridae, order nidovirales. tgev is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs. an oral lactobacillus casei (l. casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. in this l. casei vaccine, repetitive peptides expressed by l. casei (specifically the mdp and tuftsin fusion protein (mt)) were repeated times and the d antigenic site of the tgev spike (s) protein was repeated times. immunization with recombinant lactobacillus is crucial for investigations of the effect of immunization, such as the first immunization time and dose. the first immunization is more important than the last immunization in the series. the recombinant lactobacillus elicited specific systemic and mucosal immune responses. recombinant l. casei had a strong potentiating effect on the cellular immunity induced by the oral l. casei vaccine. however, during tgev infection, the systemic and local immune responses switched from th to th -based immune responses. the systemic humoral immune response was stronger than the cellular immune response after tgev infection. we found that the recombinant lactobacillus stimulated il- expression in both the systemic and mucosal immune responses against tgev infection. furthermore, the lactobacillus vaccine stimulated an anti-tgev infection th pathway. the histopathological examination showed tremendous potential for recombinant lactobacillus to enable rapid and effective treatment for tgev with an intestinal tropism in piglets. the tgev immune protection was primarily dependent on mucosal immunity. lactic acid bacteria (lab) are a group of gram-positive bacteria that includes species of lactobacillus, leuconostoc, pediococcus and streptococcus. consumed for centuries, lab has enjoyed a long and safe association with humans and animals for healthy food. over the past decade, there has been increasing interest in the use of lab as mucosal delivery vehicles. the application of lab stems from research into effective strategies to deliver vaccine antigens, which may come into contact with the mucosal tissues for the first time, such as the intranasal, oral and genital mucosal surfaces (lavelle and o'hagan ; malik et al. ). mucosal delivery of therapeutics or vaccines for chronic diseases and infections of mucosal origin could increase their potency and specificity. because the mucosal immune system builds an effective iga barrier in no more than days, contact with the mucosal tissues will neutralize the pathogenic microorganism outside of the host. there are abundant studies in the field of lab vaccines. one major advantage of the use of lab as a delivery vehicle for vaccines is that the bacteria can elicit both antigen-specific secretory immunoglobulin (ig) a and an effective systemic immune response. the specific igas have the same function as the neutralizing iggs. some candidate lab vaccines elicited antigen-specific iga responses in faeces, saliva or bronchoalveolar and intestinal lavage fluids (wells and mercenier ) . additionally, lactobacilli are probiotics that may confer health benefits to the host (bengmark and gil ; corthesy et al. ; isolauri et al. ; saarela et al. ) , and there is accumulating evidence that lactobacilli are effective at preventing intestinal disease in both humans and animals due to their ability to inhibit pathogen adhesion to the intestinal wall and prevent inflammatory processes (blum and schiffrin ; de vrese and marteau ; ouwehand ; sartor ; sheil et al. ). because the porcine digestive tract is similar to the digestive tract of human infants with respect to anatomical and histological features and digestive physiology (kararli ; oswald et al. ; tadros et al. ) , the pig has been used as an animal model to study gastrointestinal diseases of human infants (gunzer et al. ) and vaccination studies against these diseases. coronaviruses (covs) comprise a large family of enveloped, positive-stranded rna viruses that infect a broad range of animal hosts as well as humans. well-known representatives are porcine transmissible gastroenteritis virus, porcine respiratory cov, porcine epidemic diarrhoea virus, canine cov, feline cov, bovine cov, human covs, severe acute respiratory syndrome-associated cov, murine hepatitis virus (mhv), avian cov infectious bronchitis virus (ibv) and turkey cov (tcov). the most famous and critical coronavirus is the middle east respiratory syndrome virus found in africa and asia (siddell et al. ). this study investigates whether mucosal immunization is effective in stimulating a protective immune response against cov infection. transmissible gastroenteritis coronavirus (tgev), which is a member of the genus coronavirus, family coronaviridae, and order nidovirales, is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs (cavanagh ) . the viral rna consists of a single strand comprised of three major structural proteins: a phosphorylated nucleoprotein (n protein) and two glycoproteins (the membrane (m) and the spike (s) proteins) (schwegmann-wessels and herrler ). the s protein has four sites (a, b, c and d). both the a and d sites were demonstrated to induce tgev-neutralizing antibodies (di-qiu et al. ) ; however, the a site is highly glycosylated and thus is not suitable for expression in the lab prokaryotic expression vector. additionally, the different tgev sites induce different immune responses. following infection with virulent transmissible gastroenteritis coronavirus, isolated mesenteric lymph node cd + t cells mounted a specific proliferative response against infectious or inactivated purified virus upon secondary in vitro stimulation (anton et al. ) . the peptide n defines a functional t helper epitope that elicits t cells capable of collaborating with b cells specific for different tgev proteins (anton et al. ) . the most important finding is that oral immunization with a recombinant lactobacillus vaccine and infection with tgev elicit various immune responses, such as humoral immunity and cellular immunity. here, an oral lactobacillus casei vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study the mucosal immune response (jiang et al. ) . in this l. casei vaccine, repetitive peptides expressed by l. casei (specifically the mdp and tuftsin fusion protein (mt)) are repeated times and the d antigenic site of the tgev spike (s) protein is repeated six times. the pig model was developed to study intestinal mucosal immune responses (ruan and zhang ) . probiotic feed supplementation may benefit the animal host directly by preventing infection and combating the causative agent of the intestinal disorder by balancing the disrupted equilibrium of the enteric flora and augmenting the host's immune responses. however, lab vaccine has not received national law or certification for human or animal use against coronaviruses. the first clinical trial to use recombinant lab demonstrated that the containment strategy for l. lactis expressing recombinant il- was effective against crohn's disease (braat et al. ) . our laboratory has researched the lab vaccine for more than one decade, and we have developed many lab vaccines in the field of piglet diarrhoea (di-qiu et al. ; liu et al. ; qiao et al. ; yigang and yijing ) . this study is the last step to obtain new drug certification in china. this lactobacillus vaccine has been demonstrated to increase the treg population in the mouse model (jiang et al. ) . tregs effectively depressed t and b cell proliferation, and some studies demonstrated that this regulation also depressed proliferation in inflammatory bowel disease. our study investigated whether the recombinant lactobacillus vaccine gradually increased the breg and treg populations during the immunization process at the first step of immunization (unpublished data). the intestinal immune system of the pig maintains its ability to mount an active immune response against pathogens and exhibits tolerance to at least food antigens and probably commensal flora through an extremely complex network of cellular and humoral immune interactions. consequently, it is important to elucidate the immunological inductive sites of the protective mucosal immune response following oral immunization in pigs. this vaccine could induce tgev antibody immune responses in both the humoral and mucosal immune systems. mdp and tuftsin possess substantial immunopotentiating properties and can induce cellular-mediated immune responses upon oral administration in mice. however, their use in oral vaccines against tgev challenge in the pig host may have different results. furthermore, the only host (and target of the vaccine) of the transmissible gastroenteritis coronavirus is the weaning piglet. there have been no clear reports concerning whether tgev infection stimulates th or th type immune response. the relationship between humoral and cellular immune responses is not clear; moreover, whether the systemic or mucosal lymphoid response will be the primary immune response following oral immunization is unknown. at present, we are not certain which pathway the lactobacillus vaccine will provoke against tgev infection. our study is the first to analyse immunity in response to oral immunization and tgev infection. virus, bacterium and cell line the l. casei atcc strain used in this study was deposited in atcc and is a plasmid-free strain grown in man-rogosa-sharpe (mrs) medium (sigma) at °c without shaking. the recombinant l. casei (pg: - mt d) was generated in previous study (jiang et al. ) . chloramphenicol (cm) and kanamycin (sigma) were each used at a final concentration of μg/ml. tgev was previously isolated and purified in our laboratory. swine testicle (st) cells were cultured in dulbecco's modified eagle's medium (dmem, gibco) supplemented with % foetal bovine serum (fbs, gibco) at °c with % co . the virus stocks were clarified by centrifugation at ×g for min to remove cell debris, titrated using the cytopathic effect assay and then stored in aliquots at − °c until needed. tgev-seronegative crossbreed (large white) piglets were obtained from a local breeding farm after birth. the piglets were housed separately in specialized cages that were maintained in sterile stainless steel isolators (five piglets/isolator) and fed commercial sterile milk and water. four groups (n = each) of piglets were orally dosed with cfu of pg: - mt d in ml of pbs or pbs alone (jiang et al. ) ; this formulation was used to immunize piglets via an intragastric route in a different immunization protocol. the first group was immunized with pg: - mt d in ml for priming. the second group was immunized with pg: - mt d in ml for priming. the third group was immunized with pg: - mt d in ml for consecutive hours. the forth group was immunized with pg: - mt d in ml for consecutive hours. the control group was immunized with pbs. the piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. seven days after immunization, serum samples were prepared from collected blood samples. the intestinal mucus was collected by rectal swab and subsequently homogenized for min in μl of sterile pbs (ph . ) containing . mol/ l edta-na and then incubated for h at °c. clear extracts of all samples were collected by centrifugation at ×g for min and stored at − °c with protease inhibitors for subsequent analysis. enzyme-linked immunosorbent assay (elisa) plates were coated for and h at °c with full tgev and the vp protein, respectively, which were previously isolated and purified in our laboratory. cultured st cells were used as a negative antigen control. after the wells were blocked for h at °c with pbs containing % skim milk, the plates were washed three times with pbs + tween ( . %) (pbst). mucus and serum (diluted : ) samples were added to the wells in triplicate and incubated for h at °c. afterwards, the plates were washed three times with pbst, and a horseradish peroxidase-conjugated goat anti-pig igg or iga antibody (invitrogen) was added to each well ( : ) and incubated for an additional h at °c. after another round of washing, colour development was accomplished using ophenylenediamine dihydrochloride as a substrate, and the absorbance was measured at nm. naive purified spleen t cells ( × cells/ml) were cultured in -well tissue culture plates and stimulated with . μg ml − of plate-bound anti-cd (pe) antibody (abcam) in complete rpmi medium. single-cell suspensions were stimulated in culture with ionomycin ( μg ml − ) in the presence of monensin ( μm) for - h of culture. the cells were surface labelled and then fixed, permeabilized and intracellularly labelled with ifn-γ and il- antibodies as previously described (moore-connors et al. ; zhou et al. ). for th and th differentiation, the cells were stimulated in the presence of ng ml − anti-ifn (fitc) and ng ml − anti-il- (fitc) (abcam) antibodies. the cells were labelled with carboxyfluorescein succinimidylester (cfse) according to a previous protocol (jiang et al. ) . the data were acquired by gating on the cd + cell population with a facscalibur cytometer. the sequential loss of cfse fluorescence was used to measure cell division and proliferation. groups were housed in the same facility but separated by room and ventilation system. pigs in each room were confined by pens on a solid floor that was rinsed daily, fed a balanced diet ad libitum based on weight and provided free access to water. tgev-challenged pigs received a ml dose of × plaque-forming units (pfu)/ml via oral-gastric gavage on days post-inoculation (dpi). pigs in the control group were administered volume-matched virus-free cell culture media. the control, immunized and no challenge group pigs were randomly selected for necropsy on the fourth day. to assess histological changes in the intestinal tissues, both the intestine and other major organs were examined at necropsy. after h of fixation in % neutral buffered formalin, tissue sections were trimmed, processed, and embedded in paraffin, sectioned, stained with haematoxylin and eosin (h&e) and then examined for pathological changes by light microscopy using a model microscope (olympus, tokyo, japan). real-time rt-pcr (qrt-pcr) was employed to determine the amount of tgev and cytokine gene products (isgs) in rectal swab samples and the intestinal tissues using a cfx tm real-time pcr detection system (bio-rad). total rna was extracted from faecal samples and splenic and intestinal tissues using viral rna extraction and total rna extraction kits (intron) according to the manufacturer's instructions. the extracted rna was subjected to real-time qrt-pcr using a one-step sybr® qrt-pcr reagent kit (takara, shiga, japan). following reversetranscription of the viral rna at °c for min, the resulting cdnas were used for real-time pcr amplification. a standard curve was generated by plotting threshold values against serially diluted plasmid dna encoding the fragment of the tgev spike protein (lee et al. ). all determinations were performed using data from wells evaluated in duplicate to ensure reproducibility. the copy number of the experimental samples was determined by interpolating the threshold cycle values using the standard curve. real-time quantitative pcr was utilized to quantify the products of interest (trl- , − , − , il- , il- , ifn-γ and tgf-β) relative to the quantity of messenger rna (mrna) in the total rna isolated from the splenic and intestinal tissues (dirisala et al. ; kiros et al. ) ; the specific primers are listed in table . the livak method (ΔΔct method) was used to calculate the fold change compared to the β-actin gene control. gene expression data were expressed relative to unimmunized and uninfected piglets. to phenotype immune cells in the spleen and mesenteric lymph nodes, single-cell suspensions were isolated and labelled with fluorochrome-conjugated antibodies. to determine the cell type and the frequency of ifn-γ and il- -producing th and th cells, single-cell suspensions were stimulated in culture with ionomycin ( μg ml − ) in the presence of monensin ( μm) for - h. the cells were surface labelled to detect cd and then fixed, permeabilized and intracellularly labelled with ifn-γ and il- antibodies as previously described (moore-connors et al. ; zhou et al. ). comparison of the piglets' iga and igg titres was conducted by a paired t test. the th cell, cytokine expression and faecal pedv rna shedding titres among litters were compared using one way analysis of variance (anova) followed by duncan's multiple range test. the mucosal immune response of the piglets was evaluated by measuring the iga response in diluted intestinal lavage fluid post-intragastric immunization. as shown in fig. a , the newborn piglets that received ml of recombinant lactobacillus pg: - mt d had the highest mucosal iga levels after immunization. the iga levels at h in the piglets that received ml were slightly lower than the newborn piglets that received ml throughout the process. the vaccine doses also provoked systemic immunity based on the serum analysis and elicited specific igg responses from the immunized piglets (fig. b) . newborn piglets that received ml of the vaccine also had the highest specific igg level. the specific igg titre reached as high as : . finally, the antibody kinetics in the serum and intestinal lavage samples from the animals showed that the specific antibody igg and iga levels were increased on the seventh and eighth days and the titre was decreased during the last week without immunization. to analyse the effect of recombinant lactobacillus pg: - mt d on t helper cells, we evaluated t helper cell polarization. throughout the process, we utilized the model of immunization described here. as shown in fig. , the immune response balance mediated by th and th was broken in favour of th -mediated immunity. the pg: - mt d/l. casei group exhibited % protection within days of challenge with tgev (pg: - mt d/l. casei) (fig. ) . in contrast, the control group piglets immunized with pbs all died after tgev challenge. all piglets that died/were killed were emaciated and had yellow faeces coating the skin and hair. in some piglets, the intestinal lumens were filled with large amounts (approximately - ml) of yellowish foamy fluid. in other piglets, the walls of the small intestine were transparent and thin and the intestinal lumens were empty. no significant gross lesions were observed in other major organs (lung, kidney, liver and heart). formalin-fixed intestinal tissue sections from piglets treated with different treatment modalities were blindly analysed for histopathological changes associated with tgev infection. according to the histopathological analysis, the small intestine samples from the three groups (positive, negative and immunized groups) showed obvious differences. as indicated in fig. , different degrees of pathological changes were detected after infection, especially in the positive group where serious damage was observed. the representative pathological changes included intestinal villi hyperaemia, atrophy and destruction. in the pbs infection group, the jejunum villi were severe atrophied and destroyed, and the ilea exhibited severe lymphocyte proliferation in the lamina propria. the recombinant lactobacillus group showed jejuna with intact villi but low-grade hyperaemia and lymphocyte proliferation, and the ilea exhibited lymphocyte proliferation in the lamina propria. both the pbs and vaccine groups had severe inflammatory responses. the negative control piglets that were not infected showed normal histology. the sequences of the two primers were checked using the ncbi blast software, and no significant alignment with any other animal virus gene was found piglets inoculated with virulent tgev shed the virus for h, followed by profuse diarrhoea that led the piglets to the verge of death - days after inoculation. the tgev load shed in the diarrhoea was . × copies at the th hour, peaked at . × copies at the th hour and then decreased until death in the pbs group (fig. ) . the recombinant lactobacillus vaccine group (pg: - mt d) exhibited the same trend. the tgev copy number was at the th hour, and the copies reached a peak at the th hour. the copy numbers were similar and followed the same trend after reaching the peak. however, the copy numbers in the pg: - mt d group were significantly lower than in the pbs group. next, we investigated the generation of lactobacillus vaccineinduced regulatory cells after infection in piglets. as shown in immunized with pg: - mt d and pbs piglets were orally challenged with tgev. tgev-challenged pigs received a -ml dose of × plaqueforming unit (pfu)/ml via oralgastric gavage on days postinoculation. pigs (control group) were administered volumematched virus-free st cell culture media. all piglets were euthanized at days for necropsy examination fig. , there was a marked increase in the production of il- in cd + t cells. the th immune response induced by the vaccine was seriously broken in favour of th -mediated systemic and mucosal immunity post-infection. the systemic th immune response was higher than the mucosal th mediated immune response. after tgev infection, the body activated more th to protect itself in response to the infection. as shown in fig. , toll-like receptor (tlr) expression was detected in the splenic lymphocytes (sl) and mesenteric lymph node cells (ml) in the piglets in the pbs and vaccine groups. tlr- was higher in the vaccine group than in the pbs group. in contrast, there were no significant differences between tlr- and tlr- . however, the three tlrs exhibited the same trends in the mesenteric lymph node cells compared with the pbs and lactobacillus vaccine. tgev infection induced tlr expression and especially enhanced tlr- and tlr- expression, but the expression levels in the lactobacillus vaccine group were significantly lower than the levels in the pbs group. cytokine expression in the splenic lymphocytes and mesenteric lymph node cells was also analysed and compared in our study. the ifn-γ, il- and il- levels in the splenic lymphocytes from the lactobacillus vaccine group showed marked changes, whereas no significant difference was observed in the level of tgf-β. tgev infection stimulated cytokine expression in the pbs group, including ifn-γ and il- . the vaccine group did not express a notably higher level of ifn-γ and il- compared with the pbs group. the tgf-β expression level in the mesenteric lymph node cells was lower in the vaccine group than in the pbs group; the same trend was observed with ifn-γ and il- . the vaccine group provoked higher il- expression than the pbs group following tgev infection. the il- expression levels in both the splenic lymphocytes probiotics are well known to have additive effects on human health in terms of improving the gut microflora and modulating immune responses (villena et al. ) . studies have also reported that probiotic feeding results in an increased spleen mass, followed by higher levels of total serum proteins, increased globulin levels and enhanced production of secretory iga (dock et al. ) . in humans, lactobacilli colonize the distal small bowel and the large intestine. different probiotic bacteria possess various mechanisms, including adhesins and/ or coaggregation factors, which aid in adhesion and colonization (friedrich ) . during this period, immunization with recombinant lactobacillus is crucially important on the effects of immunization, such as the timing of the first immunization in the protocol and the dose. from these results, we show that the immune response in response to the first priming immunization dose is better than the response to the second immunization because the priming dose is better at initiating adhesion and colonization in the piglet. interestingly, the mucins are large glycoproteins that are the major organic components of mucus. the mucin protein content of the mucus is %, whereas the carbohydrates comprise to % by weight. intestinal mucin has been shown to inhibit the replication of rotavirus in vitro (chen et al. ; superti et al. ). additionally, a high dose of lactobacillus adversely affects the immunization. there is some evidence of diarrhoea after a double dose of the lactobacillus vaccine, but from this result, we find that the two-dose immunization is still the best immunization plan. the reason for the diarrhoea after immunization is the overdose of lactobacillus, which is a type of microbe that causes a disturbance in the intestinal microbial flora for short time. many enteric pathogens must first adhere to the intestinal epithelial cells to initiate intestinal disease. limiting access of the pathogens to intestinal epithelial cells is one strategy to prevent disease that has been investigated previously. for example, the competitive inhibition of bacterial adherence by mimicry of receptors on the apical surface of enterocytes using oral administration of sialylated glycoproteins has been shown to protect newborn calves from the enterotoxigenic e. coli strain k (mouricout et al. ). lactobacillus must colonize the gut soon after birth; therefore, the vaccine could play a role in non-specific immunity. probiotic strains with a high adherence capacity have been demonstrated to enhance the immunoglobulin a response to rotavirus (kaila et al. ) . in this study, we observed a significant increase in the anti-tgev iga titre in the intestinal tract of piglets administered recombinant l. casei. furthermore, we showed that the diarrhoea state of piglets administered l. casei was significantly lower than that of piglets administered saline. in the murine model of ifv infection, the virus moves from the upper respiratory tract to the lower respiratory tract (hori et al. ) . hrv-vaccinated and lactobacillus acidophilus-fed pigs had a significantly higher magnitude of hrv-specific iga and igg antibody-secreting cell responses in the ileum and serum igg antibody and virus neutralizing antibody titres compared to hrv-vaccinated pigs without l. acidophilus colonization (zhang et al. ) . our immunization stimulated the same systemic specific igg titres. the specific antibody response neutralized the challenged tgev, and the load of tgev in the diarrhoea was significantly decreased. the mucosal immune response formed the first barrier function to neutralize tgev. in large scale swine farm surveillance, lower piglet birth weight and higher within-litter variability in birth weight were factors associated with higher losses from birth to weaning (yuan et al. ) . during tgev infection, it is likely that the stronger piglets obtained more iga than their non-immunized counterparts and thus were more likely to survive until the intestinal villi regenerated and immunity developed. during the histopathological analysis, some damage was observed in the small intestine. for instance, the villus wall of the control groups was thin and almost transparent, probably due to atrophy. lymphocyte proliferation in the intestinal lamina propria was also found in some piglets administered an oral dose of recombinant lactobacillus, indicating that recombinant lactobacillus induced a mucosal immune responses in the piglets. taken together, our data show the tremendous potential for recombinant lactobacillus to enable rapid and effective treatment for tgev infection with intestinal tropism in piglets. we evaluated the specific t cell immune responses induced by the recombinant l. casei vaccine compared with the pbs control group in the piglets. we demonstrated that l. casei significantly enhanced the immunogenicity of the tgev vaccine as indicated by the significantly higher magnitude of specific ifn-γ-producing cd + t cell responses. there has reported that mice fed recombinant l. casei with the adjuvant mdp and tuftsin have significantly higher th and th production than control group mice (jiang et al. ) . these results were the same and indicated that l. casei had a strong potentiating effect on both the cellular and humoral immunity induced by the oral l. casei vaccine. similarly, a previous study showed that oral intake of l. fermentum cect fig. cytokines and tlr expression. the rna of splenic lymphocyte (sl) and mesenteric lymphocyte (ml) in immunized piglets, pg: - mt d and pbs groups, were used to analysed the cytokines and tlr expression. ifn-γ, il- , il- , tgf-β, tlrs expressing were detected in splenic lymphocyte (sl) and mesenteric lymph cells (ml) piglets, such as pbs and vaccine groups. *significant difference by student t test (p < . ). data shown were compared using one-way analysis of variance (anova) followed by duncan's multiple range test. representative for three independent experiments significantly enhanced serum th type cytokine production and influenza-specific iga antibody responses to an intramuscular influenza vaccine in adults (olivares et al. ) . the mesenteric lymph nodes were primarily used to analyse tgev infection and the immunoprotection provided by the lactobacillus vaccine in terms of mucosal immunization and infection. ifn-γ induction by tgev results from interactions between an outer membrane domain of el and the pbmc membrane (charley and laude ) ; however, these authors did not study the expression of il- by pbmcs. the expression of ifn-γ was higher than il- in the immunized group, and the th /th balance was broken in our study. after immunization with recombinant lactobacillus, ifn-γ played a major role in the mucosal immune response. however, after tgev infection, the systemic and local immune responses shifted from th to th . the systemic humoral immune response was stronger than the cellular immune response after tgev infection. this is the first study to demonstrate that tgev infection polarized the immune response to th immunity and that recombinant lactobacillus could weaken tgev infection in the form of th immunity. from these results, we found that the immunization did not polarize th immunity more seriously compared to the pbs control group. the proteinbased sars coronavirus vaccine boost induced similar levels of th and neutralizing antibody responses that protected vaccinated mice from subsequent sars-cov challenges but induced stronger th and ctl responses (zheng et al. ) . the uv-inactivated sars coronavirus vaccine retained its immunogenicity and promoted th type immune responses (tsunetsugu-yokota et al. ). the activation of th responses such as il- stimulate b cell proliferation, which can produce specific and nonspecific anti-infection antibodies (grodeland et al. ) ; similarly, both t and b cells have functions following lactobacillus vaccination. the production of il- by th cells results in the proliferation of mast cell growth, and il- stimulates epithelial cell growth (tukler henriksson et al. ) . the proliferation of epithelial cells is crucial for tgev infection. the th response could also stimulate the production of mucus by epithelial cells (zhang et al. ) . il- cells play an essential role in mhv-induced immunopathology, and ifn-γ is important for maintaining the immune balance between th and th responses during acute viral infection (yang et al. ). however, the th /th balance in the negative control group was different than the balance in the immunized group. tgev affects both systemic and local cellular immunity without immunization. il has been reported to have both pro-and anti-inflammatory effects (lafdil et al. ; nagata et al. ) . our results showed that the immunized piglets provoked il- expression form both the systematic and mucosal immune responses after tgev infection. compared with the mucosal immune response, il- expression in the mesenteric lymph nodes was markedly lower than the expression in the spleen cells. however, il- expression in the pbs group was lower than the expression level in the immunized groups, indicating that the lactobacillus vaccine group activated il- expression during tgev infection. swine-origin influenza a virus-infected patients exhibited rapid lymphopenia, t cell activation and a preferential loss of the th subset during the early stage of acute infection (jiang et al. ) , which was consistent with the first reports that swine-origin virus inhibited th proliferation after infection. our study also found the same phenomenon after coronavirus infection in swine compared with the immunized group. the most important finding was that the oral recombinant lactobacillus vaccine stimulated th cell proliferation. the proliferation was involved in cytokine and chemokine production, neutrophil recruitment, promotion of t cell priming and antibody production (dirisala et al. ) . th responses are protective against lethal influenza virus infection in il- deficient mice (mckinstry et al. ). in contrast, a deleterious role of il- has been proposed to contribute to the acute lung injury associated with il- -mediated neutrophil recruitment during influenza virus infection (crowe et al. ). there were significant changes in the il- and ifn-γ expression levels in the mesenteric lymph node cells compared with the pbs group. moreover, il- expression was higher than ifn-γ expression in the spleen cells. the expression of il- and ifn-γ indicated that the recombinant lactobacillus effectively inhibited inflammation after tgev infection. in this study, we also evaluated tlr- , tlr- and tlr- expression in piglets immunized with recombinant l. casei and then challenged with tgev. previously, tlr expression in pigs has been studied only at the mrna level in lymphocytes using real-time pcr because antibodies against porcine tlrs are not currently available. tgev infection did not induce tlr- , tlr- and tlr- expression in the spleen cells. however, tlr expression was significantly different in the mesenteric lymph node cells compared with the pbs and lactobacillus vaccine groups. tlr expression was extremely high in the pbs group compared with the other groups. the cytokine and tlr expression levels in the splenic lymphocytes are indicators of systemic immunity, whereas the expression in the mesenteric lymph node cells was associated with the local and mucosal immune responses. the expression levels of all tlrs in mesenteric lymph node cells were higher in the pbs group than the vaccine group, suggesting that the lactobacillus vaccine effectively inhibited tlr expression. the recombinant lactobacillus groups exhibited jejuna and ilea with lymphocyte proliferation in the lamina propria. transmissible gastroenteritis (tge) coronavirus infection resulted in antibody production from primed mesenteric lymph node cells following an in vitro boost with viral antigen (berthon et al. ); as an intestinal infectious disease, tgev would attack the intestinal tissue and local immune system. exposure of pigs to tgev or prcv results in distinct disease patterns related to differences in tissue tropism (saif ) . however, the increased frequencies of tlr- and tlr- expression in pigs may simply be due to the significantly higher counts of l. casei or mdp and tuftsin, which may translate to an increased magnitude of tlr agonists available to stimulate the host mucosal immune system. it is likely that the higher lab count in the lab plus hrv group played a more pertinent role in the significant increases in tlr and tlr expression (wen et al. ). taken together, tgev immune protection was primarily dependent on the mucosal immune response. systemic immunity did not play a key role after tgev infection. interestingly, il- expression in the vaccine group was significant higher than il- expression in the pbs group challenged with tgev, and th played an anti-inflammatory role in mucosal immunity. il- also stimulated intestinal epithelial cell differentiation and growth. in conclusion, our study suggests that the recombinant lactobacillus vaccine provokes specific mucosal and systemic immune responses to protect piglets from infection. iga played a dominant role in the mucosal immune response after tgev challenge. therefore, the histopathology and rna copy numbers directly demonstrated that the lactobacillus vaccine was effective for tgev infection. the protective efficacy was significantly higher, which would have great value in practice. moreover, although the recombinant lactobacillus vaccine-induced th immunity, the immune 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and infections transmissible gastroenteritis virus infection: a vanishing specter probiotic effects on inflammatory bowel disease effect of polyions on the infectivity of sa- rotavirus in lcc-mk cells effects of interleukin- alpha administration on intestinal ischemia and reperfusion injury, mucosal permeability, and bacterial translocation in burn and sepsis formalin-treated uv-inactivated sars coronavirus vaccine retains its immunogenicity and promotes th -type immune responses il- stimulates proliferation and expression of mucin and immunomodulatory genes in cultured conjunctival goblet cells yoghurt accelerates the recovery of defence mechanisms against streptococcus pneumoniae in protein-malnourished mice mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria toll-like receptor and innate cytokine responses induced by lactobacilli colonization and human rotavirus infection in gnotobiotic pigs interferon-gamma negatively regulates th -mediated immunopathology during mouse hepatitis virus infection construction of recombinant lactobacillus casei efficiently surface displayed and secreted porcine parvovirus vp protein and comparison of the immune responses induced by oral immunization within-litter variation in birth weight: impact of nutritional status in the sow probiotic lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs tmem a-mediated mucin secretion in il- -induced nasal epithelial cells from chronic rhinosinusitis patients studies of sars virus vaccines critical role of the interleukin- /interleukin- receptor axis in regulating host susceptibility to respiratory infection with chlamydia species acknowledgments this work was supported by the national natural science foundation of china ( ). conflict of interest the authors declare that they have no competing interests.ethical statement the piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. this article does not contain any studies with human participants performed by any of the authors. key: cord- -tguhrqvz authors: zavattaro, staci m.; hall, jeremy l.; battaglio, r. paul; hail, michael w. title: introduction: covid‐ viewpoint symposium, part ii date: - - journal: public adm rev doi: . /puar. sha: doc_id: cord_uid: tguhrqvz nan communication, political astuteness, and collaborative capacity. he offers practical suggestions, such as maintain network control and building social media skills, which are best practices in administrative structures and even more valuable in a crisis. taking a european view, bouckaert et al ( ) introduce the term -coronationalism‖ to explain the differences in response between belgium, france, germany, and italy. a nationalistic approach pushed european union ideals to the side as countries struggled with virus containment. they highlight the importance of institutional and cultural variance, points we argue are crucial for future studies about the virus response. christensen and laegrid ( ) focus also on europe, using norway as an in-depth case study. similarly to bouckaert et al ( ) , these authors point out cultural and political wills that made strong response possible. they draw attention specifically to the relationship between democratic legitimacy and government capacity, noting perception is key when it comes to crisis response. this point meshes with van der wal's ( ) recommendations for engaging stakeholders to build trust prior to cataclysmic events. charbonneau and doberstein ( ) consider work surveillance practices and their relationship to performance management with so many public servants being forced to shift to remote working from home in response to the covid- pandemic, such oversight is increasing in importance. their article presents the results of three surveys conducted during the onset of the covid- pandemic in canada that compare public servant and citizen attitudes to various cutting-edge digital surveillance tools that can be used to monitor employee work patterns. the resulting data can help governments navigate difficult questions of reasonable privacy intrusions in an increasing digitally-connected workforce. schuster et al ( ) introduce a survey instrument to study covid- via the world bank's bureaucracy lab. the survey is already deployed in several countries, and the authors are open to collaboration with others as they argue survey data are important to gather during the ongoing situation that is testing administrative capacity. in their study, dai et al ( ) take the china case and examine the crucial need for reliable, timely, and persuasive public information to gain compliance with prevention measures. using survey data of citizens throughout china, the authors find detailed pandemic information, positive risk communication, rumor refutation, and adequate supplies combined to help people engage in protective behaviors. future research could study this model in other contexts taking culture into account as bouckaert et al ( ) suggest. one of the most widely used and scientifically backed measures to slow virus spread remains social distancing, and in their essay, pedersen and favero ( ) survey americans to find what factors might influence compliance with social distancing. their results highlight again the critical role of crisis communication and reliable public information, coupled with prosocial motivation. the study provides insights into behavioral adaptations similar to dai et al ( ) , and both studies provide rich avenues for future research especially when it comes to the nudging behaviors crucial for crisis communication. this article is protected by copyright. all rights reserved. taking a bit of a turn, fay and ghadimi ( ) examine labor relations and what changed during and because of covid- . labor relations is a critical management strategy, and they outline evidence-based strategies for engaging in meaningful labor negotiations during crisis, focusing on emphasizing needs of the most vulnerable first, negotiating early and regularly with leaders, and securing protections for all members. their essay brings to light additional streams of research regarding labor negotiations during times of social and economic crisis. without question, the pandemic has exacerbated social inequalities and brought about what some are calling the dual pandemic of covid- and systemic racism (mccandless & zavattaro, ) . wright and merritt ( ) explicitly link systemic racism and the covid- response. health inequities, segregation, food insecurity, underrepresentation in the medical profession, and exclusion from full participation in democratic ideas and ideals contribute to the inequitable response to covid- . in the end, the authors outline a path forward for practice and research, the latter of which outlines an agenda for better incorporating social equity meaningfully into administrative studies. similarly to wright and merritt ( ) , gaynor and wilson ( ) use the social vulnerability index to examine the effects of racism on covid- deaths. they argue black people are historically segregated into vulnerable communities, and the pandemic is visibly highlighting these systemic inequities. as a path forward, they offer targeted universalism to develop inclusive policiesoffering a lot of potential for future research. deslatte, hatch and stokan ( ) explain the critical role local governments can play in addressing some of the inequalities. for instance, the community development block grant and energy efficiency and conservation block grant programs can be ways to equitably rebuild marginalized communities. also highlight the health inequities, martin-howard and farmbry ( ) use the social determinants of health theory to discuss strategies for mitigating these inherent inequities. using the bronx, new york as a case study, the authors examine the ways in which unequal access to adequate medical care increased community vulnerability and death among marginalized communities. like gaynor and wilson ( ) , they offer questions for future research aimed at mitigating these ingrained problems. using refugee migration as the object of performance, schomaker & bauer ( ) seek to understand patterns of administrative performance by examining networks and knowledge management within and between crises. drawing from two german public administration surveys their results demonstrate that those administrations that were structurally prepared, those which learned during preceding crises, and those that displayed high quality network cooperation with other administrations and with society more broadly performed significantly better in the respective crises. this article is protected by copyright. all rights reserved. rounding out the viewpoint symposium are contributions centering on coproduction and partnership. complex and wicked problems such as covid- require collaboration and coordination, and these essays offer insights and paths for future research. steen and brandsen ( ) note how the pandemic necessitated this kind of coordinated response, and they pose an important question that should guide research for years to come: will it last? they argue commitment to the partnerships, supportive regulatory frameworks, sustained funding, legal flexibility, and incentives should keep the collaborations moving forward, but that remains to be seen as the crisis still unfolds globally. the essay provides a clear path for examining their propositions in future research. using lessons learned from the haiti earthquake, entress, tyler and sadiq ( ) provide practical steps for building community resilience related to mass fatality management (mfm). the covid- pandemic showed the crippling holes in existing mfm plans, and the authors, using lessons learned from the earthquake, recommend increased collaboration, psychological support, and network leads when it comes to developing viable mfm plans going forward. similar to deslatte et al ( ) , wilson et al ( ) look at the local government collective action and engaging in economically focused partnerships to mitigate damaging effects from the pandemic. they offer economic development approaches requiring coordination, and like the other essays in this viewpoint symposium offer a path for future research to see if these strategies work, such as active versus passive partnerships. cheng et al ( ) explain the role that community-based organizations in zhejiang province in china played in responding to the pandemic. looking at what worked in that province, the authors offer four points for future research and practice when it comes to community-based organizations: strategically leverage strengths of community-based organizations; incentive volunteers to participate in prevention and control; provide technology that can facilitate effective response; build trust and long-term capacity for response. cultural contexts also can underlie these findings as well, so future research can examine that aspect of community-based organizations. shi et al ( ) detail the role of nonprofit organizations in providing critical response, focusing specifically on the homeless population. using interviews from four dallas, texas-area nonprofit leaders, they find each organization experienced disruptions to mission and service delivery. they also faced serious ambiguity that limited how staff could respond, forcing innovation and creativity. the authors introduce the disruptions, ambiguity, innovation, and challenges framework based on these interviews, giving practitioners immediate ideas and scholars a path forward for additional research. in their article, van den oord et al ( ) use the case of the antwerp port authority to showcase network governance's strengths and limitations. when the pandemic hit, port authority leadership needed to change network structures to succeed. the dynamism of the network allowed for brokering to take place, bringing in experts who could round out the response. network flexibility and dynamism seems to be an emerging theme ripe for future research. this article is protected by copyright. all rights reserved. finally, hu et al ( ) look at paired assistance programs in china to show differences in hierarchical versus network approaches to emergency management response and coordination. the intricacies involved in their study highlight again the need for network flexibility to foster positive response to crises. as you review these contributions, the next chapter in this pandemic will have already been written. lives will have been lost. elected officials will have been deeply criticized for their efforts. public managers will have worked seemingly endless hours, with many wearing thin and others growing disenchanted as the end seems further and further from sight. strategies will have been proven; others will have been disproven. the price of liberty and the value of security will have been weighed out more accurately in different ends of the earth. decisions will have been made with dire consequences to health or to the economy. the tradeoffs inherent in the rapid-fire nature of executive-driven policy decisions are real, and they are perceived more clearly by individuals than at any time in recent history. elected officials' choice of frames to describe and respond to the problems generate strategies and actions that will be more strongly influenced by their framing than by prevailing evidence. as the research articles appearing earlier in this issue demonstrate, elections have consequences, and the impact of this pandemic on the outcome of the u.s. general election may be its most significant enduring effect. each party seems to have a dominant frame that will guide the policy responses to be advanced as they prepare to take office in . our sincere hope is that the evidence presented here will be used to influence many of those decisions. we hope that it will stimulate research that will reshape the way we understand many of the core concepts of our discipline. as the response continues, we will continue to seek out work that makes ever increasing conceptual advancements. while we saw many patterns across the articles in our two covid- symposium issues, we also noticed some elements missing or needing stronger attention. for instance, while our contributors were intentionally global, voices from the global south need amplification regarding pandemic response. also, while some of the authors noted the cultural differences between countries and pandemic response, more could be done regarding administrative functions within those varied systems. some of the pieces necessarily took a broad view, and future research can drill further down into the relationship between culture and administrative response. finally, a collection of articles in this issue focus on social justiceand rightly sobut broadening that lens to be more intersectional and inclusive would help spur additional research. administrative burdens need to be better understood across subsets of the population as equality in the pandemic response is pursued. european coronationalism? a hot spot governing a pandemic crisis an empirical assessment of the intrusiveness and reasonableness of emerging work surveillance technologies in the public sector coproducing responses to covid - with community-based organizations: lessons from zhejiang province, china balancing governance capacity and legitimacy: how the norwegian government handled the covid - crisis as a high performer the effects of governmental and individual predictors on covid - protective behaviors in china: a path analysis model how can local governments address pandemic inequities? managing mass fatalities during covid - : lessons for promoting community resilience during global pandemics collective bargaining during times of crisis: recommendations from the covid - pandemic social vulnerability and equity: the disproportionate impact of covid - global reflection, conceptual exploration, and evidentiary assimilation: covid - viewpoint symposium introduction hybrid coordination for coping with the medical surge from the covid - pandemic: paired-assistance programs in china framing a needed discourse on health disparities and social inequities: drawing lessons from a pandemic editors' introduction: since we last spoke network of networks: preliminary lessons from the antwerp port authority on crisis management and network governance to deal with the covid- pandemic social distancing during the covid - pandemic: who are the present and future noncompliers? what drives successful administrative performance during crises? lessons from refugee migration and the covid- pandemic responding to covid - through surveys of public servants nonprofit service continuity and responses in the pandemic: disruptions, ambiguity, innovation and challenges co-production during and after the covid- pandemic: will it last? being a public manager in times of crisis the art of managing stakeholders, political masters, and collaborative networks institutional collective action during covid- : lessons in local economic development social equity and covid - : the case of african americans this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. key: cord- - mu rrql authors: fakhruddin, bapon; blanchard, kevin; ragupathy, durga title: are we there yet? the transition from response to recovery for the covid- pandemic date: - - journal: nan doi: . /j.pdisas. . sha: doc_id: cord_uid: mu rrql abstract there is no corner of the planet that has not been impacted by the rapid spread of the novel coronavirus, covid- . while the covid- pandemic has already had far-reaching socioeconomic consequences commonly associated with natural hazards (such as disruption to society, economic damage, and loss of lives), the response of governments around the world has been unparalleled and unlike anything seen before. governments are faced with a myriad of multi-dimensional effects of the pandemic, including direct impacts on public health systems and population health and indirect socioeconomic effects including disruption to every single sector of the economy and mass unemployment. there is, additionally, the growing realisation that the timescale associated with this crisis may permanently change the very foundations of societies ‘normal’ day-to-day life. as the world transitions to recovering from covid- , those developing that recovery need support in adjusting and improving their policies and measures. the situation seems dire, the stakes are high. literature about the transition between the response and recovery phase in relation to pandemics is scarce. further complication is that the pandemic will not allow countries to simply transition to the full-scale recovery, instead, a rebound from recovery to response phase is expected for a certain period until the immunization is in place. pandemics indeed force us to think beyond typical emergency management structures; the cycles of the disaster risk management in the case of biological and other natural hazards are not exactly the same and no one-size-fits-all approach may be used. still, some parallels may be drawn with the efforts to combat natural hazards and some lessons may be used from previous and the current pandemic. based on these experiences and reflections, this paper provides a set of policy directions to be considered during the transition towards, as well as throughout, this transition phase. it is suggested that meeting this global, multi-dimensional, and complex challenge will require considerable international collaboration (even convention) and macro-scale changes to global and national policies. the recovery issues are mainly going to be dominated by politics, economics and social science. necessary for an effective recovery, the pandemic response needs to be a holistic response, combined with an improved data ecosystem between the public health system and the community. we should also view this outbreak and our response to it as an opportunity to learn lessons and reaffirm our universal commitment to sustainable development and enhancing wellbeing around the world. governments are faced with a myriad of multi-dimensional effects of the pandemic, including direct impacts on public health systems and population health and indirect socioeconomic effects including disruption to every single sector of the economy and mass unemployment. there is, additionally, the growing realisation that the timescale associated with this crisis may permanently change the very foundations of societies 'normal' day-to-day life. as the world transitions to recovering from covid- , those developing that recovery need support in adjusting and improving their policies and measures. the situation seems dire, the stakes are high. literature about the transition between the response and recovery phase in relation to pandemics is scarce. further complication is that the pandemic will not allow countries to simply transition to the full-scale recovery, instead, a rebound from recovery to response phase is expected for a certain period until the immunization is in place. pandemics indeed force us to think beyond typical emergency management structures; the cycles of the disaster risk management in the case of biological and other natural hazards are not exactly the same and no one-size-fits-all approach may be used. still, some parallels may be drawn with the efforts to combat natural hazards and some lessons may be used from previous and the current pandemic. based on these experiences and reflections, this paper provides a set of policy directions to be considered during the transition towards, as well as throughout, this transition phase. it is suggested that meeting this global, multi-dimensional, and complex challenge will require considerable international collaboration (even convention) and macro-scale changes to global and national policies. the recovery issues are mainly going to be dominated by politics, economics and social science. necessary for an effective recovery, the pandemic response needs to be a holistic response, combined with an improved data ecosystem between the public health system and the community. j o u r n a l p r e -p r o o f the consequences of any new virus are always unforeseen and become more and more multisectoral as time compounds them, at all levels, from an individual to the macro level . these consequences, although uncertain, can be reduced with sound policy. however, the policy for pandemic crisis is unlike that applied to natural hazards. pandemics force us to think outside of the box, or in this case, outside the typical emergency management cycle (figure ) . in other words, while four phases to reduce the impact of a natural hazard (preparedness, response, recovery, and mitigation) are principally linear, the response and recovery phases for a pandemic are essentially non-linear. to illustrate, unlike earthquakes, cyclones or other natural hazards, which are usually one-off events occurring within a limited period, covid- or any other pandemic, tend to come in several waves over a protracted period -until the effective vaccine or herd immunity is in place such as influenza pandemic and ebola virus disease occurred in multiple waves (saunders-hastings & krewski, ) . also, natural hazards tend to impact relatively confined areas. the covid- outbreak was declared a global pandemic by the world health organization (who) on march and already in april , the virus has affected out of countries (johns hopkins university, ). a response to biological and natural hazards follows the same disaster risk management cycle -but in the case of pandemics, alert and pandemic phases (reporting the increase of the global average of cases, with the highest number in the pandemic phase) correspond to the response phase of disaster risk management, while a transition phase (reporting the significant decrease of the global average of cases) correlates with the recovery from a disaster. biological disasters present challenges for both the response and recovery stages due to their sheer complexity (jeremias & martin, n.d.) . due to the very nature of pandemics, there will be a constant change in the way we approach and implement organisation structure, tools and technologies, as well as emergency management, recovery, and business continuity plans. each new wave of a at an individual level, a person's income, access to food, other health care (outside of that related to the pandemic) are all impacted. at the macro end of the consequences, financial structures (of both public and private institutions), political stability (or instability and tensions) are impacted. j o u r n a l p r e -p r o o f pandemic is distinct, where the same approach in policy may not be applicable, and revisions after each wave may be required to minimise the new consequences (figure ). transitioning from pandemic response to recovery in a spiral fashion: there is a high probability of the second and even the third wave of a pandemic if there is no vaccine or immunization, each new wave pushing the disaster risk reduction cycle from the recovery back to response phase. in the midst of the covid- emergency, on march , zagreb, croatia was hit by a . magnitude earthquake, as the country dealt with covid- patients (walker, ) . in canada, manitoba province and ottawa city fought against flooding caused by snow melting. a month later, cyclone harold wreaked havoc on the solomon islands, fiji, vanuatu and tonga, placing extra stress on their already stretched governments, as the need for additional funds, medical supplies and food rose (lyons, ) . in vanuatu, this was further strained by the measures put in place for the covid- response (sbs, ) . while local governments in japan suspended receiving volunteers engaged in rehabilitation works in areas devastated by typhoon hagibis in , the bangladesh government prepared for both cyclones and a covid- outbreak in the cox's bazar camps which shelter some , rohingya refugees (ishiwatari et al., ) . the world is facing the same type of difficulties to manage natural hazards as pre-pandemic whilst also investing in considerable efforts to flatten the covid- curve. and yet, with rapidly changing climate and ecology, hastily marching urbanisation, and increased travel, pandemics will become j o u r n a l p r e -p r o o f more frequent and more complex. perhaps the single common thread of pandemics, cyclones, floods and earthquakes is that they become ever more intense, magnified and complex in nature. fundamentally the issues to respond to these events are political at a number of levels including within the scientific community, with competing interests between the various sciences (social, biological and health-based) particularly noticeable. this will be no different in the recovery phase. ensuring consideration at all levels on government response, policies, plans and logistics regarding the process in which they respond to other emergencies during the pandemic, whilst ensuring scientific harmony, is crucial in ensuring effective response, recovery times, and financial and political stability. the existing framework of disaster risk management policies (national, regional and global) has largely been designed to ensure a swift and effective recovery along the traditional disaster risk management cycle. the sendai framework for disaster risk reduction, agreed in , focused the attention of signatory member states on preparing for a range of hazards including those of a geophysical, hydrological, climatological and meteorological nature. importantly, it also included a strong public health focus and consideration of the risks from biological hazards (maini, et al, ) . however, the sendai framework did not distinguish the cascading impact of biological and other hazards in a systematic risk management approach. the emerging thinking is that the concept of pandemic must evolve from crisis response during discrete outbreaks to an integrated cycle of preparation, response and recovery (bedford et al., ) . the recent global risk assessment framework (graf) promises to inform and focus action within and across sectors and geographies by decision makers at local, national, regional and global levels to improve the understanding and management of current and future risks, at all spatial and temporal scales (gordon, ). despite the two-decade experience of public health crises caused by novel virus infections (such as hiv, influenza a virus subtype h n and h n , sars-cov , mers-cov and ebola), the epidemiological novelty of covid- and its rapid spread caught many governments unprepared . the response of all governments, organisations and individuals at all levels, national or internationals, has been put to the test. a number of governments around the world failed to act on their warnings about a pandemic due to not understanding the magnitude of the problem, so-called 'strong man' politicians, lack of updated or public risk registers and other various reasons (tyler and gluckman, ). as the world continues to combat the covid- pandemic impacts, rapid evaluations of the strategies of various countries provide insights for all stakeholders to adjust and improve policy options. not all strategies have yielded positive results; in fact, while some were commended as effective (such as the response from singapore, germany and new zealand), others were criticised as devastating (for example, high infection and death rates in iran, italy, south korea, and the united states) . as an illustration, singapore's approach to the covid- outbreak has been rated as effective as it managed to keep both the infection rates low and the economy strong (lai & tan, ) . one of the key success factors of singapore's efforts has been assigned to the fact that singapore has learned from their recent pandemics experience. in , singapore experienced an outbreak of hand-foot-mouth disease, which affected more than , young children, causing three deaths. later in , sars hit singapore, infecting people, of whom died of this disease. in , avian influenza h n struck singapore, affecting , people with deaths (lai, a. y., & tan, s. l., ) . one of the most important lessons that the singapore government learned from the sars epidemic was the detrimental role of j o u r n a l p r e -p r o o f the bureaucratic structure in handling fluid and unprecedented situations. recognising that the effective response and recovery for covid- depends on a large number of stakeholders ("the health of all depends on each of us", https://www.gov.sg/), the singapore government has led a well-coordinated, multi-stakeholder response and recovery, which has been praised by other countries. however, singapore paid far too little attention to their hundreds of thousands of migrants living in crowded dorms, excluded from government initiatives, among whom sars-cov is now spreading quickly (ratcliffe, ) . sadly, this was also reported in germany where there was a rise in cases where refugees were not accommodated properly (oltermann, ) . new zealand's success factors were determined leadership and bold action early in the pandemic outbreak. because of early and strong action, only a small number of new zealanders got infected, were tested, contact was traced, and they were isolated. this prevented widespread transmission overwhelming the medical system along with the inherent effects such as widespread shortages of tests and personal protective equipment. experiences from the previous pandemic crisis offer more insights, and the responses of different governments may broadly be categorised as effective or ineffective (table ) . briceño, ; lai and tan, ; and spiekermann et al., ) . lack of knowledge on how to disseminate information correctly weak community vigilance and lack of public education measures lack of collaboration between major parties with the lack of risk management integration into major sectors (e.g., health, infrastructure, tourism, environment) evidence-based decision making, with the effective use of big data lack of support to community in lockdown j o u r n a l p r e -p r o o f parallels may also be made between the effectiveness of the country's response to pandemics and to natural hazards. for instance, the response to the devastation caused by the hurricane maria in dominica and the surrounding caribbean nations was characterised by most of the ineffective responses from table . the combination of poor technology and data collection systems, inadequate reporting, lack of public awareness and inclusiveness led to poor information being disseminated to people during the hurricane; this in turn inhibited people from preparing and therefore led to inaction and placed people at greater risk to the hurricane (tonkin & taylor international limited, ). these very factors seem to also arise when reviewing the responses to covid- in countries such as iran and italy . the primary mission during the response phase is saving lives and livelihoods (jeremias & martin, n.d.) . this, as seen from the response of many governments to the covid- outbreak, relies on the communications policy. the limited capacity for vaccinating against a novel coronavirus in the early stages results in a more concerted policy direction around communication of public health guidance and instructions (including lockdown and social distancing) (dickmann et al, ) . this policy is often targeted at the most vulnerable community members or is aimed at informing the wider public audience about the risks to the most vulnerable (dickmann et al, ) . indeed, much of the work in the response phase of a biological disaster revolves around the dissemination of information at national or regional levels (figure ) . one of the tried-and-tested approaches to responding to pandemic disasters is the epidemiological process of understanding disease transmission, including by contact tracing (o'sullivan et al, ) . this type of response differs to that of a geophysical hazard in that it is concerned with individuals, rather than the wider picture (lee & riley, ) . while this strategy requires a considerable amount of resources, it has been effective in reducing the impacts of an outbreak (for example, in south korea), implemented along with wide-scale testing and containment (mccurry, ). while many countries are only just managing the multiple aspects of the response phase, the covid- outbreak will very likely subside eventually (through herd immunity or vaccination) and there will be a period of recovery. policy for the recovery from pandemics will differ from traditional recovery in a number of ways, largely due to the global scale and sheer complexity of covid- pandemics (furnival, ) . one of the initial policy manoeuvres many governments will want to make will be returning economic activity to normal (or as close to normal as possible). this effort to restart trade, manufacturing, and services will differ from those during natural hazards in both scale and complexity (table ) . table : some elements of the response and recovery phases during natural hazards (hydrometeorological and geohazards) and biological disasters (from pandemics) are the same -but each needs to be carefully considered and specified unless or until we acquire full natural or vaccine-induced immunity to covid- , or if an eventual vaccine is only partially protective, we face the threat of a new endemic disease that will fundamentally change human society. it sounds alarmist -we admit -but it is becoming distinctly possible, based on what we have learned so far. like the human immune system, whose reaction may sometimes be part of the cause of death from the viral disease -reactions of all stakeholders and the public to the pandemic can have positive as well as negative consequences. therein lies the greatest risk and, with it, the strongest need to remain calm and in control. we are now in a global, high-risk situation and the direct threat of covid- may not turn out to be our biggest problem. as the world is transitioning to recovery, the following policy considerations are important and need to be considered: picture the governments playing whack-a-mole , hammering the new waves of virus reemerging in hotspots with the yet again forced stay-at-home measures, reactivated temporary hospitals, sourcing extra healthcare personnel and personal protective equipment. this is indeed an unwanted but warranted scenario. as with fire, it is not a question of "if?" but "when?". wildland fire suppression personnel and equipment serve as an excellent model for preparedness levels and mobility of resources throughout the fire season. countries shall be in this mindset for pandemics too. covid- hotspot outbreaks may be managed in the same way, with the inclusion of testing, monitoring, and contact tracing. data-based modelling may provide a framework within which the details are hammered out among key stakeholders (government, private, and non-governmental organisations). our concept of pandemics shall move to an interdisciplinary science, with an integrated approach of medical science and public health with medical research and development, social sciences, diplomacy, biomedical science, big data, information technology, artificial intelligence, statistics, meteorology, biotechnology, ecology and so on -combined to provide an integrated cycle of prevention, preparation, response and recovery. the international community should recognise the opportunity afforded them through already agreed policy frameworks such as the sendai framework for disaster risk reduction, united nations' sustainable development goals, and climate change conventions and agreements. these policy tools have been largely agreed and ratified in each of the countries impacted and as such, many of the processes to respond, monitor and recover are already established within domestic legislations. building recovery around these frameworks can also provide a greater level of cohesion among member states who may be at different stages of the pandemic. we have seen a number of countries reducing the level of public funding for their healthcare systems or internationally focused biological observation and scanning initiatives. these funding reductions have been cited as one reason the covid- pandemic has been so destructive. governments and international organisations should use the opportunity of j o u r n a l p r e -p r o o f recovery and demand from their public to invest in measures to ensure future novel viruses or biological threats are identified early. beyond health risks, depending on the characteristics of the pathogen, pandemics cause risks and related disasters in multiple sectors such as agriculture, public transport, logistics, finance and security. a very lethal pathogen could cause a total global shutdown, resulting in famine or another unforeseeable secondary disaster. because each new pathogen usually has unknown characteristics, the response decision making is highly flawed and error-prone. the pandemic response needs to be a holistic response. multi-sector pandemic planning and active drills with representatives from different sectors can help countries and cities to prepare for such complex chains of decisions and consequences. international communication, cooperation, collaboration, and even established convention should be considered, reconsidered and strengthened for prevention of pandemics. leadership (in terms of financial support and policy development) will need to come from established international organisations such as the world bank, asian development bank and united nations, and groupings like the g and g . while advanced organisations and groups will need to lead these efforts (they have the financial capacity and resources to do so), the inclusion of developing countries and non-traditional agents (including nongovernmental organisations) is necessary and ultimately, inevitable. while a number of countries in recent years have taken domestic policies that have gone against international cooperation and some responses to the covid- pandemic have seen bilateral spats between countries, the scale and complexity of this crisis calls for an international recovery that will only be achieved through global cooperation at all levels. also, strengthening epidemiology, public health and laboratory capacity in low-and middle-income countries is essential though collaboration. j o u r n a l p r e -p r o o f cascading problems. in fact, the director-general of the who mentioned an infodemic, referring to unprecedented misinformation that spread faster and more easily than the virus, hampering a public health response. improved information flow between the public health system and the community is necessary for an effective recovery. the use of data from people is becoming strictly controlled, whereas contact tracing is needed to better understand how infections spread. a data ecosystem is critical to ensure a stable transition from the response to the recovery phase (fakhruddin, ) . a system shall be used, where communities feed information into the public health system and the feedback loop offers a fast and direct way to provide people with details of potential actions they can take. some have called the covid- outbreak a dress rehearsal for climate change. large-scale, global impacts on the economy and our day-to-day lives, those living in poverty being among the worst impacted, have been evidenced during the covid- outbreak but are also predicted (and in some cases, already happening) in relation to our changing climate. the international community, as well as individual countries, should use the lessons we are learning through the outbreak, to ensure we are better prepared for the changes happening already and to limit or stop the increasing levels of risk caused by climate change. the sendai framework for disaster risk reduction: renewing the global commitment to people's resilience, health, and wellbeing a new twenty-first century science for effective epidemic response what to expect after sendai: looking forward to more effective disaster risk reduction building resilience against biological hazards and pandemics: covid- and its implications for the sendai framework review and analysis of current responses to covid- in indonesia: period of biological risks to public health: lessons from an international conference to inform the development of national risk communication strategies: report of an international conference on risk communication strategies before, during, and after public health emergencies a data ecosystem to defeat covid- how can society recover from covid- ? alliance manchester business school is the response over? the transition from response to recovery in the health sector post-haiyan shifting the paradigm: introducing the global risk assessment framework (graf) managing disasters amid covid- pandemic: approaches of response to flood disasters & martin (n.d.), h. bio-hazard disaster risk governance through multi-agency cooperation dashboard data retrieved on bridging the gap between evidence and policy for infectious diseases: how models can aid public health decisionmaking disease modeling for public health: added value, challenges, and institutional constraints impact of disasters and disaster risk management in singapore: a case study of singapore's experience in fighting the sars epidemic public health and disasters cyclone harold batters fiji on path of destruction through pacific. the guardian the sendai framework for disaster risk reduction and its indicators-where does health fit in? test, trace, contain: how south korea flattened its coronavirus curve. the guardian refugees in german centre fear lack of protection as covid- cases soar. the guardian from sars to pandemic influenza: the framing of high-risk populations uncertainties in biological responses that influence hazard and risk approaches to the regulation of endocrine active substances. integrated environmental assessment and management we're in a prison': singapore's migrant workers suffer as covid- surges back. the guardian reviewing the history of pandemic influenza: understanding patterns of emergence and transmission vanuatu needs aid after cyclone harold, but there are fears it could cause a coronavirus emergency the disasterknowledge matrix -reframing and evaluating the knowledge challenges in disaster risk reduction governance, technology and citizen behavior in pandemic: lessons from covid- in east asia coronavirus: governments knew a pandemic was a threathere's why they weren how korea responded to flattening the curve on covid- provision of coordination and standardization services of early warning systems in dominica zagreb hit by earthquake while in coronavirus lockdown. the guardian key: cord- -m axi k authors: leigh, laurasona; taylor, colleen; glassman, tavis; thompson, amy; sheu, jiunn-jye title: a cross-sectional examination on the factors related to emergency nurses’ motivation to protect themselves against an ebola infection date: - - journal: j emerg nurs doi: . /j.jen. . . sha: doc_id: cord_uid: m axi k introduction: the - west african ebola outbreak impacted the united states. due to the sporadic occurrence of the ebola infection, there is insufficient research regarding how the united states emergency nurses provide care to patients potentially infected with the ebola virus and the nurses’ motivation to protect themselves when providing care to these patients. this study was aimed to investigate the predictors of emergency room nurse’s protection motivation. method: a cross-sectional design was employed in the study. a survey developed based on a modified protection motivation theory was administered to randomly selected emergency room nurses who are members of the emergency nurses association. descriptive statistics, nonparametric kruskal-wallis h-test (as well as post hoc dunn-bonferroni test), spearman rho correlation, and stepwise multiple linear regression were conducted for data analysis. result: protection motivation was found in two components: proactive and passive protection motivation. the regression analysis indicated that response efficacy (β=. , p<. ) and self-efficacy (β=. , p<. ) significantly predict emergency nurses’ proactive protection motivation, while perceived vulnerability (β=. , p<. ), response cost (β=. , p=. ) and knowledge (β=-. , p<. ) significantly predict emergency nurses’ passive protection motivation. conclusion: results indicate the need for interventions to improve emergency nurses’ response efficacy, self-efficacy, and knowledge, while simultaneously reducing the nurses’ perceived vulnerability and response cost. such interventions would be expected to proactively motivate nurses to protect themselves when providing care to patients that exhibit the signs and symptoms of an ebola infection and reduce their passive protection motivation. contribution to emergency nursing practice: • only approximately % of emergency nurses felt prepared to provide care to patients potentially infected with ebola, even though on average . hours of training on controlling an ebola infection was reported and % of emergency nurses cited that their place of employment provided accommodations, i.e. adequate patient placement for individuals potentially infected by the virus. • response efficacy and self-efficacy positively predicted emergency nurses' proactive protection motivation to provide care to potential patients of an ebola infection. emergency care leaders are encouraged to provide continuous educational trainings and simulations that will increase emergency room nurse's capacity, skills, resources, and confidence, which might improve their ability to perform the recommended response. • perceived vulnerability, response cost and knowledge have an impact on emergency room nurse's passive protection motivation. emergency care leaders are encouraged to provide continuous trainings on the pathophysiology of the ebola infection and the use of relevant protective equipment to make nurses feel less vulnerable. in the meantime, health system administrators are encouraged to reduce nurse's response cost associated with ensuring job security, and providing family and/or childcare support, professional practice insurance coverage, and paid leave for incidents due to ebola infection and other emerging and highly communicable diseases, especially those that are associated with significant morbidity and mortality. the sporadic occurrence of this infection has resulted in limited research on the care of potential patients with an ebola infection within countries such as the united states, since the country has had a very low prevalence rate during past outbreaks. additionally, there is insufficient research concerning how healthcare professionals treat patients potentially infected with an ebola infection. in most healthcare settings, the emergency room is the first point of entry and nurses have an increased risk of exposure to the virus, especially nurses who often come in direct and prolonged contact with patients while providing care. according to the centers for disease control and prevention (cdc), nurses often experience blood and body fluid exposure and have an annual exposure prevalence rate ranging from < % to %. furthermore, during the to ebola outbreak, two of the four united states ebola- infected cases were healthcare professionals who were exposed while providing care to an imported case from west africa. the purpose of this study was to use a modified protection motivation theory (pmt) to explore united states emergency nurses' motivation to protect themselves against patients with an ebola infection they may encounter at work and to identify its associated factors and predictors. pmt focuses on the cognitive meditational processes, which involves the maladaptive and adaptive responses. both responses can be processed as the threat appraisal and coping appraisal. in this study, the modified pmt model as shown in figure [insert figure ] was used to investigate a social cognitive account of protective behavior in an attempt to provide clarity on the area of fear appeals and explain attitude and behavior change through matching cognitive processes people use to evaluate threats and select coping alternatives. , protection motivation is an intermediate variable that functions to arouse, sustain and direct protective health behavior within individuals. similar to the intention to perform a behavior, it has a positive and negative linear function. this includes whether the threat was considered severe (perceived severity), one's perception of her/his vulnerability (perceived vulnerability), effectiveness of the recommended response (response efficacy) and the confidence an individual has in her/his ability to perform the recommended response (self- efficacy). the negative function is the cost of conducting the recommended response (response cost). the constructs perceived vulnerability and perceived severity are part of the threat appraisal, which results in fear. this means the more an individual feels vulnerable and takes the threat seriously their fear increases, which leads to a greater threat appraisal. to enhance the study, two additional constructs, knowledge and outcome expectation, were included to strengthen the predictability. knowledge construct is found in other health behavior theories such as the integrated behavioral model (ibm). within the ibm, intention to perform a behavior is considered the most important determinant of a behavior, however knowledge is needed to carry out the behavior. the construct outcome expectation is found in the social cognitive theory. within this theory, the expected outcome is the belief that multiple consequences might result from the behaviors a person chooses to perform. thus, for this study, it was hypothesized that each of the emergency nurses' psychological variables (i.e. self-efficacy, response efficacy, response cost, knowledge, outcome expectations, perceived vulnerability, perceived severity, fear and protection motivation) are significantly correlated with and predictive of their motivation to protect themselves against an ebola infection. in summary, this study used a modified pmt model to examine the emergency room nurses' motivation to protect themselves and determine their apprehension to provide care to potential patients who may have an ebola infection, and their related factors. a cross-sectional research design was used to examine emergency nurse's motivation to protect themselves when providing care to a potential patient with an ebola infection during a single point in time. the research protocol was approved by the emergency nurses association, institute for emergency nursing research (ienr) director and the university of toledo institutional review board (# ). in , the united states had approximately , emergency nurses association (ena) members. a power analysis using g*power was conducted and a sample size of was estimated to achieve a satisfactory statistical power. multiple linear regression was selected as the statistical test for sample size estimation and a projected power of . , type i error of . and a conservative effect size of . were entered into the estimation. a systematic literature review was conducted to determine the journal articles that have used pmt as the theoretical backbone in conjugation with examining an infectious disease (unpublished data). , based on the review, the survey items were developed using prior articles on ebola and other infectious diseases, - then refined with the assistance of a focus group. the focus group (n= ) was conducted to gather primary qualitative data from ten emergency nurses working at a university teaching hospital. based on the focus group discussion, the survey questions were modified to remove or include additional questions. the survey was administered online using qualtrics. upon obtaining permission from the review board, a randomized mailing list of emergency nurses within the united states was obtained from ena. to reduce external validity threats and increase response rates, best practices in survey research were used. these include using the three-wave mailing process to maximize response rates. a systematic review of electronic survey has shown that non-monetary incentives, the use of a university letterhead and personalization of cover letters might increase the response rate. descriptive statistics (frequencies, mean and standard deviations), nonparametric stepwise multiple linear regression were performed using statistical package for the social sciences (spss). these methods were chosen due to their ability to identify associations more conservatively without normality assumptions. the bonferroni correction for multiple tests was applied to adjust the significance levels of kruskal-wallis h tests and spearman rho correlation. the significant variables were considered a priority in the stepwise multiple linear regression. incomplete answer of a particular question was treated as missing data and was excluded from the statistical analysis involving the particular question. no participant missed all questions. of the randomly selected participants, emergency nurses completed the online survey. majority of the participants were caucasians ( . %), females ( . %), between the ages of and ( . %), who are registered nurses ( . %) that work in an urban setting ( . %). most of the participants had a bachelor's degree ( . %) and on average had practiced emergency nursing for . years (sd= . years), with . hours per week (sd= . hours) of direct care to patients. of the emergency nurses that responded, . % indicated that they received less than hours of training at their place of employment on controlling an ebola infection. the bivariate analyses for each of the demographic characteristics and each investigated pmt construct are documented in table p<. ) was highly and negatively correlated with passive protection motivation. stepwise multiple linear regression was conducted for each outcome variable (proactive protection motivation and passive protection motivation) to estimate their predictors and their proportion of variance. all the psychological explanatory variables were entered into the regression model and the stepwise procedure selected and removed the predictors based on their significance levels. response efficacy (β=. , p<. ) and self-efficacy (β=. , p<. ) were significant predictors for proactive protection motivation and account for . % of variance. perceived vulnerability (β=. , p<. ), response cost (β=. , p=. ) and knowledge (β=-. , p<. ) were significant predictors for passive protection motivation and account for . % of variance as illustrated in table [insert table ]. the results from the study revealed a statistically significant relationship between the outcome variables and the modified pmt variables, which rejects the null hypotheses that the psychological variables do not correlate with protection motivation as well as there being no significant predictors for the nurses' motivation to protect themselves against potential patients with an ebola infection. the outcome variable, protection motivation, was divided into ( ) proactive protection motivation and ( ) passive protection motivation based on pca. while the bivariate analyses showed multiple paired associations, the multiple linear regression was able to further identify statistically significant predictors after controlling other explanatory variables. based on the results from the multivariate analysis, response efficacy and self-efficacy positively predicted emergency nurses' proactive protection motivation. both response efficacy and self-efficacy are part of the coping appraisal in pmt. response efficacy assesses the belief that the recommended coping response might be effective in reducing their threat and self- efficacy assesses the perceived ability to perform the recommended coping response. both constructs influence the nurses' motivation to conduct the recommended coping response. the regression coefficients from these two constructs indicate that higher self-efficacy and/or higher response efficacy can lead to an increase in the nurses' proactive protection motivation. these findings are consistent with previous studies that used pmt as their theoretical framework. coping strategies available for participants have a stronger effect on whether they will conduct the protective behavior and high response efficacy strengthens their intention to protect themselves and reinforces their belief that the protective behavior is effective. , for passive protection motivation, the regression analysis indicated that perceived vulnerability positively, response cost positively and knowledge negatively predicted passive protection motivation. perceived vulnerability assesses the likelihood of contracting the disease, response cost assesses the cost associated with performing the recommended protective behavior and knowledge is the amount of information needed to carry out the behavior. , the more vulnerable a nurse believes he/she is, the more likely they would exhibit passive protection motivation behavior. perceived vulnerability was the strongest predictor of passive protection motivation. vulnerability is associated with fear of the disease and believing that the individual has a higher likelihood of contracting and being exposed to the disease. previous researchers have examined behavioral intention and vulnerability relating to the individual hearing about the threat, assessing how dangerous the disease is and estimating their personal vulnerability before determining whether to perform the protective behavior. added to perceived vulnerability, the cost associated with the preventive behavior and less knowledge they have about the disease led to an increase in passive protection motivation. limited knowledge about the ebola virus and the protective behaviors determine whether a nurse will actively perform the protective behavior or possibly avoid their place of employment due to potential patients they might encounter. according to the american nurses association survey of rns (n= , ), health and safety at their work place were cited as influencing concerns on whether nurses will continue working in the field and the type of nursing work they choose to perform. in addition, media messages and incorrect information can lead to heightened perception of risk, which could increases fear and perceived vulnerability. the cdc reported that high frequency of risk-elevating messages in news coverage can increase public concern and perception and contribute to social amplification of risk, which leads to the spread of misinformation. continuous re-education, training and demonstrations of preventive recommendation and non-pharmacological interventions will not only increase nurses' confidence in providing care to potential patients but it might also reduce passive protection motivation. there were some limitations in this study. the random sample of emergency nurses are members of ena. the ena members may not demographically represent all emergency nurses in the united states, which presented a potential threat to the external validity of the findings. however, it can be hypothesized that ena members might be professionally dedicated and experienced, and the current findings is a liberal estimate of the nurses' motivation to protect themselves when providing care to potential patients with an ebola infection. additionally, the timing of the ebola outbreak might have influenced the nurse's motivation towards engaging in protective practices while providing care to patients possibly infected with the disease. the outbreak occurred over a year before the survey was distributed and the nurses' heightened awareness of the disease might have reduced considerably, which affected the response rate. the study response rate was % ( / , ), which yielded a power of %. the power analysis indicated a strong probability that the research might only commit a small type ii error. previous studies that surveyed emergency nurses and other healthcare professionals achieved high and low response rates depending on the research topic and their specialty. studies that focus on stress, burnout and depression among nurses had a response rate of %, substance abuse study had response rate of %, research focused on exposure to various common diseases had response rates between % to % and work place injury research had response rates between % and %, while a violence surveillance study had a response rate of . %. this study identified that perceived vulnerability, response cost and knowledge have an impact on whether the nurses exhibit passive protection motivation behavior. thus, it is recommended that administrators provide continuous training and simulations for nurses. administrators need to provide continuous education about the disease, its method of transmission and current cdc personal protective equipment (ppe) recommendations when providing care to ebola infected patients. rebmann et al., identified that fewer than % of healthcare personnel correctly don and doff a ppe needed to provide care to an ebola patient, while hand hygiene compliance was between % and % among triage nurses in an emergency department. continuous training and education acquired by nurses would reduce their perceived vulnerability towards the disease and increase their self-efficacy and response efficacy towards the protective behavior such as using the recommended equipment and guidelines. such educational intervention also improved ppe compliance from % to %. in instances where the infectious agent is new such as with the current covid- pandemic, nurses can use prior education and training to direct their current protective behaviors. additionally, emergency care leaders need to have updated protective equipment such as adequate respirators, disposable impermeable gown/coverall, disposable exam gloves with extended cuffs, disposable boot covers and disposable apron available in their hospitals or clinics. the availability of ppes has emerged as a major barrier to nurses and other health care professionals, with regards to their self-efficacy and response efficacy towards their protective behaviors in the current covid- pandemic. furthermore, administrators should be aware of the influencing factors and barriers that prevent the use of ppe among nurses. a focus group study indicated that emergency situations, availability of equipment, negative influence of protective equipment on nurses, patient discomfort, being busy, implementing guidelines being time consuming and physicians influence were cited as barriers that may influence a nurse's compliance with standard precautions, even if they received continuous training. although these factors were identified by participants in the focus group as barriers to the use of ppe among nurses, these findings may not be supported for other communicable infectious diseases. the transmission route, knowledge and outcome expectations as well as the mortality and morbidity associated with an infectious disease may have an influence on nurses' proactive protection motivation, which could limit their engagement in the recommended protective health behaviors. more specifically, nurses' perceived severity and perceived vulnerability towards an infectious disease may have a stronger impact than their response efficacy and self-efficacy, which might enable them to be proactive in their motivation to engage in protective health behaviors. during the current covid- pandemic, multiple anecdotal evidence is emerging in support of nurses being proactive in their motivation to engage in protective health behaviors. adequate accommodations such as having an available single patient room with a private bathroom and providing dedicated medical equipment should be made accessible, while also adhering to the hospitals environmental infection control guide. reducing the response cost such as making the protective equipment readily available and up to date can limit the nurses' passive protection motivation behavior. finally, having a standard operating procedure (sop) for receiving patients with a potential ebola infection is also recommended. to our knowledge, this is the first study to examine emergency nurses' motivation to . the type i error was adjusted by the bonferroni correction for multiple tests: * p<. , ** p<. , *** p<. understanding the dynamics of ebola epidemics the natural history of ebola virus in . world health organization. ebola in the democratic republic of the congo ebola hemorrhagic fever: known cases and outbreaks of ebola hemorrhagic fever, in chronological order nurses' working conditions: implications for infectious disease consistent infection prevention: vital during routine and emerging infectious diseases care effects of components of protection-motivation theory on adaptive and maladaptive coping with a health threat prediction and intervention in health-related behavior: a meta-analytic review of protection motivation theory a meta-analysis of research on protection protection motivation theory predicting health behavior: research and practice with social cognitive models theory of reasoned action, theory of planned behavior and the integrated behavioral model how individuals, environments, and health behaviors interact: social cognitive theory toward a new classification of nonexperimental quantitative research reporting and interpreting effect size in quantitative a national survey of emergency nurses and avian influenza threat knowledge regarding ebola hemorrhagic fever among private dental practitioners in tricity, india: a cross-sectional questionnaire study ebola risk perception in germany australian hajj pilgrims' knowledge, attitude and perception about ebola development of a public health assessment tool to prevent lyme disease: tool construction and validation focus groups: a practical guide for applied research current concepts in validity and reliability for psychometric instruments: theory and application california: sage: thousand oaks understanding the error of our ways; mapping the concepts of validity and reliability methods to increase response to postal and electronic questionnaires factors influencing nurses' compliance with standard precautions in order to avoid occupational exposure to microorganisms: a focus group study recommendations for hospitalized patients under investigation (puis) for ebola virus disease key: cord- -qqohijqi authors: smith, jacqueline; sadeyen, jean-remy; cavanagh, david; kaiser, pete; burt, david w. title: the early immune response to infection of chickens with infectious bronchitis virus (ibv) in susceptible and resistant birds date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: qqohijqi background: infectious bronchitis is a highly contagious respiratory disease which causes tracheal lesions and also affects the reproductive tract and is responsible for large economic losses to the poultry industry every year. this is due to both mortality (either directly provoked by ibv itself or due to subsequent bacterial infection) and lost egg production. the virus is difficult to control by vaccination, so new methods to curb the impact of the disease need to be sought. here, we seek to identify genes conferring resistance to this coronavirus, which could help in selective breeding programs to rear chickens which do not succumb to the effects of this disease. methods: whole genome gene expression microarrays were used to analyse the gene expression differences, which occur upon infection of birds with infectious bronchitis virus (ibv). tracheal tissue was examined from control and infected birds at , and days post-infection in birds known to be either susceptible or resistant to the virus. the host innate immune response was evaluated over these days and differences between the susceptible and resistant lines examined. results: genes and biological pathways involved in the early host response to ibv infection were determined andgene expression differences between susceptible and resistant birds were identified. potential candidate genes for resistance to ibv are highlighted. conclusions: the early host response to ibv is analysed and potential candidate genes for disease resistance are identified. these putative resistance genes can be used as targets for future genetic and functional studies to prove a causative link with resistance to ibv. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. infectious bronchitis (ib) is a highly contagious respiratory disease of chickens first described in the usa in the 's [ ] [ ] [ ] . clinical signs include: coughing, sneezing, rales and nasal discharge. the disease can also affect the reproductive organs, which leads to a decrease in egg quality and production, thus making it a major cause of economic losses within the poultry industry [ ] . the causative virus, infectious bronchitis virus (ibv) is a coronavirus, which is an enveloped virus with a single positive-stranded rna genome, which replicates in the host cell cytoplasm [ ] . proteins encoded by ibv include the viral rna polymerase, structural spike proteins, membrane and nucleocapsid and various other regulatory proteins. the spike glycoprotein mediates cell attachment and plays a significant role in host cell specificity [ ] . the existence of many different ibv serotypes, which are not cross-protective means that control of ib, is very difficult. mortality is usually fairly low (~ %), however some strains of the virus can also cause nephritis meaning that, depending on strain, mortality can be greater than % [ , ] or even up to % with some australian isolates [ ] . ibv infection leaves birds more susceptible to colibacillosis [ ] and subsequent bacterial infections can also lead to a high level of mortality [ ] . currently, attenuated live vaccines are used in broilers and pullets, and killed vaccines are used in layers and breeders [ ] . however, virus control is very difficult, as there are only a few vaccine types and many different strains of ibv. the virus also continues to mutate rapidly, generating more virulent strains of the disease [ ] [ ] [ ] . coronaviruses have now also been detected in other avian species such as turkey, duck, goose, pheasant, guinea fowl, teal, pigeon, peafowl and partridge [ ] . the extent to which the virus affects the host is highly dependent on the chicken breed [ ] and the mhc b locus is known to play a role in susceptibility to the virus [ ] . in this study we attempt to identify non-mhc genes, which may be involved in resistance to ibv. no genetic analyses have thus far been undertaken in order to try and do this and no quantitative trait loci or genes associated with resistance have been determined, so far. based on differential gene expression in susceptible and resistant lines of chickens, we identify potential candidate genes for disease resistance towards ibv (virulent m strain). building on the previous work by dar et al. [ ] and wang et al. [ ] we used affymetrix wholegenome chicken microarrays to examine the tracheal gene expression profiles of a line of birds known to be susceptible to ibv infection (line i) and a line known to show resistance (line n). we determined the early host response to infection and propose possible candidate genes for involvement in disease resistance towards ibv. understanding how coronaviruses infect the host and identifying genes involved in resistance is important not only for the poultry industry but also has important implications for human health, as diseases such as sars are also caused by coronaviruses [ , ] . all animal work was conducted according to uk home office guidelines and approved by the roslin institute animal welfare and ethical review body. the lines used in these experiments are an ibv susceptible lineline i (inbred white leghorn strain) [ ] and an ibv resistant lineline n (non-inbred cornell strain). line i was developed at east lansing in the usa in the s [ ] and line n at cornell, usa in the s [ ] . the lines have since been maintained at the institute for animal health in compton, uk. twoweek-old chicks from each line ( i and n) were separated into two experimental rooms, with ad libitum access to food and water. in one room, birds ( from each line) were infected with log cid ( cid ) of virulent ibv-m strain in a total of μl of . % bsa in pbs equally by intra nasal and ocular routes. in the other room, control birds ( from each line) received ul pbs via the same route. trachea samples (upper half ) were collected at , and days postinfection ( individual birds from each line at each time point). the trachea of infected and control birds from each line were analysed for viral load using taqman real-time quantitative rt-pcr assays. tissue samples (~ mg) were stabilized in rnalater (ambion, life technologies, paisley, uk) and disrupted using a bead mill (retsch mm , retsch, haan, germany) at hz for min. total rna was prepared using an rneasy kit (qiagen, crawley, uk) extraction method as per the manufacturer's protocol. samples were resuspended in a final volume of μl of rnasefree water. concentrations of the samples were calculated by measuring od and od on a spectrophotometer (nanodrop, thermo scientific, paisley, uk). quality of the rna was checked on a bioanalyser (agilent technologies, south queensferry, uk). an rna integrity number (rin) > proved the integrity of the rna. biotinylated fragmented crna was hybridized to the affymetrix chicken genome array. this array contains comprehensive coverage of , transcripts corresponding to over , chicken genes. the chicken genome array also contains probe sets for detecting transcripts from avian viruses. for each experimental group (control and infected birds in each of the two lines at each of , and dpi), three biological replicates ( rna pools from birds) were hybridized. thus, arrays were used in total. hybridization was performed at °c for hours in a hybridization oven with constant rotation ( rpm). the microarrays were then automatically washed and stained with streptavidin-phycoerythrin conjugate (sape; invitrogen, paisley, uk) in a genechip fluidics station (affymetrix, santa clara, ca). fluorescence intensities were scanned with a genearray scanner (affymetrix, santa clara, ca). the scanned images were inspected and analyzed using established quality control measures. array data have been submitted to array express (http://www.ebi.ac.uk/arrayexpress/) under the accession number e-tabm- . gene expression data generated from the genechip operating software (gcos) was normalised using the plier (probe logarithmic intensity error) method [ ] within the affymetrix expression console software package. this normalised data was then analysed using the limma and farms [ ] packages within r in bioconductor [ ] . probes with a false discovery rate (fdr) value < . and a fold change ≥ . were deemed to be biologically significant. in order to determine which biological pathways are involved in the responses to viral infection, we analysed our differentially-expressed (de) genes using pathway express [ , ] which uses kegg pathways [ ] to pictorially display up/down regulation of genes. (nb. these diagrams are based on the human pathways and so are not completely representative of the chicken pathways). genes differentially expressed during the host response (fdr < . ) were analysed against a reference background consisting of all genes expressed in the experiment. factors considered by pathway express include the magnitude of a gene's expression change and its position and interactions in any given pathway, thus including an 'impact factor' when calculating statistically significant pathways. anything with a p-value < . is deemed significant when using this software. use of the ingenuity pathway analysis (ipa) program [ ] revealed which canonical pathways are being switched on by ibv infection in the host (with benjamini-hochberg multiple testing correction) and allowed us to analyze the gene interaction networks involved in the host response. genes were clustered by similar expression pattern and analysed for enriched go-terms and transcription factor binding sites (tfbs) using expander (v . ) [ ]. normalised expression data from control samples were compared with infected samples to examine the host response to ibv infection. enrichment analysis of particular go terms or tfbs within clusters was done using the tango and prima functions, respectively, within the expander package. taqman real-time quantitative rt-pcr (qrt-pcr) was used to quantify viral rna levels and for confirmation of the microarray results for the mrna levels of selected genes. this was performed on replicate pools of samples ( birds). primers (sigma) and probe (pe applied biosystems, warrington, uk) ( table ) were designed using primer express (pe applied biosystems). briefly, the assays were performed using μl of total rna and the taqman fast universal pcr master mix and one-step rt-pcr mastermix reagents (pe applied biosystems) in a μl reaction. amplification and detection of specific products were performed using the applied biosystems fast real-time pcr system with the following cycle profile: one cycle at °c for min and °c for sec, followed by cycles at °c for sec and °c for sec. data are expressed in terms of the cycle threshold (ct) value, normalised for each sample using the ct value of s rrna product for the same sample, as well described previously [ ] [ ] [ ] . final results are shown as -ct using the normalised value, or as fold-change from uninfected controls. taqman real-time quantitative rt-pcr analysis was used to measure viral load in trachea samples from both control and infected birds from both lines i and n. tracheal tissue was chosen for examination in this study as the target of ibv is the epithelial surface of the respiratory tract. viral rna was detected in infected birds, but no significant difference in viral load was detected between lines at any of the days , or post infection (fig. ) . this would indicate that the resistance to the virus seen in line n is due to how the birds respond to the virus once it has entered the body and is not a measure of how the birds can prevent initial infection by the virus itself. when resistance to ibv infection was originally determined in these lines, it was noted that they were equally susceptible to infection, but a variation in outcome was seen. in line n, % of birds showed air sac lesions whereas % of i birds presented lesions. mortality was in line n, but % within line i birds. it was hypothesized that the different lines were producing different immunological responses upon infection [ ] . gene expression differences found in the susceptible i line between infected and control birds over days , and post infection were analysed, with a view to examining the innate host response to infection by ibv. genes seen to be induced during the host response to infection include c s, irf , stat , mx , tlr and ctss as previously recognised by guo et al. [ ] . we also identified ifit , oasl, sca , lyg , isg - , ddx , ifih , irf , table s . to elucidate which biological pathways are being perturbed during the host response to ibv infection, we analysed our data using pathway express [ ] . the resulting pathway diagrams are extremely useful in establishing which gene networks are involved in a particular experimental response. as seen in fig. , genes involved in antigen presentation and the toll-like receptor (tlr) pathway are up-regulated. tlrs identify pathogen associated molecular patterns (pamps) and are crucial to the innate immune system. in this study tlr is shown to be induced at dpi. tlr recognizes double-stranded rna intermediates produced during viral replication and has previously been shown to be induced in the trachea at this time after ibv infection [ ] . another pathway involved is the phosphatidylinositol signalling pathway (table ) . phosphatidylinositol kinases are known to play an important role in the viral life cycle after infection of the host and pi kb is known to be exploited by coronaviruses for viral entry. the product of pi kb catalysis is phosphatidylinositol -phosphate (pi p) and coronavirus entry into the host is mediated by the pi p lipid microenvironment [ ] . genes involved in the complement system are also highlighted as being up-regulated in response to ibv infection. complement-mediated lysis of viruses is an important facet of the host innate immune system and its role in defence against viral infection [ ] as reflected in the induction of these genes in this study. use of ingenuity pathway analysis (ipa) software also allowed us to determine which biological systems are active during the host response. up-regulated genes are seen to be part of the canonical biological pathways shown in fig. a . biological processes involving pattern recognition receptors and interferon signalling feature heavily. the interferon response is a powerful antiviral mechanism, which has previously been shown to be involved in the host response after ibv infection. a very early induction of ifn-γ has been reported in splenocytes [ ] , and in peripheral blood mononuclear cells (pbmcs) and lung leukocytes [ ] . ifnb expression has also been reported in trachea between and dpi [ ] . we do not see this increase in expression of interferon genes (due to the absence of data earlier than dpi), but we do see the downstream effects, with increased expression of many interferon-induced genes. specific physiological processes activated upon ibv infection can also be seen in fig. b . the stimulation of various different immune cells is seen along with the indication of reproductive abnormality, which would reflect the problems seen with egg-laying upon ibv infection. in order to cluster genes seen to be involved in the host response to infectious bronchitis into groups with similar expression profiles and probably sharing similar functions or gene regulatory pathways, we utilised the click algorithm within the expander program [ ] . figure a shows the expression profile of genes upregulated during the response to virus. the expander program was also used to analyse the gene ontology (go) functional annotations of the genes being differentially expressed. figure b shows the biological process terms, which are significantly enriched in the genes responding during the host response to infection. as would be expected, these include terms like 'innate immune response' and 'antigen processing and presentation'. 'nad + adp-ribosyltransferase activity' and 'phosphoinositide binding' are also highlighted. transcription factor binding sites present in de genes which are significantly over-represented were also predicted. figure c shows that genes up-regulated during the host response have a high proportion of irf and isre binding sites. irf is a transcriptional activator, which binds to the interferon-stimulated response element (isre) in ifn promoters and functions as a molecular switch for antiviral activity. analysis of the gene expression differences between infected and control birds across the two lines has provided us with information on how these lines differ in their response to infection. examination of the gene expression profiles in the control birds of the two different lines also allowed us to identify genes, which are inherently different between the susceptible and resistant birds. it can be seen that there are numerous genes, which show large expression differences between the two lines, even before infection. dramatic differences in gene expression of certain genes, including ddt, sri, blb , hscb, bf , bf , suclg , mx and sri, which are more highly expressed in the resistant n line table s shows all de probes) so, it can be seen that these are genes which have inherently different expression levels between susceptible and resistant birds, even before infection occurs. we therefore postulate that some of these genes may play an important role in disease resistance. the potential interactome of ibv has recently been investigated by stable isotope labelling with amino acids in cell culture (silac) coupled to a green fluorescent proteinnanotrap pull-down methodology [ ] . host proteins, which bind to the ibv n protein were identified, some of the genes for which, we see as being inherently expressed at higher levels in susceptible birds in this study. these genes include myh , caprin , dhx , hnrnph , rpl a, fmr , c orf , hnrpdl, sfrs , rpl , npm and rpsa. this may therefore be one of the reasons why line i is more susceptible to ibv infectionthere are more host proteins to which the virus binds, compared with the resistant line n. upon infection, differences in response are also seen between the two lines. interestingly, apart from cd and cd at dpi and fkbp at dpi, all other differential gene expression between the lines is seen at dpi in this study (additional file : table s ). cd is a glycoprotein found on the surface of many immune cells including cd +, cd +, b lymphocytes and natural killer cells and is a marker of cell activation. it functions in cell adhesion, signal transduction and calcium signalling. cd is found on the surface of immune cells such as t helper cells, monocytes, macrophages and dendritic cells. it is a membrane glycoprotein which interacts with mhcii antigens. the protein functions to initiate or augment the early phase of t-cell activation. the protein encoded by fkbp is a member of the immunophilin protein family, which play a role in immuno-regulation and basic cellular processes involving protein folding and trafficking. early defence by the host is a key mechanism for combatting viral infection, and induction of ifnb and other innate genes in response to ibv infection has been shown to peak around - hr post infection [ ] . in this study, genes more highly expressed (or less down-regulated) in the resistant n line at dpi include a number of collagen genes (col a , col a , col a , col a , col a and col a ) and other genes such as acan, fstl , comp, eif a, stat and igfbp . genes seen to be more highly expressed (or less down-regulated) in the susceptible i line include rbm , mafb, nnk , ccn , mgat and thrap . one consequence of ibv infection is the production of poor quality, misshapen eggs by infected birds [ ] . some of the genes previously identified as being important for the creation of a healthy eggshell are seen to be more highly expressed by the resistant n line birds after infection in this study. these genes include col a , creld , hsp b , p hb and erp [ ] . for a full list of genes differentially expressed between the two lines in trachea ( de probes) see additional file : table s . ipa analysis of genes showing different inherent expression between lines i and n shows that the molecular functions of these genes is primarily concerned with their involvement in cell death and cell adhesion (fig. ) , two processes previously shown to be significant in infected kidneys [ ] . when the differential host responses to infection are examined, it is seen that genes involved in proliferation of t-lymphocytes and genes concerned with cell attachment and cytoplasmic organization are more highly expressed in the resistant line n. other processes significantly involved are apoptosis and necrosis (fig. a) , which have been previously documented in ibv-infected vero cells by liu et al. [ ] . one of the most perturbed biological networks noted in this analysis is that involving genes related to connective tissue disorders and involve many collagen genes. these genes are more highly expressed in susceptible line i birds compared to resistant line n birds (fig. b) suggesting that ibv infection might cause more disorder of eggshell formation in this line [ ] . the production of poor quality eggs by ibv infected birds may, in part be a reflection of the expression of these kinds of gene networks compared to that seen in resistant birds. twenty-one genes were selected for qrt-pcr validation ( table ) . these genes were chosen based on their involvement in the host response and whether they were differentially expressed between the susceptible and resistant lines (either inherently or during the course of infection). of the genes tested, showed comparable higher expression in response to infection in the resistant than in the susceptible line c inherently higher expression in the resistant line differential expression to that determined by the arrays. however, the results for ifnar and igfbp were not confirmed (additional file : figure s ). besides knowing that the mhc b locus has a bearing on disease resistance, the lack of any genetic information or identified qtl meant that we had to rely upon the gene expression differences we saw between susceptible and resistant lines to give us clues as to genes potentially involved in resistance to ibv infection. identifying genes which were expressed at different levels in the two lines of birds highlighted b-locus genes (blb , bf , bf , b-g) as well as bringing to our attention various other non-mhc genes which, due to their known biology, could be candidates for being involved in resistance to ibv infection (table ) . mx , c s, irf , tlr , c r, ccli , isg - and ifitm are all strongly induced during the host response to ibv infection. they are all innate immune genes which could potentially have a role in determining susceptibility to the virus. mx and ifitm are already established as anti-viral molecules [ ] [ ] [ ] . cd , cd , fkbp and stat all show a higher level of expression during the host response in the resistant birds compared to that of the susceptible birds, indicating their involvement in the host defence mechanism. cd and cd , with their role as receptors on immune cells, as described above, are obvious candidates, along with fkbp as an immune-regulator. stat is activated by various cytokines and growth factors and functions in cellular processes such as cell growth and apoptosis. even before infection, many genes are seen to be highly differentially expressed between lines i and n. oasl is an interferon-induced molecule known to have anti-viral activity against certain viruses such as hepatitis c virus. ddt is highly homologous to the macrophage migration inhibition factor, mif. we have also shown it to be highly differentially expressed in other chicken lines, which are susceptible or resistant to marek's disease virus [ ] . ifnar is an obvious candidate prediction, as the interferon response is central to the host's defence against ibv infection. tpd l , bcl l , faim and ciapin are all known to be involved in regulation of apoptosis, a process seen to be important during ibv infection. hscb, sri, and suclg , although not having an obvious potential biological role in disease resistance, are highly differentially expressed between susceptible and resistant lines and should thus be considered as potential candidates. resistance to ibv infection is brought about by the immune response after the virus has entered the host and is not due to prevention of initial viral infection. there is a small initial innate response at dpi, with much more gene expression seen at and dpi. analysis of genes being activated or inhibited upon infection shows that the biological pathways primarily affected during ibv infection include mapk signalling, those involved in the interferon response and those involving pattern recognition receptors. susceptible and resistant lines show a differential host response mostly at dpi. there are also genes which are inherently different between the two lines studied, including many genes, which control the apoptotic potential of the host. these differences seen in gene expression levels, allow us to postulate on many candidate genes for disease resistance. some potential candidates for involvement in disease resistance include genes already known to confer resistance to other viral infections (mhc-b locus genes, mx , oasl and ifitm ), genes involved in apoptotic processes (tpd l , bcl l , faim and ciapin ) and others which could be potential candidates due to their known biology (e.g. ddt and cd ). array data have been submitted to array express (http://www.ebi.ac.uk/arrayexpress/) under the accession number e-tabm- . additional file : table s . gene expression seen during the host response to ibv infection in the trachea of susceptible birds. (xlsx kb) additional file : table s . gene expression differences found to be inherent between susceptible and resistant lines in the trachea. (xls kb) additional file : table s . genes found to be differentially expressed between susceptible and resistant lines in response to ibv infection in the trachea. 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of minerals or in the shell mineralization process transcriptome analysis of chicken kidney tissues following coronavirus avian infectious bronchitis virus infection induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus effects of avian infectious bronchitis virus antigen on eggshell formation and immunoreaction in hen oviduct interferons: cell signalling, immune modulation, antiviral response and virus countermeasures mx proteins: mediators of innate resistance to rna viruses ifitms restrict the replication of multiple pathogenic viruses submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported by the biotechnology and biological science research council (bbsrc), as part of grant numbers bb/d / , bb/ d / and bb/d / . the authors would like to thank alison downing (edinburgh genomics, edinburgh, uk) for excellent technical assistance with the affymetrix microarray experiments. authors' contributions js performed the arrays, analysed the results and wrote the manuscript; dc carried out challenge experiments, j-rs prepared rna, measured viral load and performed qrt-pcr; db and pk conceived and supervised the project and revised the manuscript. all authors read and approved the final manuscript. key: cord- - sctqkwr authors: alcamí, josé; joseph munné, joan; muñoz-fernández, maría Ángeles; esteban, mariano title: current situation in the development of a preventive hiv vaccine date: - - journal: enfermedades infecciosas y microbiología clínica doi: . /s - x( ) - sha: doc_id: cord_uid: sctqkwr the uncontrolled progression of the aids epidemic has made the development of an efficacious human immunodeficiency virus (hiv) vaccine a major objective of scientific research. no effective preventive vaccine against hiv is currently available and sterilizing immunity has not yet been achieved in animal models. this review analyses the major challenges in developing an aids vaccine, in particular the mechanisms involved in viral escape from the immune response, and summarizes the results obtained with the different prototypes of therapeutic and preventive vaccines. finally, social, economic and healthcare aspects of research into hiv vaccines and current controversies regarding the development of clinical trials are discussed. in the year , the aids pandemic was responsible for more than three million deaths, and five million people are calculated to have contracted the virus during this period. this takes the number of infected people throughout the world to million, with million deaths registered since the origin of the pandemic was identified . the geographical and economic differences in this disease are obvious, with more than % of cases and deaths by aids occurring in the third world ( % in africa), especially among young adults, with a gradual increase in women. it is dramatic to see how, in sub-saharian africa, the epidemic continues to spread and, in many countries, the high percentage of people infected with aids has devastating effects on families and on the productive economy. the explosion of this epidemic in developing countries has raised the need to take urgent preventive measures and provide expanded access to antiretroviral therapy. however, in some parts of the world, these measures, although essential, are probably insufficient to curb the epidemic, so that the development of a vaccine is the only available possibility of controlling it. therefore, the development of an efficacious hiv vaccine is not only an area of aids research which has yet to be resolved but also an urgent need for developing countries. this awareness has led to a considerable increase in financing the search for an aids vaccine. to obtain an effective hiv vaccine represents a major challenge and priority scientific objective for public and private institutions, governments and ngos [ ] [ ] [ ] (table ) . this chapter analyses prototype vaccines being developed and the results obtained, the scientific difficulties in developing a vaccine, especially with regard to the mechanisms of viral escape from the immune response. furthermore, as this research is set in the social, economic and healthcare context of the aids pandemic, current controversies surrounding clinical trials with the different types of vaccine are addressed in this review. situación actual en el desarrollo de una vacuna preventiva frente al vih el avance de la epidemia de sida ha convertido la obtención de una vacuna eficaz frente al virus de la inmunodeficiencia humana (vih) como un objetivo científico prioritario. en el momento actual no disponemos de una vacuna preventiva frente a la infección por el vih y en ningún modelo animal se ha conseguido la protección frente a la infección. en esta revisión se analizan las dificultades existentes en el desarrollo de una vacuna contra el sida, en especial los mecanismos de escape viral a la respuesta inmunitaria y se describen los prototipos de vacunas preventivas y terapéuticas en desarrollo y los resultados obtenidos. por otra parte se sitúa esta investigación en el contexto sanitario, económico y social de la pandemia de sida y se analizan las polémicas in order to develop a vaccine, it is necessary to know the genes of the pathogen involved in the induction of a specific immune response, and to use experimental models to test the efficacy of the virus. despite the fact that, in the past, there have been important advances in the control of infectious diseases by vaccination, many of the mechanisms used by the pathogen to take over cell machinery are unknown. the same applies to mechanisms of escape from the immune system and ways of boosting an immune response capable of eliminating the infected cell. in scientific terms, obtaining an efficacious vaccine capable of preventing hiv infection faces a series of challenges: the immune response begins with the recognition by cd lymphocytes via their receptor (tcr) of foreign antigens in the class-ii hla groove presented by cells specialized in antigen processing. antigen recognition activates the different effector mechanisms of the immune system: cytokine and chemokine synthesis, production of antibodies by b lymphocytes, activation of cd lymphocytes and generation of cytotoxic cd lymphocytes. these last events represent the main mechanism involved in killing of virus-infected cells and to initiate this process cytotoxic lymphocytes must recognize the antigenic determinants of the virus lodged in the class-i hla groove of infected cells . as a consequence of the polymorphism of the hla system both in antigen presenting cells and target cells different peptides (that are anchored in class ii and class i hla molecules respectively) are selected according to individual hla haplotypes. in many viruses there are "major immunodominant determinants" (epitopes) which induce a potent response by the immune system. the efficacy of this response depends on two characteristics: first, they correspond to epitopes or domains of the viral proteins which are conserved among the different isolates, even in the context of highly variable viruses; second, these major determinants are capable of binding to the grooves of most hla haplotypes. the existence and identification of these major immunogenic determinants is crucial when developing a vaccine, since they make up viral targets par excellence by being "universal" in two senses: as epitopes conserved in the viral protein among different isolates and as epitopes susceptible to antigenic presentation by most subjects regardless of their hla haplotype. however, in the case of hiv, no similar dominant epitopes have been found to date, which represents a very important limitation when designing a vaccine. the presence of these major immunogenicity determinants in viruses with tremendous genetic variability, such as hiv- , makes it practically impossible to define these epitopes empirically and experimentally. nevertheless, bioinformatics can provide us with predictive methods which make identification easier . the objective of a vaccine is to induce an efficacious, memory-type immune response which allows the immune system to react against the infectious agent by preventing its spread. for this objective to be reached, it is essential to know which immunological effectors are efficacious in the control of the infection in order to define a series of surrogate immunological parameters which enable us to evaluate whether a vaccine preparation is efficient or not. the intense immune response of hiv-infected patients is reported to encompass practically all the effector mechanisms of the immune system. this response is relatively wide, since it is developed against numerous epitopes, and practically all the viral proteins, both structural and regulatory, are recognized as foreign antigens. however, controversy still surrounds the role of "protection" played by each of these components of the antiviral response. below, we describe the type of immune response generated against hiv infection. hiv infection induces an intense antibody response against practically all the regulatory and structural proteins of hiv . some of these antibodies have neutralizing capacity in vitro and in in vivo adoptive immunotherapy experiments , . however, the production of antibodies with neutralizing capacity is scarce and viral escape from these antibodies is rapid . furthermore, in the immunization models developed to date, high levels of neutralizing antibodies are not consistently obtained and their presence is not systematically associated with protection. these data raise severe concerns about the role of the humoral response in the control of hiv infection , . nevertheless, almost all preventive vaccines induce neutralizing antibodies and their role as a surrogate protection marker is clearly demonstrated in other diseases. therefore, "a priori", a preventive hiv vaccine should induce broad-spectrum neutralizing antibodies and this is one of the huge challenges currently facing the development of an aids vaccine. recent studies which define the loca- tion of neutralization epitopes, antibody structure and mechanism of action represent an important advance to define the characteristics that a given vaccine must have to induce neutralizing antibodies. most studies agree that the combined response of cd and cd is probably the most important protective mechanism against hiv . study of the cellular response has shown that in seropositive patients there is a clonal expansion of cd and cd lymphocytes which are active against hiv. this expansion is particularly intense in patients with primary infection and correlates with the control of viral replication , . there are also reports showing an intense cd and cd response in some patients during immune reconstitution after antiretroviral therapy, especially in those with a good immunological status before starting therapy , . the same phenomenon has been described in patients with structured treatment interruptions who spontaneously control viral replication . although it is difficult to draw conclusions as to a cause-effect relationship between the appearance of a specific type of immune response and the control of viral replication, all the data seem to suggest that both helper and cytotoxic immune responses are essential to contain viral replication in the early stages of the disease, when the immune system is relatively undamaged. the most conclusive experimental data on the role of the cellular response in the control of viral replication come from studies in which selective depletion of cd lymphocytes in macaques infected with siv leads to a huge increase in viremia and accelerated evolution to aids . hiv transmission occurs mainly via the mucosa. the large quantity of cd + lymphocytes in genito-rectal lymphoid tissue represent a major reservoir for the replication of hiv or siv, even when the infection is contracted intravenously. the gut-associated lymphoid tissue system (galt) is set up by activated b and t lymphocytes and dendritic cells which migrate through the lymphatic system and bloodstream to distant lymph nodes where they become resident. thus, the induction of a strong immune response in mucosa is probably a necessary requirement for a vaccine to be efficient against hiv. each family of viruses develops different escape mechanisms to avoid elimination by the immune system. a vaccine must face the same escape mechanisms and, to be successful, must induce a series of immune responses capable of overcoming them. the rate of variability of hiv is due to the high error rate of reverse transcriptase (one substitution per - nucleotides and round of copy). in consequence there is wide intersubtype and intrasubtype variability but the immunological relevance for vaccine design of this genetic disparity is a matter of debate. several investigations have shown that genetic differences among hiv subtypes do not correlate with immunotypes. in fact, several genetic subtypes could share common protective epitopes and more than one immunotype can be found in the same genetic subtype. in general, neutralizing antibodies seem to be more strain-specific, whereas cellular immune responses have a broader spectrum. this lack of fidelity generates a high diversity in viral proteins which allows escape from the control of specific immune response. therefore, hiv shares with other rna viruses a common escape mechanism related to their high variability that allow the virus finding holes in the immune repertoire. together with the variability generated by the high error rate of reverse transcriptase other mechanisms such as genetic recombination, which produces new subtypes and "mosaic" viruses among different subtypes, are also involved in the generation of hiv variants. several molecular epidemiology studies have stressed the rapid dissemination of hiv variants and have described the distribution of several subtypes or recombinant viruses in different parts of the world. this could be an obstacle to the development of a universal vaccine . one central aspect of hiv infection which is not totally understood is the reason why viral replication is not controlled despite the potent immune responses elicited in primary infection. although several explanations have been put forward, the most widely reported is viral escape through mutations in the viral epitopes recognized by the different effector mechanisms of the immune system . escape from ctl response is due to ad hoc mutations of the viral epitopes which interact with the groove of the hla molecules. it has been shown that mutations in critical residues generate viral escape in both animal models and patients with primary infection and this event results in loss in the ctl response and parallel increase in viremia , . however, in the chronic phase of infection, there is no clear correlation between the presence of specific ctl and the elimination or persistence of a given viral variant . in addition to merely quantitative data, functional analysis have shown qualitative differences between ctl from progressors and non-progressors, such as the expression of perforins , production of cytokines and chemokines, and reduced activity of the t-cell receptor against viral epitopes presented in the hla complex . these data suggest that qualitative aspects of ctl may be also important in the control of viral replication. a general strategy for maximizing the efficacy of a vaccine would be to obtain a cytotoxic response against a large number of epitopes from several proteins. however, recent studies suggest that a more targeted approach can be more effective. thus, ctl against non-structural proteins (tat, nef) are more efficient in controlling infection but more prone to viral escape and do not last as long as ctl against the structural proteins gag and pol . for a sterilizing vaccine, the objective would be to induce an intense ctl response against early proteins, whereas immunization against structural proteins would generate a response which would control viral replication thus attenuating infection. another problem which may be a serious genetic resistance barrier is the adaptation of the virus to the hla haplotype of the infected patient. in this situation peptides from viral mutants generated would reduce their affinity to hla thus decreasing recognition by tcr and generating a greater resistance to ctl response . the structure of the viral envelope in its native form hides the domains of interaction with viral coreceptors due to the trimeric structure and folding of the protein (oligomeric exclusion and entropic masking) . exposure of these conserved epitopes which are identified by neutralizing antibodies takes place at the moment of interaction between the viral and cellular membranes, a setting in which antibodies efficacy is lower given their low accessibility to neutralization epitopes. a second, more classic escape mechanism is epitopic mutation in the hypervariable regions found in the external domain of the viral envelope. nevertheless, recent studies show that escape from these antibodies does not always require mutation in amino acid residues but takes place by glycosylation of the residues and formation of carbohydrate structures on viral gp known as "glycan shields" that represent authentic barriers to the action of neutralizing antibodies . one of the most spectacular studies published during the last year shows how, during evolution in a specific patient, the viral envelopes gradually become resistant to all types of neutralization by neutralizing antibodies via accumulation of the previously mentioned escape mechanisms . both in animal models and in patients with primary infection through sexual contact the establishment of hiv infection is a very rapid process . in a few hours, the lymphoid cells of the rectal and vaginal submucosa become infected and, in seven days, the infection spreads to systemic lymph nodes where it reaches viral and proviral loads similar to levels found in chronic infection . the speed at which these reservoirs appear, before a specific immune response is triggered, represents a major obstacle to the control of viral replication since once established hiv infection will "persists" in lymphocytes despite immune response . hiv can infect target cells in a latent form. in this situation no viral proteins are expressed on the membrane of infected cells thus allowing escape from immune surveillance. furthermore, reactivation-reinfection processes take place in lymphoid organs, which provide an ideal cellular microenvironment for the process of infection: dendritic cells express in their membrane a lectin (dc-sign) which interacts with the virions and lymphocytes and enhances hiv infection . antigenic recognition by lymphocytes and the presence of cytokines in this microenvironment in turn increase infection of target cells and promote viral replication. as confirmation of these data, hiv-specific lymphocyte clones have been shown to be infected at higher proportion, which implies a preferential immunosuppression of the hiv-specific responses . it must be stressed that the continuous generation of new, latently infected cells from the active viral replication compartment generates a "continuous archive" of changes produced in the virus throughout the disease, by including treatment-resistant mutated genomes and variants of the immune escape. the latent compartment is therefore not static and in some way hiv stores its "history" in latently infected cells thus contributing to viral diversity as a mechanism of escape from antiretroviral therapy and vaccines. attenuated virus vaccines are without doubt the most efficacious because the germ carries out a limited series of replication cycles and simulates a low-level infection which induces the whole spectrum of antiviral response in a physiological setting. in the case of lentiviruses, one of the most spectacular findings was that which showed that a defective nef-deleted siv virus induced a protective response against the challenge with highly pathogenic aggressive viable viruses . these experimental data had a natural correlate in the "sydney cohort", made up of patients infected through blood transfusion from a seropositive donor and who, after years of infection, had an excellent clinical and immunological status. the cloning and characterization of the virus in these patients and the donor showed that it presented deletions in the nef gene and in critical regulatory sequences of the ltr region . these findings led to the proposal of attenuated hiv vaccines similar to defective siv mutants. however, it must be stressed that attenuated vaccines are usually used against viruses which do not persist or, alternatively, the attenuated virus used as vaccine is not capable of persisting in the host. this is not the case for nef-defective viruses which not only infect, but also replicate and persist in the host, with the risk of drifting towards more aggressive variants in the vaccinated subject. the first alarming data came from vaccination of newborn maca ques in which, in contrast with was found in adults, the innocuous virus rapidly induced aggressive infection and death by immunodeficiency . furthermore, prolonged follow-up of patients from the sydney cohort enabled us to observe an immunological deterioration and blips of viremia in some subjects . similarly, some adult maca ques vaccinated with the defective siv virus developed aids from the virus they had been vaccinated with, which suffered reversions of the mutant phenotype . therefore, the use of vaccines from defective viruses has been ruled out, and this approach has been explicitly excluded in guidelines and recommendations of regulatory agencies. inactivated viruses have scarcely been used as preventive vaccines. on the contrary, this is the most widely used model in therapeutic vaccines of which remune ® is the prototype. these viral preparations are composed of complete virions or particles whose envelope has been eliminated, which are then inactivated using different chemical methods and administered in conjunction with freund's incomplete adjuvant . the first hiv vaccines were based on the hepatitis b immunization model. they were composed of recombinant proteins gp and gp produced by genetic engineer-ing or using vaccinia virus as expression vectors. in preclinical studies and in phase i and phase ii clinical trials, the preparation was safe and induced antibody synthesis against the viral envelope , but these antibodies were incapable of neutralizing wild-type variants "in vitro" . in spite of the evidence against the efficacy of this prototype, phase iii trials were continued (see below). other trials have used the regulatory protein tat in toxoid form, which has provided good protection results in macaque studies, although its role remains controversial . peptide vaccines have little immunogenic capacity, since, in many cases, the antibodies do not recognize the primary structure of the aminoacid sequence, but rather secondary and tertiary structures in the target proteins which are not simulated by the peptides. therefore, peptides are generally used in combination with other vaccine preparations such as viral vectors or dna in order to induce complementary immunization . the advantages of these combinations are low toxicity, the possibility of preparing peptide "cocktails" which cover a wide range of viral isolates in proteins presenting high variability, and the use of "mixed peptides" which, by including t and b immunodominant epitopes induce cellular and humoral responses. these systems use viruses or bacteria into whose genome hiv genes are inserted in such a way that their proteins are expressed during the course of replication of the vectors in the host cell. the most developed models are those which use poxvirus (vaccinia, canarypox, modified ankara virus/mva) and adenovirus . other experimental approaches use bacteria (bcg, salmonella) and rna viruses (coronavirus, vsv, sfv, reovirus, poliovirus, influenza) including also lentivirus . some of these systems are limited by the risk that exogenous genetic information from the vector can be integrated in the host genome. the advantage of these viral and bacterial systems lies in the possibility of inserting several viral genes in their genomes and their capacity to express high levels of viral proteins. strong antigen expression can in turn induce a potent and prolonged immune stimulation, particularly of cellular immune responses, against these proteins. the vaccine prototypes currently being developed include the genes gag, pol, env and nef in different combinations , , different priming-boosting strategies and vaccine doses. these types of preparation have failed as preventive vaccines in animal models, since they have not achieved protective immunity, probably due to the fact that the humoral response induced against hiv proteins is erratic and of reduced potency. they do, however, induce a potent cellular response which makes viral load stabilize at low levels , . in the most optimistic scenario it has been suggested that this response could be enough to "attenuate" the infection and transforming vaccinated patients who become infected into "long-term survivors". new vectors, such as bcg, salmonella and poliovirus are expected to induce greater humoral and cellular immunity in the mucosa by means of oral administration, thus improving the efficacy of these vaccines. the observation that "naked dna" is capable of inducing an immune response against several viruses and in different animal models broke new ground in the development of vaccines . in infection models with siv and shiv, it has been observed that, as with microbial vectors, immunization with dna is capable of inducing an immune response which, although it does not protect against infection, can often attenuate viral replication and clinical symptoms . the main limitation of dna vectors is that the intensity of the immune response induced is low, therefore they are generally used in combination with viral vectors. a disadvantage of this type of vaccine is the potential long-term secondary effects owing to chromosomal integration processes. adjuvants are preparations which boost the immune response of vaccine antigens by different mechanisms. traditional adjuvants, such as freund's, are bacterial lysates which, by inducing a non-specific inflammatory response, "recruit" immune cells at the injection site. others, such as iscom or liposomes improve the presentation of antigens. recent studies have demonstrated the efficacy of interleukins, especially these activating th responses (interleukins and ) or chemokines, in boosting the response induced by attenuated-vectors or naked-dna vaccines . successive inoculation with an interval of some weeks using two different vectors expressing the same hiv antigen (prime/booster) has been shown to induce a stronger cellular immune response against hiv antigens than when the same vector is administered in two doses. these procedures, which boost specific cd t cell induction, were developed in the murine malaria system by showing that this increase correlates with protection against the pathogen . one of the best systems reported is based on recombinant poxviruses, especially the attenuated vaccinia virus ankara (mva). this vector must be administered at the second immunization (booster), whereas in the first inoculation (priming), dna, capsids and other viral protein-expressing vectors can be used indiscriminately. the most promising prime/booster combinations include dna/pox, sfv/pox, and adeno/pox. there is currently no available preventive hiv vaccine. in fact, previously described strategies have failed because no one single animal has been protected from infection in any experimental model. table . this trial has raised controversy over whether it should be carried out, since both the experimental results and the immune response induced by these vaccine preparations have been very limited , . the most advanced pre-clinical protocols of the new vaccine prototypes include that being developed by aventis pasteur in uganda, which uses a canarypox vector for the expression of viral structural proteins . also in uganda, january saw the start of a phase i trial combining dna + mva (strain a) . a similar phase i clinical trial sponsored by iavi and kavi is being carried out in kenya. unfortunately, it seems unlikely these assays go on due to the low proportion of individuals in which a relevant immune response has been elicited with the vaccine preparations tested. in europe, through the eurovacs initiative a clinical phase i trial using poxviral-based vaccine ny-vav has been completed by juin . another phase i trial based on the combination dna/nyvac expressing gp , gag, pol and nef of subtype c was launched also in juin . a comparative assay between nyvac-c and mva-c is planned in . this latter immunogen will be generated in spain at the national centre of biotechnology. complete and updated information on the situation of existing vaccines and clinical trials is available at: www. hvtn.org/trials. obtaining an aids vaccine has become a global enterprise - . this is very positive because it has increased social awareness of the disease and financial support. it is also important to note that priority given to vaccine research and the development of new approaches appears at a time when we know much more about the pathogenesis of aids than ten years ago. nevertheless, it must be remembered that demonstrating the usefulness of a vaccine is a long and expensive process. therefore, a critical aspect of the problem is to define strategies and criteria for the different phases in vaccine development: type of vaccine, objectives of the vaccine, experimental design, animal models, pre-clinical and laboratory analysis, phase i and ii trials and requirements and infrastructure for phase iii trials. given the healthcare, social and political priorities, this subject is sometimes affected by serious concerns outside the scientific world. these include the social pressure from international organizations and countries devastated by the epidemic and financial pressure from the pharmaceutical companies. although some of these motives are understandable, given the severity of the situation, these attitudes can also distort the scientific process. below, we summarize the key questions in the search for an "aids vaccine". some scientists doubt that an efficacious aids vaccine can be found . the reason is the difficulty in obtaining what has been defined as "sterilizing immunity" against retroviruses. if we analyze the mechanisms of action of vaccines, in most cases they do not achieve "sterilizing im-munity", since they do not prevent infection but rather the persistence of the microorganism and development of disease: the germ infects, but the immune response prevents it from spreading and destroying new infected cells, thus helping to eradicate the infection. in the case of siv and hiv infection, we know that, after the first inoculum, infection takes place in a short period of time and an important reservoir of cells from the lymphoid system become infected. in some of these initially infected cells, the virus replicates actively, but in others it remains in a state of latency as an integrated provirus in the genome of the host cell. therefore, despite the immune response induced by vaccines, the virus can "persist" in the reservoirs from where it can replicate continuously. a particularly controversial area is the "final objective" of the vaccine: some people argue that if it is not possible to induce "sterilizing immunity" to prevent infection, will it be enough to have an immune response capable of controlling the level of viral replication to sufficiently low levels that allow the immune system to escape from huge destruction. the objective would not be so much to prevent infection but to attenuate it, in such a way that the infected patients become "long-term survivors" capable of living with the virus. another area of debate is the level of protection which must be "reached" by an aids vaccine. in contrast with the high efficacy of protection in most vaccines (above % of vaccinated patients) different authors propose that a partial protection rate of - % should be considered as "sufficient". this reduction in the final objectives to be attained by an hiv vaccine is arguable. on one hand, it is doubtful that "attenuation of the infection" will be a definitive phenomenon in the medium-long term. both in animal models and in isolated cases in which an infection has been caused by a defective virus, this virus increases its virulence in the long-term. on the other hand, although it is true that the establishment of a low viral load after primary infection is a good prognostic factor in the medium term, this does not guarantee that patients who present low levels of viral load after vaccination will behave as long-term survivors. the fact that some scientists set the "sufficient" efficacy of a vaccine at - % protection level can also be criticized. this could, perhaps, be considered a realistic stance and, even in specific prevalence rates in specific risk groups, a vaccine of this type could be efficacious, but we do not know its real impact on the evolution of the epidemic in the medium term. we must not forget that one of the mechanisms of vaccine efficacy is due to the "population or epidemiological impact" in the decrease in the prevalence of the infection and the consequent reduced possibility of transmitting the germ among the general population. this epidemiological impact of the vaccine would not exist with the proposed protection rates. some authors suggest that variability among subtypes represents an important obstacle for the development of a universal vaccine and that "ad hoc" vaccines should be manufactured based on the subtypes circulating in each region . however, the new vaccine prototypes use other viral genes (env, nef, gag, pol, tat) as targets which have a much lower variability than the envelope. in fact, different studies show that the immune response induced by vaccination against a specific hiv subtype is capable of acting against other subtypes , . how, when and where is the efficacy of the different vaccines to be evaluated? the efficacy of an aids vaccine must be evaluated in populations with a high rate of infection in order to obtain significant differences between the control group and the vaccinated group in the shortest time possible. this means that almost all the trials are carried out in africa and southeast asia, where annual seroconversion rate in the most affected areas is approximately % of the population . carrying out trials in developing countries raises a series of ethical issues: . it is essential that the studies comply with all ethical requirements and that patients' rights are guaranteed . . the vaccines tried must have satisfied the scientific and medical requisites of potency and safety which are necessary in any medicine tried on humans. . vaccine trials need a wide-ranging follow-up infrastructure which can guarantee patient follow-up. therefore, it is essential to develop healthcare structures and reference centers with the following objectives: recruitment and follow-up of volunteers, extraction, freeze and storage of blood according to standard procedures, and assessment of immunological parameters such as lymphoid populations, cytokine production and neutralizing antibodies. if this requisite is not met, the analysis of results could be skewed and/or incomplete, thus making it impossible to draw conclusions . . one demand by governments is that if a vaccine is efficacious, free access to the vaccine must be guaranteed to the country where the evaluation was carried out. . according to the ethical guidelines of unaids, lifelong antiretroviral therapy must be administered to any person infected during the clinical trial . an important problem, now the center of social and scientific controversy, is to define the requirements a vaccine preparation must fulfill to start a phase iii clinical trial. the journal "science" has been the forum for a series of letters from prestigious scientists criticizing investment strategy in the development of an aids vaccine and the initiation of phase iii clinical trials , . the strict scientific position defends that there are no consistent data on the efficacy of current vaccine prototypes to carry out phase iii clinical trials. consequently, such investment should be concentrated in basic research in order to get a better understanding on the mechanisms of protective immune responses and to develop new relevant animal models. faced with this stance, a more humanist position bases the start of phase iii trials on the catastrophic situation in developing countries and on the counterargument that, if there are no adequate animal models, it will be anyway necessary to carry out all the phases of the studies, including phase iii, in humans to obtain a definitive response. despite the reticence and pessimism of a large part of the scientific community, the general impression is that phase iii trials will be carried out. it is important to remember the cost and effort involved in these trials, which require the follow-up of , patients for at least five years to obtain conclusive results. therefore, with regard to aids vaccines, we are living in difficult times in which a huge economic investment will be necessary so that the scientific community can generate, develop and evaluate all the vaccine prototypes imaginable in animal models in order to find the holy grail of vaccines. as a reference, in case the european union decided to start a program of phase i and ii clinical trials with a reduced number of vaccine prototypes already generated in european laboratories an investment of . billions euros in the following years should be required. with this objective in mind, the development of vaccine research centers has been proposed . these centers would combine: (i) a critical mass of investigators, (ii) their sole dedication to the development of prototype hiv vaccines, (iii) a long-term commitment by academic, governmental and private institutions, (iv) sufficient resources and (v) continuous exchange of information and collaboration with the private sector. as a consequence of this policy the main leader organizations (nih, iavi, anrs, eu, gates foundation...) should finance vaccine development centers and would coordinate their work. the prototypes considered interesting would be prepared under the conditions of good manufacturing practice for use in humans and would enter a previously defined process of pre-clinical studies and phase i, ii and iii clinical trials. all the prototypes would meet the minimum requirements for clinical application, which would mean not only defining these criteria but also involving the regulatory authorities (fda, emea) in their development. the evaluation of prototypes also requires the definition of those immunological markers which must be used to evaluate their potential efficacy. this in turn would mean developing standardized and reproducible trials to evaluate the humoral and cellular responses to hiv and the approval of laboratories which would carry out these immunological determinations. lastly, the necessary healthcare structures should be set up to carry out the trials in clinical phases in developing countries, and the ethical criteria to be fulfilled in these trials should be defined. given the large number of current prototypes (table ), the application of homogeneous evaluation criteria is the only way to reach consistent conclusions which can be extrapolated to all situations. nevertheless, it is important to be aware that this search is full of unanswered questions and that it can fail despite all the efforts made. as it may not be possible to develop a vaccine it may be time to convey this terrible possibility to society. the history of vaccines is defined by the words "empiricism" and "success". no intervention has saved as many lives throughout the history of medicine as vaccines. these successes were often the fruit of the most basic empiricism. however, at present, empiricism cannot serve as the basis of success in the scientific development of an aids vaccine. to conclude, in recent years, the development of an aids vaccine has changed radically due to different factors: the devastating growth of the epidemic, social awareness, fi-nancial investment and, in particular, a better understanding of the pathogenesis. all these new elements enable us to face this challenge rationally and with adequate resources. only scientific effort combined with unprecedented solidarity will allow us to decide whether it is possible to find a vaccine against hiv and whether its application will be sufficient to curb the current aids pandemic. genève: who the global alliance for vaccines and immunization: a millennial challenge accelerating the development and future availability of hiv- vaccines: why, when, where, and how? challenges and opportunities for development of an aids vaccines hiv vaccines - mhc-restricted cytotoxic t-cells: studies on the biological role of major transplantation antigens determining t-cell restriction-specificity, function and responsiveness genomewide conserved epitope profiles of hiv- predicted by biophysical properties of mhc binding peptides inmunidad humoral en la infección por el vih antibodies and resistance to natural hiv infection protection of macaques against vaginal transmission of a pathogenic hiv- /siv chimeric virus by passive infusion of neutralizing antibodies rapid evolution of the neutralizing antibody response to hiv type infection neutralizing antibody-independent containment of immunodeficiency virus challenges by dna priming and recombinant pox virus booster immunizations hiv- neutralizing antibodies: how full is the bottle? antibodies, viruses and vaccines hiv vaccine design and the neutralizing antibody problem cellular immune responses to hiv. nature vigorous hiv- -specific cd + t cell responses associated with control of viremia quantitation of hiv- -specific cytotoxic t lymphocytes and plasma load of viral rna immune control of hiv- after early treatment of acute infection hiv- -specific cd + t cells are detectable in most individuals with active hiv- infection, but decline with prolonged viral suppression the challenge of immune control of immunodeficiency virus effects of in vivo cd + t cell depletion on virus replication in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine molecular epidemiology of hiv- genetic forms and its significance for vaccine development and therapy immunopathogenesis and immunotherapy in aids virus infections antiviral pressure exerted by hiv- -specific cytotoxic t lymphocytes (ctls) during primary infection demonstrated by rapid selection of ctl escape virus acute phase citotoxic t lymphocyte escape is a hallmarg of simian immunodeficiency virus infection lack of strong immune selection pressure by the immunodominant, hla-a* -restricted cytotoxic t lymphocyte response in chronic human immunodeficiency virus- infection hiv-specific cd + t cell proliferation is coupled to perforin expression and is maintained in nonprogressors ctl ontogeny and viral escape: implications for hiv- vaccine design hiv-tailored to fit hiv- evades antibody-mediated neutralization through conformational masking of receptor-binding sites antibody neutralization and escape by hiv- rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus population biology of hiv- infection: viral and cd + t cell demographics and dynamics in lymphatic tissues the challenge of viral reservoirs in hiv- infection dc-sign, a dendritic cell-specific hiv- -binding protein that enhances trans-infection of t cells hiv preferentially infects hiv-specific cd + t cells protective effects of a live attenuated siv vaccine with a deletion in the nef gene genomic structure of an attenuated quasi species of hiv- from a blood transfusion donor and recipients live-attenuated, multiply deleted siv causes aids in infants and adult macaques declining cd t-cell counts in a person infected with nef-deleted hiv- pathogenic conversion of live attenuated siv vaccine is associated with expression of truncated evaluation of hiv- immunogen, an immunologic modifier, administered to patients infected whith hiv having to × /l cd cell counts: a randomized controlled trial advancing aidsvax to phase . safety, immunogencity, and plans for phase immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type tat as one key to hiv-induced immune pathogenesis and tat toxoid as an important component of a vaccine neutralization of hiv- by secretory iga induced by oral immunization with a new macromolecular multicomponent peptide vaccine candidate immunization with a modified vaccinia virus expressing simian immunodeficiency virus (siv) gag-pol primes for an anamnestic gag-specific cytotoxic t-lymphocyte response and is associated with reduction of viremia after siv challenge replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity protective immune responses induced by secretion of a chimeric soluble protein from a recombinant mycobacterium bovis bacillus calmette-guérin vector candidate vaccine for human immunodeficiency virus type in small animals lentiviral vectors: turning a deadly foe into a therapeutic agent hiv- dna vaccines and chemokines augmentation of immune responses to hiv- and simian immunodeficiency virus dna vaccines by il- /ig plasmid administration in rhesus monkeys il- and il- act in synergy to clear vaccinia virus infection: involvement of innate and adaptive components of the immune system priming with recombinant influenza virus followed by administration of recombinant vaccinia virus induces cd + t-cell-mediated protective immunity against malaria preventive hiv vaccine development in thailand aidsvax flop leaves vaccine field unscathed public health. a sound rationale needed for phase iii hiv- vaccine trials policy rebuttal. hiv vaccine trial justified a canarypox vaccine expressing multiple human immunodeficiency virus type genes given alone or with rgp elicits broad and durable cd + cytotoxic t lymphocyte responses in seronegative volunteers effective induction of simian immunodeficiency virus-specific cytotoxic t lymphocytes in macaques by using a multiepitope gene and dna primemodified vaccinia virus ankara boost vaccination regimen why an hiv vaccine is not currently within our grasp. xi croi clade b-based hiv- vaccines elicit cross-clade cytotoxic t lymphocyte reactivities in uninfected volunteers a human immunodeficiency virus prime-boost immunization regimen in humans induces antibodies that show interclade cross-reactivity and neutralize several x -, r -, and dualtropic clade b and c primary isolates the highest attainable standard: ethical issues in aids vaccines some important issues in the planning of phase iii hiv vaccine efficacy trials unaids. ethical considerations in hiv preventive vaccine research the needfor a global hiv vaccine enterprise our laboratories are funded by redes temáticas cooperativas de investigación en sida (ris g / ) y de genética clínica y molecular (rig / ), "fundación para la investigación y la prevención del sida en españa" (fipse / ; / ; / ; / ), "programa nacional de salud" (saf - y - - ) and comunidad de madrid. we are grateful to florencia etcheverry for her help in revising the bibliography of this manuscript. key: cord- -h o yqv authors: nan title: oral communications and posters date: - - journal: inflamm res doi: . /bf sha: doc_id: cord_uid: h o yqv nan the drosophila host defense is a multifaceted process which involves reprogramming of gene expression, activation of proteolytic cascades in the blood and uptake of microrganismsby professionalphagocytes. most of the recent studies have focused on challenge-induced expression of antimicrobial peptides and have addressed the following questions : ( ) which genes are upregulated during various types of bacterial, fungal or viral infections and what are their functions? ( ),what is the nature of the intracellular signalling cascades which lead togene transcription during these infections; ( ) how does drosophila recognize infections and does it discriminate between various types of aggressors (e.g. fungal versus bacterial or viral) to mount an appropriate response. over the last ten years we have gained significant insights into these various aspects and the presentation will review our current understanding of the drosophila immune response and put it into a phylogenetic perspective, namely by drawing some stringent parallels with the mammalian innate immune response. there is strong evidence that autoimmunity to myelin antigens plays a major role in the development of multiple sclerosis. several myelin-derived autoantigenic targets have been described and include myelin basic protein (mbp), proteolipid protein and myelin oligodendrocyte glycoprotein. there has been a particular focus on mbp for at least two reasons: mbp-specific cd + tcell receptors (tcrs) have been found in multiple sclerosis brains, and cells presenting an immunodominant mbp( - ) peptide in complex with hla-dr b have been shown to be present in multiple sclerosis lesions. also, humanized mice expressing the hla-dr b gene and a human t-cell receptor (tcr) that recognises the mbp - peptide in the context of hla-dr b either spontaneously or after immunization with mbp - develop experimental autoimmune encephalomyelitis, which has several features in common with multiple sclerosis. this talk will focus on, how humanized mice recently has been used to study the interplay between genetic and environmental risk factors in multiple sclerosis. to resolve the pathogenic mechanisms of rheumatoid arthritis (ra) we need to identify the causative genetic and environmental factors. this has however proven to be complex, with many factors and genes interacting. inbred animals are useful for studies of the identification of genes associated with complex traits and diseases such as ra. animal models offer a possibility to better define the traits, and to segregate the genes in a controlled way enabling linkage analysis. there are several arthritis models, which each may reflect various variants of the heterogeneity of ra in humans. examples are collagen induced arthritis (cia) and pristane induced arthritis (pia), which both fulfills the clinical diagnostic criteria for ra. both diseases are genetically complex and the susceptibility is, as ra, dependent on many polymorphic genes operating in concert. so far genes in this concert have been identified; the mhc class ii ab gene in the mouse ( ) and the ncf gene in the rat ( ) and in the mouse ( ) . the ncf protein is a part of the nadph oxidase complex mediating oxidative burst. the discovery of the ncf polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive t cells. mice with the deficient ncf allele are more susceptible to cia, and also developed a chronic form of arthritis. interestingly, the immune response to cii was enhanced by the ncf deficiency linking the ncf pathway to the adaptive immune response. oxidation of t cell membranes seem to be a key event in the pathogenic mechanism as reduction of t cell membranes induces arthritis in rats ( ). on the basis of these findings a new type of therapy myasthenia gravis is a prototypic autoimmune disease, caused in most cases by autoantibodies to the muscle acetylcholine receptor (achr) at the neuromuscular junction. the antibodies reduce the number of achr leading to failure of neuromuscular transmission and muscle weakness. the achr antibodies as measured in conventional immunoprecipitation assays are igg, high affinity, polyclonal and specific for human achr. they reduce the numbers of achr by antigenic modulation and by complement-mediated damage to the neuromuscular junction. myasthenia gravis has a very intriguing relationship with the thymus gland. in many younger patients, the thymus is hyperplastic with immune cell infiltrates and germinal centre formation. around the germinal centres, within the medulla, there are rare muscle-like cells called myoid cells that express achrs. there are many b cells and plasma cells and thymic lymphocyte preparations synthesise achr antibodies. the possibility that the thymic achr induces the germinal centre formation and achr antibody synthesis is supported by much evidence. some patients, however, have thymic tumours and in these the role of the thymus is less clear. moreover, older patients with typical myasthenia usually have thymic tissue which is normal for their age. there are up to % of myasthenia patients that do not have the typical achr antibodies. some of these have instead antibodies to muscle specific kinase, a receptor tyrosine kinase that is restricted to the neuromuscular junction. the pathogenic mechanisms by which the antibodies cause disease are not yet clearly identified and the evidence will be discussed. finally, among the patients who have neither achr nor musk antibodies by conventional testing, we have evidence for lower affinity antibodies to achr which can now be detected using molecular approaches which will be described. arry- (azd ) is an inhibitor of mek / currently in development for cancer. phase determined the msd ( mg) and the safety of the compound given continuously. in decreasing frequency, common treatment-related side effects were rash, diarrhea, nausea, peripheral edema, and vomiting. paired pre-and postdose tumor biopsies showed a reduction in perk (- %) and proliferative index (- %). the trough plasma concentration ( ng/ml) corresponded to~ % inhibition of perk. about % of pts had stable disease after months. these results demonstrate that arry- (azd ) is well-tolerated, hits its target, and produces a high incidence of long-lasting stable disease. there are several on-going phase studies, in melanoma, colorectal, lung and pancreatic cancer. arry- is another potent, selective mek / inhibitor, currently in development for inflammatory diseases. when human whole blood was stimulated with tpa, arry- inhibited tnfa, il- b and il- (ic s of , and nm, respectively). % inhibition of perk required nm. arry- was highly efficacious in cia and aia rat models, with ed s of and~ mg/ kg, respectively. in normal volunteers arry- was well-tolerated and there was a dose-proportional increase in drug exposure. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpa-induced tnfa and il b. an on-going multiple ascending dose clinical study is further exploring the pharmacokinetics, pharmacodynamics and tolerability of arry- monotherapy. in addition, we have initiated a clinical trial designed to evaluate arry- in combination with methotrexate in patients with rheumatoid arthritis. rho kinases (rock) are involved in many physiological and pathological processes including smooth muscle contraction, cytoskeletal arrangement, cell adhesion, migration and proliferation.rocks prominent role in cytoskeletal architecture suggests that rock inhibitors should have therapeutic impact in oncology and fibrotic diseases which require cytoskeletal rearrangement to progress.we have synthesized small molecule inhibitors of rock which are specific for the rock- isoform.these rock- inhibitors, typified by slx- and slx- , are potent (ic < nm), selective for rock- (> fold selectivity for rock- over rock- ) and exhibit good oral bioavailability.this talk will focus on several areas in oncology and fibrotic disease where the ability to demonstrate an in vitro effect on the cytoskeleton translates into activity in the disease model in vivo.slx- inhibits cell proliferation and migration in several tumor cell lines including ht- , panc- and mda-mb- . moreover in xenograft studies using nude mice, slx- significantly inhibited tumor growth with these same cell lines. in liver fibrosis, slx- prevents the differentiation of rat primary hepatic stellate cells into myofibroblasts and inhibits the proliferation of myofibroblasts as well as inhibiting hepatic steatosis in an atherosclerosis model.slx- is an effective antifibrotic agent in the kidney unilateral urethral obstruction model and inhibits renal fibroblast differentiation and proliferation.these data suggest that rock- selective inhibition of cytoskeletal modification in key cell types (e.g. tumor cells, stellate cells and fibroblasts) by compounds such as slx- and slx- will provide effective treatment for oncology and fibrotic disease. cyclooxygenases (cox) catalyze the first step in the synthesis of prostaglandins (pg) from arachidonic acid.cox- is constitutively expressed.the cox- gene is an immediate early-response gene that is induced by variety of mitogenic and inflammatory stimuli.levels of cox- are increased in both inflamed and malignant tissues.in inflamed tissues, there is both pharmacological and genetic evidence that targeting cox- can either improve (e.g., osteoarthritis) or exacerbate symptoms (e.g., inflammatory bowel disease).multiple lines of evidence suggest that cox- plays a significant role in carcinogenesis.the most specific data that support a cause-and effect relationship between cox- and tumorigenesis come from genetic studies.overexpression of cox- has been observed to drive tumor formation whereas cox- deficiency protects against several tumor types.selective cox- inhibitors protect against the formation and growth of experimental tumors.moreover, selective cox- inhibitors are active in preventing colorectal adenomas in humans.increased amounts of cox- -derived pge are found in both inflamed and neoplastic tissues.the fact that pge can stimulate cell proliferation, inhibit apoptosis and induce angiogenesis fits with evidence that induction of cox- contributes to both wound healing and tumor growth.taken together, it seems likely that cox- induction contributes to wound healing in response to injury but reduces the threshold for carcinogenesis. ( ), k hagihara( ), t nishikawa( ), j song( ), a matsumura ( ) ( ) health care center, osaka university, japan ( ) osaka university graduate school of medicine, japan it is still less known about the actual pathogenic role of il- in the inflammatory status. to know the pathogenic role of il- and the efficacy of il- blockade in inflammation, a humanized anti-il- receptor antibody, tocilizumab, was used for the treatment in chronic inflammatory diseases, such as castlemans disease, rheumatoid arthritis and crohns disease. since il- blocking therapy improved the clinical symptoms and the laboratory findings, the il- function in inflammation could be analyzed for the induction of inflammatory molecules, such as serum amyloid a (saa). saamrna induction, saa promoter activity and assembling of transcriptional factors on saa promoter were analyzed by the real time rt-pcr, gel shift assay and dna affinity chromatography in hepatocyte stimulated with the proinflammatory cytokines, il- , il- and tnf-alpha. in result, il- was an essential cytokine in induction of saamrna through the activation of stat which constructed the complex with nf-kappab p and a cofactor p . although there was no stat consensus region on saa promoter, stat bound at the site of nf-kappab re. the above research proved that il- signal is essential on the synergistic induction of saa via newly discovered stat transcriptional mechanism, suggesting the presence of this stat mechanism in inflammation, and confirming the normalization of serum saa level by il- blocking therapy in inflammatory diseases. this research method develops a subsequent therapy for serious aa amyloidosis by inhibition of saa production, and elucidates the cytokine mechanism on immunopathogenesis of chronic inflammatory diseases. takashi wada( ), k matsushima( ), s kaneko ( ) ( ) kanazawa university, japan ( ) university of tokyo, japan accumulating evidence indicates that chemokine/chemokine receptor system plays a key role in the pathogenesis of various renal diseases via leukocyte migration. pathophysiological impacts of chemokines have shed light on molecular mechanisms of leukocyte trafficking and their activation in the inflammatory aspects of progressive renal injury.locally expressed chemokines are proven to be capable of inciting leukocyte migration to the kidney, resulting in initiating and promoting chronic kidney diseases.the possible positive amplification loop from cxc chemokines to cc chemokines may contribute to progressive renal injury, resulting in sclerosis/fibrosis.it is of note that monocyte chemoattractant protein (mcp)- / monocyte chemotactic and activating factor (mcaf)/ ccl , a prototype of cc chemokines, promotes and escalates chronic kidney diseases with any etiologies via the infiltration and activation of monocytes/macrophages, proteinuria and collagen synthesis.interactions between infiltrated inflammatory cells and resident renal cells eventually lead to the progression of fibrosis. new insights into renal fibrosis have been uncovered by the regulation of fibrocytes dependent on chemokine system. in addition, recent studies demonstrate that chemokines have been expanding their universe beyond leukocyte migration to the kidney, including homeostasis, development and repair of the kidney.the selective intervention of chemokines might have the therapeutic potential to alter inflammatory responses, thereby halting the progression of renal injury. we focus on recent progresses on the role of chemokines and their cognate receptors in renal injury in this symposium. ( ), p lacamera ( ), b shea ( ), g campanella ( ), b karimi-shah ( ) , n kim ( ), z zhao( ), v polosukhin ( ), y xu( ), t blackwell ( ) aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses remain to be fully identified.here we demonstrate that lysophosphatidic acid (lpa) is induced by lung injury in the bleomycin model of pulmonary fibrosis, and that mice deficient for one of its receptors, lpa , are dramatically protected from pulmonary fibrosis and mortality following bleomycin challenge. the absence of lpa markedly reduced fibroblast responses to the chemotactic activity present in the airspaces following bleomycin, and attenuated the subsequent accumulation of fibroblasts in the lung.the increase in vascular permeability caused by lung injury was also markedly reduced in lpa -deficient mice, whereas bleomycin-induced leukocyte recruitment was preserved.these results demonstrate that lpa links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, two of the wound-healing responses that are thought to be inappropriately excessive when injury leads to fibrosis rather than repair.lpa therefore represents a new target for lung diseases in which aberrant responses to injury contribute to the development of fibrosis, such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. we have reported that inflammation is detrimental for survival of new hippocampal neurons early after they have been born. our data now show that microglia activation, as an indicator of inflammation, is not pro-or antineurogenic per se but the net outcome is probably dependent on the balance between secreted molecules with pro-and antiinflammatory action. we have found that a substantial fraction of the new hippocampal neurons formed after status epilepticus survive despite chronic inflammation. we have started to explore the role of tnf-alpha for adult neurogenesis. infusion of an antibody to tnf-alpha was shown to reduce survival of new striatal and hippocampal neurons generated after stroke, probably by interfering with action of the ligand on the tnf-r receptor. we have shown that tnf-r is a negative regulator of progenitor proliferation in basal and insult-induced hippocampal neurogenesis. we have also used patch-clamp technique to explore whether a pathological environment influences the synaptic properties of new granule cells. rats were exposed to either a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus which is associated with inflammation. we found that new granule cells in runners and status epilepticus animals had similar intrinsic membrane properties. in contrast, the new neurons which had developed in the physiological and pathological tissue environments differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. the role of inflammation for these differences is currently being explored. proteinase-activated receptor- (par- ) is cleaved within its aminoterminal extracellular domain by serine protei-nases such as trypsin, unmasking a new aminoterminus starting with sligkv that binds intramolecularly and activates the receptor. par- is implicated in innate defense responses associated with lung inflammation. we showed that par- is expressed by human alveolar (a ) and bronchial ( hbe) epithelial cell lines, and is activated by trypsin and by the activating synthetic peptide sligkv-nh . in cystic fibrosis patients, airspaces are invaded by polymorphonuclear neutrophils that release elastase and cathepsin g, two serine proteinases, and by pseudomonas aeruginosa that secretes an elastolytic metalloproteinase.we demonstrated that these three proteinases do not activate par- , but rather disarm this receptor, preventing its further activation by trypsin but not by sligkv-nh . preincubation of a par- transfected cell line with either proteinases leads to the disappearance of the cleavage/activation epitope. proteolysis by these three proteinases of synthetic peptides representing the aminoterminal extracellular domain encompassing the cleavage/activation sequence of par- , generate fragments that would not by themselves act as receptor-activating ligands and that would not yield receptor-activating peptides upon further proteolysis by trypsin. our data indicate that neutrophiland pathogen-derived proteinases can potentially silence the function of par- in the respiratory tract, thereby altering the host innate defense mechanisms. caspase- -dependent killing of host cells and to disrupt intestinal barrier function, which, at least in the case of giardiasis, ultimately causes lymphocyte-dependent intestinal malfunction, and the production of diarrheal symptoms. ongoing research is investigating whether par agonists and microbial pathogens may cause epithelial apoptosis, increased permeability, and overall epithelial malfunction in the gastrointestinal tract, via common or intersecting pathways. the intestinal epithelium is exposed to a variety of proteases in both health and disease, including digestive proteases such as trypsin.given that protease-activated receptor (par ) responds to trypsin and is expressed on intestinal epithelial cells, we investigated the effect of trypsin on intestinal epithelial barrier function.scbn, caco-ii and t epithelial cells were grown to confluence on filter supports and mounted in ussing chambers to study short circuit current (isc) and transepithelial resistance (rte).cell monolayers were incubated with inhibitors of transcellular ion transport in order to isolate the contribution of the paracellular pathway to rte.apical exposure to serine proteases including trypsin, elastase and chymotrypsin caused a rapid and sustained increase in rte and decreased the transepithelial flux of a mw dextran.interestingly, the effect of trypsin could not be replicated by activators of pars , and , suggesting that the effect on rte was not due to activation of pars.subsequent experiments showed that trypsin activated phosphatidylinositol-dependent phospholipase c.a trypsin-induced increase in intracellular calcium was not involved.inhibition of pkc-zeta, but not of typical pkcs, also blocked the response.our data point to a role for postprandial trypsin that extends beyond that of a digestive enzyme; it is also a participant in cellular pathways that control tight junction permeability. physiologically, the trypsin-induced increase in resistance could augment transcellular transport by reducing passive paracellular back-diffusion of ions. further studies will assess how these pathways might be disrupted in the barrier dysfunction characteristic of intestinal inflammation. clustering of inflammatory bowel disease in large families and the observation of an increased concordance between monozygotic twins suggests heritable components in these disorders. the high concordance in monozygotic twins (> %), which is not seen in dizygotic twins (< %) points to strong contribution of genetic susceptibility to the overall risk for disease. ibd represents a "complex disease" and may involve a large number of interacting disease genes. crohns disease has become an example for the successful molecular exploration of a polygenic etiology. crohns disease was not known before . incidence has increased since now leading to a lifetime prevalence of up to . % in western industrialized countries. the current hypotheses propose unknown trigger factors in the life style of western industrialized nations that interact with a polygenic susceptibility. it appears that increased expression and production of tnf and an enhanced state of activation of the nfkb system are main drivers of the mucosal inflammatory reaction. the exploration of inflammatory pathophysiology of crohns disease using full genome, cdna and oligonucleotide based arrays, respectively, has generated large sets of genes that are differentially expressed between inflamed mucosa and normal controls. while this may lead to new targets for a pathophysiology oriented therapy, it appears, however, that the dissection of the inflammatory pathophysiology does not allow to identify the multifactorial etiology of the disease. genome-wide linkage analysis has demonstrated eight confirmed susceptibility regions with the one on chromosome being most consistent between different populations. in three coding variations in the card gene were identified that are highly associated with development of the disease. all variants affect a part of the gene that codes for the leucin rich part of the protein, that appears to be involved in bacteria induced activation of nfkb in macrophages and epithelial cells. interestingly, the three disease associated snps are never found on the same haplotype. in compound heterozygotes or homozygotes they result in a rr of > to develop crohns disease as an adult. a particular subphenotype with localization of the disease in the ileocecal region is highly associated with the variants in the card gene. variations in the card gene do not fully explain the linkage finding in the pericentromeric region of chromosome . after stratification for card variants, the broad linkage peak is reduced to two more defined peaks on p and q, respectively. while the exploration of these regions has led to several association signals that are subject to further fine mapping a further disease gene progress has been greater in the other linkage regions (i.e. on chromosomes and , respectively). dlg- is the example of a low-risk susceptibility gene with a modest associated odds ratio ( . - . ) . interestingly, the associa-tion signal appears to be confined to young males. slc a / which encode the kation-transporters octn and have been suggested to represent the disease gene in the + kb haplotype block on chromosome q . mdr has also been implicated as a disease gene in ibd. although the human association studies have resulted in highly controversial findings a knockout mouse with a colitis phenotype makes mdr likely as a low risk susceptibility gene. with the advent of high-density, genome wide association studies enormous progress has been made to discover the remaining disease genes. recently a k illumina scan has been published identifying il- r as a further disease gene. we used a genome wide candidate gene approach (with appr. . csnps) to identify atg l as a further disease genes. both genes were confirmed and a further regulatory snp involving ptger was annotated by a belgian genome wide scan. by the time of presentation three further genome wide snp scans in crohn disease will most likely have entered the public domain. the further exploration of crohns disease (and other inflammatory conditions of barrier organs) will have to annotate the function and pathophysiologies based on genetic risk maps that are completed with amazing speed. the creation of a medical systems biology of disease will lead to new models and eventually new therapies. the chemokine receptor ccr plays a pivotal role in mediating the migration of t cells to the gastrointestinal mucosa. the ligand for ccr , teck, s highly expressed in the gi tract. the pathogenicity of intestinal ccr +/ cd + t cells has been demonstrated in animal models and this cell population is substantially increased in the peripheral circulation of crohns and celiac disease patients. ccx -b is a highly selective and potent, orally bioavailable, small molecule antagonist of ccr .the compound proved to be highly efficacious in the tnf-aare and mdr a-/-murine models of inflammatory bowel disease (ibd). in phase i trials, ccx -b was well-tolerated, and no drug-related saes were reported.a -day placebo-controlled phase ii study was recently completed in patients with moderate to severe crohns disease. ccx -b was shown to be both safe and to have encouraging clinical results: % of patients on ccx -b (cdai ! , crp > . mg/l) exhibited a -point drop in cdai compared to % on placebo. furthermore, a crp decrease of mg/l was seen in the ccx -b group compared to placebo. colonic biopsy samples were analyzed for expression of several pro-inflammatory cytokines. a mean decrease from baseline in the concentrations of tnf-alpha, il- p , ifn-gamma, and the chemokine rantes was shown in the ccx -b group while levels remained stable in the placebo group. ccx -b is the first chemokine-based inhibitor of leukocyte trafficking to be tested in ibd. the compound shows anti-inflammatory activity and encouraging evidence for clinical benefit in the treatment of crohns disease. the activating receptor nkg d seems to be implicated in the pathogenesis of several autoimmune diseases in humans and in animal models for type diabetes and multiple sclerosis. the aim of this study was to asses the role of nkg d in a model of inflammatory bowel disease, where cd +cd -t cells from balb/c mice are adoptively transferred to scid mice, and to evaluate the therapeutic effect of an anti-nkg d antibody therapy. the expression of nkg d was evaluated by flow cytometry, immunohistochemistry and by pcr. we found a marked up-regulation of nkg d on the cell surface as well as increased levels of nkg d mrna in cd + t cells from colitic scid mice as compared to normal balb/c mice. we next studied the effect of anti-nkg d antibody (cx ) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. cx treatment decreased the cellsurface expression of nkg d and prophylactic administration of cx attenuated the development of colitis significantly. a moderate reduction in the severity of disease was observed after cx administration to mildly colitic animals, whereas cx did not attenuate severe colitis. thus, nkg d may be involved in the pathogenesis of colitis in this model, particularly in the early phases, since the expression of nkg d in cd + t cells increased markedly during the development of disease and since administration of cx early but not late in the course attenuated the disease severity. proteins are used already for more than a century in the treatment of disease. the first generation were proteins derived from animals such as antisera used to treat infectious diseases as diphtheria and tetanus and later bovine and porcine insulin for the treatment ofdiabetes. the second generation were natural proteins from human source like the plasma derived clotting factors and human growth hormone. the development of the recombinant dna and cell fusion technology in the seventies of the th century opened up the possibilities to produce human proteins and monoclonal antibodies in unlimited amount in microbial and mammalian host cells. in human insulin was introduced as the first recombinant dna derived biopharmaceutical and since than more than have gained approval. the pipeline contains many more potential biopharmaceuticals and at present in new drug applications concerns a biotechnology derived product. a major problem of therapeutic proteins is the induction of antibodies. for foreign proteins such as the murine derived monoclonal antibodies thisimmunogenicity was to be expected. however the humanization of monoclonal antibodies has reduced but not solved the problem of immunogenicity. and also the proteins which are homologues of endogenousfactors such as gm-csf, interferons etc. induce antibodies, sometimes even in the majority of patients. by definition we are immune tolerant to products which are copies of endogenous proteins. the products not necessarily need to be exact copies of the natural proteins to share this immune tolerance. when human therapeutic proteins induce antibodies, they are breaking b cell tolerance, which starts with the activation of autoreactive b-cells. presenting the self-epitopes in an array form is very potent activator of these b-cells. this explains why aggregates of human proteins are the most important factor in induction of antibodies. these aggregates may not be immediately present in the product, but may appear during storage making stability and formulation an important issue in predicting the immunogenicity. there are only a few studies in experimental model systems on the properties of the aggregates which break b-cell tolerance, indicating that only multiple order aggregates (>trimers) are involved. we study the capacity of a protein product to break b-cell tolerance in mice made transgenic for the specific protein. these mice are immune tolerant and there is a good correlation of an immune response in these mice and in patients. although these models have helped to identify the factors important for breaking b-cell tolerance and also have been useful in improving the formulation of products, there is not yet enough experience to use them as absolute predictors of immunogenicity of human proteins. they also allow to study the involvement of tcells in breaking b cell tolerance. all data obtained untilnow indicate this process to be t-cell independent. contact information: dr huub schellekens, central laboratory animal institute, utrecht university, utrecht, the netherlands e-mail: h.schellekens@gdl.uu.nl biomonitor aps, and institute for inflammation research (iir), rigshospitalet, copenhagen, denmark using recombinant technology, one can now produce protein drugs which are almost identical with naturally occurring human proteins, including antibodies (abs). many have assumed that these drugs may be administered with little or no risk of triggering specific t-and/or b-lymphocyte reactivities, because patients according to immunological dogma are tolerant towards their own proteins. unfortunately, this is not the case, and even socalled % human biologicals are potentially immunogenic ( ) ( ) . i shall discuss two examples: ) recombinant human cytokines (ifn-beta- a and - b), and ) anticytokine ab constructs (anti-tumor necrosis factor (tnf)-alpha). ifn-beta has been used for treatment of patients with multiple sclerosis since the early nineties. though initially neglected as a clinical problem, ifn-beta like many other human proteins is indeed immunogenic, especially those produced by recombinant gene technologies. the reported frequencies and titers of anti-ifn-beta ab vary considerably depending upon ifn-beta preparations and administration, and the types of assays being used ( - ). it took more than years of clinical use before abmediated decrease in bioactivity of ifn-beta, a condition in which the clinical effect of continued injection of rec. ifn-beta is minimized or abrogated, was universally recognized ( , ). ) anti-tnf-alpha human ab constructs tnf-alpha is an inflammatory cytokine of central pathogenic importance in many immunoinflammatory conditions, and measures to diminish production and/or effects of tnf-alpha have long been a goal in the treatment of these conditions. currently, there are three approved and two other anti-tnf-alpha biopharmaceuticals in clinical use. unfortunately, response failure is frequently encountered. thus, - % of patients are primary non-or lowresponders to the anti-tnf constructs, and secondary response failure is commonplace, mostly due to induction of anti-abs. several different methods have been used to assess circulating levels of anti-tnf drugs as well as anti-abs. most of these have been based on elisa technology, with their inherent problems of false positivity, susceptibility to nonspecific interference, etc. interferon beta (ifnbeta) has been an important step forward in the treatment of multiple sclerosis(ms), an inflammatory disease of the human central nervous system. however, one of the problems of ifnbeta is its immunogenicity; a substantial percentage of ms patients treated this recombinant protein develop anti-ifnß antibodies, primarily of the igg class. the level of these antibodies tends to be low in the first month or two and peaks by six to eighteen months after initiation of therapy. most studies of these antibodies have measured their ability to neutralize ifnbetas effect in vitro, using assays in which sera from ms patients inhibit the protective effect of ifnbeta on viral killing of target cells. this antibody population is called neutralizing antibodies (nabs). tests measuring binding of antibodies to ifnß in vitro are called binding antibody (bab) assay. anti-ifnbeta antibodies detected by bab assays are present in a high percentage of ms patients, and can occur at low levels without any apparent adverse effect on ifnbeta bioactivity. the distinction between babs and nabs is artificial, and all binding antibodies are likely neutralizing, if the neutralizing assay system is adequately sensitive; i.e., the development of babs and nabs is a continuum with the assay systems simply measuring the strength of the antibody response. in many treated patients, the anti-ifnbeta antibody response is strong, despite the resemblance of the injected protein to the human homologue, and high levels of neutralizing antibodies develop. high levels of anti-ifnbeta antibodies with high affinity results in loss of ifnbeta bioactivity, a phenomenon which has been called antibody-mediated decreased bioactivity or adb. adb can be considered the in vivo correlate of the neutralizing effect of the anti-ifnß antibody population, while the nab assay measures the in vitro neutralization of this population of immunoglobulins in the serum. the three ifnbeta preparations have different incidences of nabs and different patterns of appearance and disappearance over time of nabs. because there is no direct correlation between nab levels and bioactivity at moderate levels of nab, in vivo bioactivity assays for ifnbeta have become increasingly utilized. in a large multicenter study in the us, called the insight study, bioactivity as measured by ifnbeta induced upregulation of the ifn-response genes mxa, viperin, and ifit , was shown to be highly correlated with nab levels, confirming a single center study (pachner, a.r., pak,e., narayan, k., multiplex analysis of expression of three ifnbeta-inducible genes in antibody-positive ms patients, neurology, : - , ) . multiple studies, including a large multicenter study in denmark and a recent study from our center using high resolution mri of the brain once a month, have demonstrated that nabs abolish the salutary effects of ifnbeta on clinical aspects of ms, especially inflammation. recent guidelines for european neurologists recommend stopping ifnbeta in nab-positive patients. in order to maintain bioactivity of this important medication for ms, some neurologists have attempted to use immunosuppressives either to prevent the development of nabs, or to treat them once they have developed. however, at this point in time, there is no clearly optimal way to treat nabs. major efforts have been underway to decrease the immunogenicity of ifnbeta and a new formulation of one of the higher immunogenicity products has been recently developed and tested. proteinase-activated receptors (pars). endogenous serine proteinases such as thrombin, mast cell tryptase, trypsins, kallikreins, cathepsin g, for example, as well as exogenous proteases released by mites or bacteria are involved in cutaneous inflammation, host defense or tumor cell regulation. thus, the expression of pars on keratinocytes, endothelial cells, nerves, and immune cells suggest important role of pars as a part of the communication system in the skin during inflammation and the immune response. for example, par activates nf-kb in keratinocytes and endothelial cells, stimulates the release of chemokines and cytokines, and is involved in proliferation and differentiation. on sensory nerves, this receptor controls neurogenic inflammation by modulating edema and extravasation via release of neuropeptides into the inflammatory site. par and par also modulate leukocyte-endothelial interactions in the skin, thereby regulating inflammatory responses such as leukocyte trafficking through the vessel wall. they also stimulate signal transduction pathways involved in cutaneous inflammation. in sum, this novel receptor family requires a paradigm shift in thinking about the role of proteases in cutaneous biology and disease. novel compounds regulating protease and par function may be beneficial for the treatment of several skin diseases such as atopic dermatitis, psoriasis or pruritus. serine proteinases are upregulated in arthritic joints where their enzymatic activity participates in the destruction of articular soft tissues. in addition to their degradative functions, serine proteinases can also act as signalling molecules by activating members of the gprotein coupled receptor family called the proteinase activated receptors (pars). these receptors are known to regulate tissue inflammation and pain, although their function in joints is unclear. our study examined the effect of par activation in joint inflammation and pain. male c bl/ mice received an intra-articular injection of either the par activating peptide aypgkf-nh or the inactive peptide yapgkf-nh ( mg) into the right knee. knee joint blood flow was then measured in these mice by laser doppler perfusion imaging while joint diameter measurements gave an indication of tissue oedema. mechanical allodynia was also assessed in these animals by application of von frey filaments onto the plantar surface of the ipsilateral hindpaw and a pain score was calculated. intra-articular injection of the par activating peptide caused knee joint blood flow to gradually increase by up to % over the succeeding hrs. knee joint swelling was also observed as well as the development of a mechanical allodynia. all responses could be blocked by pre-treatment with the selective par antagonist pepducin p pal ( mg i.p.). the control peptide yapgkf-nh had no discernible effect on joint inflammation or pain. these experiments show that peripheral activation of par receptors in mice knees causes joint inflammation and pain. vincent lagente ( ), e boichot ( ) ( ) air liquide, centre de recherche claude-delorme, jouy en josas, france ( ) inserm u , universitØ de rennes matrix metalloproteinases (mmps) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. these led to the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue may be of interest for future treatment. among metalloproteinases (mmps) family, macrophage elastase (mmp- ) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease (copd). pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. pulmonary fibrosis is associated with deposition of extracellular matrix (ecm) components in the lung interstitium. the excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-mmp- /timp- ratio in broncholaveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day after bleomycin administration. finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of extensive lung destruction and fibrosis. in addition to their degradative properties, proteases can act as signalling molecules that send specific messages to cells.recent work has demonstrated that proteases are able to signal to peripheral sensory neurons, thereby participating to neurogenic inflammation processes and to the transmission or inhibition of pain messages. serine proteases cleaving specifically at an arginin site are able to activate protease-activated receptors (pars), which then send specific messages to cells. we have demonstrated that members of the par family (par , par and par ) are present on peripheral sensory neurons, where they can be activated by different proteases.the activation of par and par in isolated sensory neurons provokes calcium mobilization and the release of substance p and cgrp, while the activation of par inhibited bradykinin-and capsaicin-induced calcium signal, and neuropeptide release.thrombin and pancreatic trypsin caused inflammation respectively through a par and par -dependent mechanism involving the release of neuropeptide.the extrapancreatic form of trypsin (mesotrypsin or trypsin iv) also caused neurogenic inflammation through a par and par dependent mechanism, and causes inflammatory hyperalgesia and allodynia, through a par -dependent mechanism.in contrast, activation of par on peripheral sensory neurons inhibited inflammatory hyperalgesia and allodynia. taken together, these results provide evidences that proteases can interfere with inflammatory and pain mechanisms through the activation of pars on peripheral sensory neurons.determining the role of each individual proteases and their receptors in sensory neuron signalling and above all inflammatory and pain mechanisms constitutes an important challenge to raise new anti-inflammatory and analgesic drugs. introduction: a scoring system for disseminated intravascular coagulation (dic) in humans has been proposed by the international society on thrombosis and haemostasis (isth). it was the objective of this study to develop and validate a similar scoring system for dic in dogs in order to establish the dog as a spontaneous animal model. methods: for the developmental study, consecutive dogs admitted to the intensive care unit (icu) were enrolled prospectively (group a). blood samples were collected daily and a broad panel of coagulation assays performed. diagnosis of dic was based on the expert opinion of one human physician and two veterinarians. a multiple logistic regression model was developed with the coagulation parameters as explaining variables for the diagnosis of dic. integrity and diagnostic accuracy was subsequently evaluated in a separate prospective study according to the stard criteria. the validation study prospectively enrolled consecutive dogs (group b). results: dogs were excluded from group a where / dogs ( %) had dic. final multiple logistic regression model was based on aptt, pt, d-dimer and fibrinogen and had a very high diagnostic sensitivity and specificity. diagnostic accuracy of the model was sustained by prospective evaluation in group b. conclusion: based on generally available assays, it was possible to design an objective diagnostic model for canine dic, which has both a high sensitivity and specificity. such a model will provide basis for treatment optimization and make it possible to conduct multicentered therapy studies with a minimum risk of systematic misclassification of patients. in , a coagulation independent change in light transmittance (biphasic waveform [bpw] ) was reported in automated activated partial thromboplastin time assays (aptt) in patients with disseminated intravascular coagulation (dic). a calcium-dependant precipitate of creactive protein and very-low-density-lipoprotein was causing the bpw. our group recently identified this phenomenon in dogs also. initially, bpw was introduced as a complementary tool to assist diagnosing dic. however, recent studies reported that bpw may have a stronger potential as a prognostic marker for survival. the aims of the study were to prospectively investigate (a) the diagnostic significance of bpw regarding dic and (b) the significance of bpw to outcome, in dogs with diseases known to predispose for dic. the study was performed as a prospective, observational study including consecutive dogs with a final diagnosis known to predispose for dic ( % were finally diagnosed with dic). outcome was -day survival. bpw was assessed by means of a hirudin-modified aptt assay (kjelgaard-hansen et al., jvim : ; - ) . relative risk according to bpw (rr [ % confidence interval]) for (a) a dic diagnosis and (b) -day mortality, were assessed. -day mortality in the study population was %. % were bpw positive. bpw was not a significant diagnostic factor for dic (rr= . [ . ; . ] ), but strongly so for outcome (rr= . [ . ; . ] ) with a % ( / ) mortality amongst bpw positive dogs. in conclusion, bpw was observed in dogs predisposed to dic, with a strong potential as a risk factor for outcome, a finding in line with recent findings in humans. ( ), b hideo ( ) ( ) department of molecular pathology, kumamoto university, japan ( ) department of gastroenterological surgery, kumamoto university, japan aeromonas species are facultative anaerobic gramnegative rods that are ubiquitous, waterborne bacilli, most commonly implicated as causative agents of gastroenteritis. aeromonas infections often develop sepsis and disseminated intravascular coagulation syndrome (dic) is a life-threatening complication of sepsis patients, causing multiple organ failure.however, a mechanism leading to coagulation induction in the bacterial infection has not been known. to study the dic induction by aeromonas species infection, we investigated coagulation activity of a serine protease (asp) from aeromonas sobria, predominantly isolated in patients blood. proteolytically active asp shortened both activated partial thromboplastin time and prothrombin time of human plasma in a dose-dependent manner starting at an enzyme concentration of nm. asp activated human prothrombin, releasing hydrolytic activity for thrombinspecific substrate boc-val-pro-arg-mca, but no enzymatic activity was produced from coagulation factors ix and x. analysis by sds-page revealed that asp released a prothrombin fragment with a molecular weight identical with that of f¿-thrombin in an incubation timedependent manner. western blotting using biotinylated phe-pro-arg-chloromethylketone, a thrombin inhibitor, showed that asp produced an enzymatically active fragment whose molecular weight was same as that of f¿-thrombin. prothrombin incubated with asp but not the protease itself caused platelet aggregation. these results indicate that asp activates prothrombin, producing f¿-thrombin that converts fibrinogen to fibrin clot, and suggest that asp coagulation-inducing activity contributes to dic development in sepsis caused by aeromonas sobria infection. the present study shows a link between inflammation and coagulation mediated by a bacterial protease. hemolytic episodes are often associated to high amounts of free heme in circulation (up to um) and the development of an inflammatory response that may develop to a chronic inflammation. our group has shown that free heme is a prototypical proinflammatory molecule, able to induce neutrophil migration, actin cytoskeleton reorganization and nadph oxidasederived reactive oxygen species (ros) generation, as well as pkc activation and interlukin- expression (graÅa-souza et al., ) . moreover, free heme inhibits human neutrophil spontaneous apoptosis, a feature that is closely related to the impairment of resolution of inflammation and consequent promotion of chronic inflammatory status. heme protective effect requires nadph oxidase-derived ros and involves the activation of mapk, pi k and nf-kb signaling cascades as well as heme oxygenase (ho) activity (arruda et al., ) . more recently, we have shown that heme antiapoptotic effect is closely related to the maintainance of mitochondrial stability, inhibition of bax insertion into mitochondria and a dramatic increase on bcl-xl/bad protein ratio in a ros-dependent manner, requiring the same signaling pathways that regulate heme anti-apoptotic effect. these findings attest to a prominent role of free heme in the onset of inflammation associated to hemolytic episodes as well as the statement of chronic inflammation related to these disorders. the recent advance on the study of free heme as a proinflammatory molecule brings up hope for the development of new strategies to ameliorate acute and chronic inflammation found during hemolytic episodes.financial support: faperj, cnpq, capes. ( ) ( ) university of melbourne, victoria, australia ( ) monash university, victoria, australia we have previously demonstrated that mice lacking the anti-oxidative enzyme, glutathione peroxidase (gpx ), show significantly larger infarcts after stroke. recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. nevertheless, the involvement of oxidative stress in leukocyte recruitment and subsequent regulated cell injury is yet to be elucidated. to explore this, gpx -/-mice were subjected to transient mid-cerebral artery occlusion (mcao) followed by cerebral intravital microscopy, for assessment of leukocyte-endothelium interactions in intact cerebral microvasculature. after hr mcao, leukocyte-endothelium interaction was significantly reduced in gpx -/-mice compared to wt counterparts during the second hour of reperfusion. laser doppler and direct measurement of blood flow in pial postcapillary venules revealed a reduction of reperfusion in gpx -/-mice following transient mcao. this suggests that the reduction in nutritive blood flow following stroke in gpx -/-mice may explain the enhanced injury in these mice as well as the reduced leukocyte-endothelium interaction. furthermore, matrix metalloproteinase- (mmp ) which has previously been shown to be implicated in endothelial dysfunction and the pathogenesis of stroke was found to be up-regulated in gpx -/-mice to a greater extent than in wt mice after mcao, suggesting a role for oxidative stress in cerebral microvascular injury. the data present here suggests oxidative stress may be one of the factors that contribute to reduced post-ischemic perfusion, via the disruption of the endothelial function as indicated by the increased level of mmp . chris bolton( ), c paul( ), s barker ( ), r mongru ( ) ( ) william harvey research institute, london, uk ( ) university west of england, bristol ( ) queen mary university of london adrenomedullin (am) acts as a vasodilator in many vascular beds including the cerebral circulation where the peptide is produced in larger amounts than in the periphery.in vitro work has shown that am beneficially regulates blood-brain barrier (bbb) characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation.our preliminary studies in acute experimental autoimmune encephalomyelitis (eae), a model of the human disease multiple sclerosis (ms), have demonstrated significant elevations in am peptide levels corresponding with am mrna changes during late, neurological disease where am production may be linked to the restoration of bbb function. however, am is not exclusively produced as result of am gene upregulation. furthermore, am peptide levels do not always match am mrna changes during other disease phases of eae.the current study has investigated, more closely, the relationship between am gene expression and subsequent levels of associated peptides. am mrna levels were determined, by rt-pcr, in the cerebellum, medulla-pons and spinal cord of normal and eae-inoculated lewis rats at the height of disease. am and proadrenomedullin peptide (pamp) levels were measured in the tissues by radioimmunoassay.all tissues examined showed an increase in am gene mrna compared to control levels.am and pamp changes were observed in the samples and differences between the peptide profiles were recorded.an understanding of alterations in the generation of am and related peptides during neuroinflammation may provide insight into mechanisms affecting bbb permeability and be of relevance to the changes in neurovascular function seen during ms. platelet-activating factor (paf) contributes to the robust inflammatory responses in acute phase and spread of secondary injury. although, paf is believed to be a potent edematous but non-painful mediator in peripheral tissues, we recently demonstrated that paf may be a mediator of noxious signaling in spinal cord in case of neuronal injury. paf-induced tactile allodynia may be mediated by atp, glutamate and the generation of nitric oxide (no). the present study elucidated down-stream signaling pathway for paf-induced tactile allodynia. paf-and glutamate-induced tactile allodynia was blocked by the pretreatment with no scavengers and inhibitors of no synthase, soluble guanylate cyclase or cgmp-dependent protein kinase (pkg). recent evidence attributes the generation of pain to specific disfunctions of inhibitory glycinergic neurotransmission. to explore the target molecule for induction of tactile allodynia, the effect of knockdown of glycine receptors containing the a subunit (glyr a ) by sirna spinally transfected with hvj-e vector was examined. in mice spinally transferred with sirna for glyr a , the reduction of glyr a was demonstrated in superficial layer of dorsal horn by immunohistochemical analysis. pcpt-cgmp, paf, glutamate failed to induce tactile allodynia in mice spinally transferred with sirna of glyr a , while these compounds produced tactile allodynia in mice transferred with mutant sirna of glyr a as a control. glycine tranporter inhibitors ameriolated paf-and pcpt-cgmp-induced allodynia. these results suggest that glutamate-no-cgmp-pkg pathway plays a key role for paf-induced tactile allodynia in spinal cord and glyr a may be a target molecule for pkg to induce allodynia. ( ), r leite( ), ys bakhle ( ) ( ) federal university of minas gerais, belo horizonte, brazil ( ) medical college of georgia, augusta, usa ( ) imperial college, london, uk selective cyclooxygenase inhibitors (coxibs) induce a characteristic increase in mechanical nociceptive threshold, referred to as "hypoalgesia", in inflammatory pain induced by carrageenan in rat paws.we have here assessed the role of the cytoskeleton in this hypoalgesia induced by celecoxib (cx). male holtzman rats ( - g; - animals/group) were injected in the right hind paw (ipl) with a range of cytoskeletal inhibitors (selective inhibitors of microtubules (taxol, nocodazole, colchicine), of actin microfilaments (latrunculin b, cytochalasin b) or of intermediary filaments (acrylamide) (pico to nanomoles per paw) and min later given cx ( mg/kg, s.c.). after a further min, rats were injected (ipl) with the inflammatory stimulus, carrageenan ( mg/paw). mechanical pain threshold was hourly measured over the next h, using the randall-sellitto method. the cxinduced hypoalgesia was reversed by low doses of latrunculin b or cytochalasin (latrunculin % reversal = . nanomoles), higher doses of microtubule inhibitors (taxol % reversal = . nanomoles) with no effect of acrylamide ( up to nanomoles).we conclude that ) local changes in (paw) cytoskeleton occurred during cxinduced hypoalgesia and ) actin microfilaments were the cytoskeletal components most critically involved in this hypoalgesia.financial support: cnpq, fapemig and capes there are reports regarding the up-regulation of cyclooxygenase isoenzyme particularly inducible isoform i.e. cox- in brain during neurodegenerative or neuropsychiatric disorders.in the present study, we examined the effect of nimesulide (a preferential cox- inhibitor) in subchronic immobilization stress. mice were subjected to immobilization stress for hrs daily for a period of seven days. nimesulide ( . mg/kg, i.p.) was administered daily for days before challenging them to immobilization stress. behavioral analysis revealed the hyperlocomotor activity and increased anxious response. subchronic stress decreased % retention of memory and also caused hyperalgesic response in mice. biochemical analysis revealed that chronic immobilization stress significantly increased lipid peroxidation and nitrite levels and decreased the reduced glutathione and adrenal ascorbic acid levels. chronic treatment with nimesulide significantly attenuated the immobilization stress-induced behavioral and biochemical alterations. these results suggested that the use of nimesulide could be a useful neuroprotective strategy in the treatment of stress. there is accumulated evidence for ngf role as a peripheral pain mediator. ngf is upregulated in diverse inflammatory conditions and evokes hyperalgesia when injected in humans and rats. ngf increase was also observed in temporomandibular join (tmj) after cfa injection, indicating its possible involvement in local hyperalgesic states. therefore, the objective here was to evaluate if ngf participate in the tmj nociception. to test this hypothesis, the ngf was injected into the tmj alone or after carrageenan (cg) and the spontaneous nociceptive behavior of head flinches was counted for up min. further evidence for the ngf nociceptive activity was obtained quantifying the local production of ngf after cg injection, by elisa, and the fos-like immunolabeling in the trigeminal sensory nucleus (including the caudalis, interpolaris and oralis) after ngf injection. injections were performed in . ul. ngf ( . , and ug) injected in the tmj challenged h prior by cg ( ug) induces a dose-dependent increase in the number of head flinches. this increase was reduced by k a ( and ug), indicating a trka receptor-mediated effect. we detected a significant increase in the ngf production and h after the tmj cg ( ug) injection. the tmj injection of ngf ( ug) alone did not induce detectable spontaneous nociceptive behavior. however, the ngf ( ug) injection induces a significant increase in the fos like immunolabeling (fli) in the sensory trigeminal nucleus compared to the saline injection. these results indicate that the ngf participates in the nociceptive activity in the tmj, specially in inflammatory conditions. mif was reported as a key cytokine in the pathogenesis of rheumatoid arthritis (ra) several years ago, but it now clear that mif is also involved in the pathogenesis of systemic lupus erythematosus (sle) and atherosclerosis. mif-deficient lupus-prone mrl/lpr mice exhibit prolonged survival and reduced renal and skin disease compared to mif-expressing mice. similarly, mif-deficient atheroma-pone ldlr-deficient or apoe-deficient mice are significantly protected from disease and antimif mab therapy is beneficial. ra and sle are each characterised both by an increased prevalence of atherosclerotic vascular disease and by overexpression of mif. given the effects of mif on atherosclerosis it can be hypothesised that mif overexpression participates in the risk of atherosclerotic vascular disease in ra and sle. recent data have provided insights into mechanisms of action for mif relevant to all these concepts. firstly, the newly described role of mif in the selective recruitment of monocyte-macrophage lineage cells is of particular relevance to ra, sle, and atherosclerosis, with evidence that mif mediates macrophage recruitment in sle and atherosclerosis. secondly, glucocorticoid (gc) therapy is possible risk factor for atherosclerosis in patients with ra and sle, and it is now clear that gc increase the expression and release of mif, potentially implicating mif in gc-related increases in atherosclerosis in ra and sle. specific therapeutic targeting of mif in ra and sle may address not only primary disease pathways but also the increased risk of atherosclerosis in these diseases. to enter inflamed tissues, leukocytes must undergo adhesion molecule-mediated interactions with the endothelial surface of vessels at the site of inflammation.cytokines such as tumour necrosis factor (tnf) are established as important mediators capable of promoting leukocyte-endothelial cell interactions.however, in inflammatory diseases such as atherosclerosis and rheumatoid arthritis, elevated expression of another cytokine, macrophage migration inhibitory factor (mif) occurs, yet the role of this cytokine in leukocyte recruitment is unknown.therefore we explored the ability of mif to regulate leukocyte recruitment.this was achieved using intravital microscopy to examine the intact microvasculature in mice following local mif treatment. these experiments showed that mif induced leukocyte adhesion and transmigration in vivo, resulting in accumulation of predominantly cd +/f / -ve/cd c-ve monocyte/ macrophage lineage cells.mif did not induce upregulation of adhesion molecules p-selectin and vcam- , although their constitutive expression contributed to recruitment.in contrast, mif-induced recruitment was blocked by antibodies to the monocyte-specific chemokine, ccl /mcp- , and its receptor ccr , and in response to anti-cxcr .this was supported by in vitro experiments showing that mif induced ccl /mcp- release from cultured murine endothelial cells.finally, mice lacking cd , the putative mif binding molecule, did not respond to mif.these data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues, and indicate the involvement of a pathway involving a complicated chemokine/chemokine receptor pathway with contribution from cd .this function may be critical to the ability of mif to promote diseases in which macrophages are key participants. gm-csf and m-csf (csf- ) can enhance macrophage lineage numbers as well as modulate their differentiation and function. of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. what the critical actions are of these csfs on macrophages during inflammatory reactions are unknown. to address this issue, adherent macrophages (gm-bmm and bmm) were first derived from bone marrow precursors by gm-csf and m-csf, respectively, and stimulated in vitro with lps to measure secreted cytokine production, as well as nf-kb and ap- activities. gm-bmm preferentially produced tnfa, il- , il- and il- while, conversely, bmm generated more il- and ccl ; strikingly the latter population could not produce detectable il- and il- . following lps stimulation, gm-bmm displayed rapid ikba degradation, rela nuclear translocation and nf-kb dna binding relative to bmm, as well as a faster and enhanced ap- activation. each macrophage population was also pre-treated with the other csf prior to lps stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and nf-kb activity. thus gm-csf and m-csf demonstrate at the level of macrophage cytokine production different and even competing responses with implications for their respective roles in inflammation including a possible dampening role for m-csf. granulocyte macrophage-colony stimulating factor (gm-csf), initially discovered for its role in the differentiation of haematopoietic cells into granulocytes and macrophages, can also affect mature cell function and may be considered proinflammatory. gm-csf is able to prime macrophages for increased pro-inflammatory responses, including the increased release of tnfa and il- following stimulation with, for example, lps. in addition, gm-csf has been shown in vivo, using murine disease models, to play a key role in a number of inflammatory diseases. gm-csf-/-mice have been shown to be resistant to several diseases, including arthritis, and, most notably, blockade of gm-csf with a neutralizing monoclonal antibody was effective at ameliorating arthritis when given either prophylactically or therapeutically. t cells appear to be the major cell type responsible for gm-csf production required for arthritis, and gm-csf appears important in the effector phase of disease, subsequent to t cell activation. blockade of gm-csf results in fewer inflammatory cells, particularly macrophages, and cytokines such as tnfa, at the site of inflammation. these findings suggest that blockade of gm-csf may be an effective treatment in a range of inflammatory diseases. the autoimmune disease type diabetes mellitus (t dm) is thought to be mediated by autoreactive t cells recognizing islet autoantigens, including gad , ia- and proinsulin. this disease arises on a distinctive genetic background, mapping most notably to the mhc, and is also open to strong environmental influence. to investigate the pathogenesis of the disease, and in particular the prevailing paradigm that islet autoreactive t cells are important, we have developed an approach to epitope identification that is mhc allele and autoantigen specific, and operates for both cd and cd t cells. utilizing this, we have uncovered populations of islet antigen-specific t cells that have the immunological credentials to be both pathogenic (eg th , tc ) and protective (treg) in the disease. we have cloned some of these cell types, enabling us to analyse their function and provide an insight that will be important for an understanding of disease mechanisms, as well as guiding novel therapeutic interventions. tcr transgenic targeting b: - cause diabetes . knockouts of the insulin gene (expressed in thymus as well as islets) accelerates diabetes while knockout of insulin gene (islet expression) prevents % of diabetes . dual insulin knockout with transgenic insulin with altered peptide (b :a) prevents all diabetes . islets with native b: - sequence, but not altered sequence when transplanted into knockouts restore anti-insulin autoimmunity and diabetes transfer by t cells .anti-b - t cells have conserved valpha and jalpha chain usage but no conservation n region or beta chain . alpha chain as transgene sufficient to engender anti-insulin autoantibodies . kay and coworkers demonstrate insulin reactivity "upstream" of igrp and igrp reactivity nonessential.future studies in nod directed at deleting specific conserved alpha chains to test diabetes prevention and develop therapeutic.in man we can now identify at birth genetic risk as high as % of activating anti-islet autoimmunity with mhc analysis and restricted heterogeneity suggesting dominant target.insulin autoantibodies in prospective studies such as daisy usually appear initially and levels are related to progression to diabetes.analysis of cadaveric donors is underway to elucidate primary targets. (t d) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the islets of the pancreas. the holy grail of autoimmune disease prevention is negative vaccination against autoantigens to induce disease-specific immune tolerance. this has been achieved in rodents by administering autoantigen via a tolerogenic route (mucosal), cell type (stem cell or resting dendritic cell), mode (with blockade of t-cell co-stimulation molecules) or form (as an altered peptide ligand). compelling evidence demonstrates that proinsulin is the key autoantigen that drives beta-cell destruction in the non-obese diabetic (nod) mouse model of t d, and possibly in humans. proinsulin/ insulin dna, protein or t-cell epitope peptides administered in a tolerogenic manner to the nod mouse can delay or prevent the development of diabetes, via one or more mechanisms (deletion or anergy of effector t cells, induction of regulatory t cells). administration of autoantigen via the mucosal route, which induces anti-diabetic regulatory t cells in the rodent, is the most immediately translatable approach to humans. initial human trials of vaccination with oral autoantigens lacked evidence of bioeffect, probably due to inadequate dosage in end-stage disease. recently, however, the first evidence for a therapeutic effect of mucosal autoantigen has been seen in trials of oral and nasal insulin in islet autoantibodypositive individuals at risk for t d. combination autoantigen-specific vaccination also shows promise in combination with non-specific immunotherapy in established t d. leukocyte extravazation is an integral process both physiologically (immunosurveillance) and pathophysiologically (inflammation). the initial paradigm of a -step process comprising tethering/rolling, activation, firm adhesion, and diapedesis, each involving specific adhesion molecules, has repeatedly been modified in the light of more recent findings. additionally, organ-specific differences regarding the role of distinct molecules were established. finally, the skin became a good "model" to study due to its accessability and availability of powerful animal models. in-vitro adhesion assays, flow-chamber systems, intravital microscopy, animal models for delayed-type hypersensitivity, and transplantation approaches have successfully been employed to investigate leukocyte extravazation. numerous molecular interactions such as the cutaneous lymphocyte-associated antigen and sialyl-lewisx, or icam- and lfa- , have been proven sufficiently relevant to make them candidates for potential therapies. with the anti lfa- antibody efalizumab, approved for the treatment of psoriasis, the first therapeutic agent specifically targeting leukocyte extravazation is already on the market; other compounds are under development. moreover, novel data suggest that well-established anti-inflamamtory therapies such as fumarates also influence this process, thus contributing to their clinical efficacy. ongoing research aks for adopting a more "dynamic" view on leukocyte extravazation as several molecules obviously perform multiple tasks throughout this process rather than being limited to just one step of this multi-step cascade; this is particularly true for the so-called junctional adhesuíon molecules which obviously mediate more than just diapedesis. finally, similarities between leukocyte extravazation and hematogenic metastases are emerging. consequently, certain anti-inflammatory compounds may turn out to also exhibit striking anti-metastatic efficacy, and vice versa. department of dermatology, heinrich-heine-university, düsseldorf, germany atopic dermatitis, psoriasis vulagaris and cutaneous lupus erythematosus represent chronic inflammatory skin diseases showing distinct clinical phenotypes but sharing one aspect. the recruitment of pathogenic leukocyte subsets into the skin represents a prerequisite for their initiation and maintenance. during recent years, our knowledge of the immunopathogenesis of chronic inflammatory skin diseases increased significantly. with regard to the recruitment pathways of leukocytes, a superfamily of small cytokine-like proteins so called chemokines has attracted significant attention. here the complex interactions within the chemokine ligand-receptor network are introduced, the involvement of chemokines in memory t and dendritic cell trafficking is outlined and current concepts of their role in the immunopathogenesis of atopic dermatitis, psorasis vulgaris and cutaneous lupus erythematosus are summarized. the skin serves as a unique organ for studying general principles of inflammation because of its easy accessibility for clinical evaluation and tissue sampling. a network of pro-inflammatory cytokines including il- and tnf-a is known to play a key role in the pathogenesis of cutaneous inflammatory diseases through activation of specific signalling pathways. recently, progress in understanding the underlying mechanisms regulating inflammatory signalling pathways in the immunopathogenesis in skin carcinomas, psoriasis vulgaris and atopic dermatitis has been made. kinases have been identified to play a crucial role in regulating the expression and activation of inflammatory mediators in these inflammatory skin diseases. mitogen-activated protein kinases (mapks) are a family of serine/threonine protein kinases that mediate a wide variety of cellular behaviours in response to external stress signals. increased activity of mapks, in particular p mapk, and their involvement in the regulation of synthesis of inflammatory mediators at the transcriptional and translational level has recently been demonstrated. progress in our understanding of inflammatory signalling pathways has identified new targets for treating inflammatory diseases, but the challenge is to place a value on one target relative to another and to evolve strategies to target them. a careful examination of different signalling pathways in various inflammatory conditions is therefore needed. this presentation gathers recent advances in signal transduction in skin inflammation focusing interleukin- , tnf-µ, p mapk, msk / , mk , nf-kappab and ap- . histamine is an important inflammatory mediator in humans, and despite their relatively modest efficacy antihistamines are frequently used to treat allergic conditions, as well as other histamine-mediated reactions such as pruritus. in contrast, antihistamines are of very limited use for controlling other conditions where histamine production is abundant, including asthma. the discovery of the histamine h receptor (h r) prompted us to reinvestigate the role of histamine in pulmonary allergic responses, as well as in pruritus. h r deficient mice and mice treated with h r antagonists exhibited decreased allergic lung inflammation in several models, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in th responses. ex vivo restimulation of primed t cells showed decreases in th cytokine production, and in vitro experiments suggest that decreased cytokine and chemokine production by dendritic cells after blockade of the h r was responsible for the the t cell effects. the influence of h r on allergic or histamine-induced pruritus was explored in mice using selective histamine receptor antagonists and h r deficient mice. the h r was found to mediate the majority of histamine-mediated and allergic itching, while the contibution by the h r was minor. surprisingly the h r effect was independent of mast cells or other hematopoetic cells. this work suggests that the h r can modulate both allergic responses via its influence on t cell activation, and pruritus through mechanisms that are independent of hematopoetic cells. the studies show that the h r mediates previously uncharacterized effects of histamine and highlight the therapeutic potential of h r antagonists. ( ), bsp reddy ( ) ( ) nizams institute of medical sciences, hyderabad; india ( ) genix pharma, india rupatidine, carries the majority of the histamine h receptor -blocking activity and has been introducedfor the treatment of allergic rhinitis and urticaria. objectives: the aim of this study was to compare the effect of two by measure of inhibition of histamine induced wheal and flare response. methodology: male volunteers were enrolled after written informed consent before to ethic committee approved protocol. in this randomised, double-blind, single oral dose, cross overstudy, they were randomized to receive either mg rupatidineformulation after overnight fast. washout was days. wheal and flare were induced on the forearm of the trial subjects, by histamine intradermally injection while the subject was lying comfortably with arm resting on the bed. ten minutes later, wheal and flare were visualized under a bright lamp. histamine induced wheal and flare skin test was performed before and regularly to hours after drug administration. results: administration of reference and test formulations of rupatidine, significantly inhibited the histamine induced cutaneous response in all the subjects. the least square mean ratio (%) t vs r for peak activity imax-% (maximum inhibition of histamine induced wheal and flare response); area under the activity time curve (auc - mmsq/hr and auc - %/hr) both for untransformed and log transformed data were found to be within - % of % ci limits both formulations well tolerated. conclusions: it can thus be concluded that be concluded that test formulation of rupatidine tablet is bioequivalent to reference rupatidine tablet ( ), h yoshimura( ), k ohara ( ), y mastui ( ), h hara ( ), h inoue ( ), h kitasato ( ), c yokoyama( ), s narumiya ( ), m majima ( ) ( ) kitasato university school of medicine, kanagawa, japan ( ) tokyo dental medical colledge, tokyo, japan ( ) kyoto university school of medicine, kyoto, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported the magnitude of cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitorsconstitute revascularization. we hypothesized that txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggrega- inflamm. res., supplement ( ) tion.to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb model. recovery from acute hind limb ischemia, as assessed by the ratio between the treated ischemic limb and the untreated control right limb was assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out(ipko) and thromboxane (tx) a receptor (tp) knock out(tpko). blood recovery in tp-/-significantly delayed compared to wt and ipko. immunohistochemical studiesrevealed that tp-/-mice were less stained against pecam positive cells compared to wt and ipkoplasma sdf- and vegf concentration were significantly reduced in tp-/-mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. purpose: the s calcium-binding proteins, a , a and a are constitutively expressed in neutrophils and induced in activated macrophages. high levels are found in sera from patients with infection and several chronic inflammatory diseases. the calgranulin complex, a /a is anti-microbial; a has oxidant-scavenging functions. a is chemotactic for monocytes, and recruits leukocytes in vivo by activating mast cells (mc). effects of these mediators on mc and monocyte function were compared. methods: human pbmc or murine mc were activated in vitro with s and mediator release and cytokine induction (assessed by quantitative rt-pcr/elisa), determined. a cys to ala a mutant was used to determine whether effects on mc are mediated by redox. immunohistochemistry was used to demonstrate s s in asthmatic lung. the s s were expressed in asthmatic lung, particularly in eosinophils and alveolar macrophages. strong reactivity occurred with an antibody recognising predominantly the hypochlorite-oxidised from of a . a , a or the a /a complex had relatively low ability to induce il , tnf, il , and chemokines mrna from pbmc compared to a . only a induced significant levels of il ; none induced il or gm-csf mrna compared to lps. in contrast to a which is activating, a significantly inhibited mc degranulation provoked by ige cross-linking; suppression was dependent on cys . conclusions: the cytokine profile generated by a in mc and monocytes strongly supports a role the pathogenesis of asthma. in contrast, results strongly support a role from a in oxidant defence, particularly to hypobromite generated by activated eosinophils. ( ), d mankuta( ), g gleich ( ), f levi-schaffer ( ) ( ) hebrew university of jerusalem, israel ( ) hadassah university hospital, israel ( ) university of utah, usa the onset, amplitude and termination of allergic responses is regulated at the mast cell/eosinophil interface. eosinophil major basic protein (mbp), which activates mast cells in the late-chronic phase of allergic inflammation, is a central determinant in this interface. characterized more than two decades ago, the exact nature of this activation has not been clarified as yet. here we demonstrate that mbp exerts its activating effect on human mast cells and basophils through cd and hematopoietic cell kinase (hck). a genome-wide analysis showed that hck displays shifts in mrna levels specifically upon mbp-induced mast cell activation. hck also shows a unique priming pattern prior to this activation. cd is phosphorylated specifically upon activation with mbp and deploys a signaling complex that critically depends on hck. extracellular neutralization of cd interferes with mbp entry into the cell, and this as well as rna silencing of hck results in defective mbp-induced activation. finally, cd neutralization abrogates mbp-induced anaphylaxis in-vivo. these findings picture for the first time a chronic-phase specific pathway mediating eosinophil-induced mast cell activation with critical consequences for the therapy of chronic allergic inflammation. alexander robinson ( ), d kashanin ( ), f odowd( ), v williams( ), g walsh ( ) ( ) cellix ltd, institute of molecular medicine, dublin, ireland ( ) university of aberdeen, scotland, uk leukocyte adhesion to endothelial cell bound proteins, such as icam- and vcam- , is an initial step of the inflammatory response. we have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. - dynes/cm ). the adhesion profiles of resting and pmastimulated peripheral blood lymphocytes (pbls) were recorded, with respect to vcam- , icam- , and bsa. images at each shear stress level were captured using a digital camera, and analysed using our in-house ducocell software package. distinct morphological changes in pma-stimulated pbls, compared to non-stimulated cells, can be observed. these include a less rounded appearance of the pma-stimulated pbls, and evidence of "uropod" formation, which anchor the t cell to the endothelium as part of the migration process. levels of adhesion to vcam- are high ( - %, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and pma-stimulated pbls.however, there is a distinct difference between the adhesion levels of non-stimulated and pma-stimulated pbls to icam- , with pma-stimulated cells showing a higher affinity for icam- than nonstimulated cells (approx. % and %, respectively).pbl adhesion to bsa is negligible. we present a novel in vitro microfluidic pump system that can simulate leukocyte adhesion to the endothelium under flow conditions. this platform is a more efficient and economical system compared to those currently available, due to reduced material costs and style of construction. introduction: pulmonary aspiration of gastric contents is a common complication observed in icu patients and a potential trigger of ards. in this study we evaluated the course of lung inflammation induced by intranasal instillation of gastric juice (gj). methods: gj was obtained from donor rats (ph . ). male c bl/ were instilled with ml/kg of gj. after or h, the animals were sacrificed and lung and balf were collected. control group consisted of non-manipulated mice. . ae . , sg h: . ae . ; pg/ml). discussion: gj aspiration induced an initial adherence of pmn to lung tissue that is correlated with increased tnf-a/il- ratio in balf at the nd h. the reduction of mpo activity is correlated with the decrease in tnf-a/il- ratio. the late increase of pmn in balf might be a consequence of the early production of tnf-a. the results are suggestive that the treatment of patients exposed to acid aspiration should be focused in the initial period of the insult and in the blockage of tnf-a. objectives: intestinal i/r is implicated as a prime initiating event in the development of acute respiratory distress syndrome (ards) after trauma and hemorrhagic shock. we investigated the effects of lps challenge to mice previously submitted to i-i/r, a two-hit model of acute lung injury. methods: male c bl/ mice were subjected to min of intestinal ischemia and challenged with . mg/kg of intranasal lps at the th hour of reperfusion (two-hit). balf and culture of lung explants were performed h after lps challenge. mice subjected to i-i/r or lps alone were used as controls. results: two-hit mice showed marked increase in lung evans blue dye leakage compared to i-i/r ( . ae . vs . ae . , mg/mg). lung mpo was increased ( . ae . vs . ae . ; od nm) whereas the neutrophil recruitment to balf was inhibited in the two-hit group compared to lps group ( . ae . vs . ae . ; x e cells/mouse). the levels of nox-in the two-hit group were significantly increased when compared to i-i/ r controls in balf ( . ae . vs . ae . ; mm) and in lung explants ( . ae . vs . ae . ; mm/mg of tissue). conclusions: intestinal i/r predisposes the animal to an exacerbated response to a low dose lps insult. the exacerbated production of nitric oxide observed in the two-hit group may cause endothelial damage, thereby explaining the major increase in vascular permeability in the two-hit group. the results are suggestive that patients exposed to systemic inflammatory response might develop ards when in contact with secondary inflammatory stimuli. nitric oxide may play an important role in this process. ( ) ( ) novartis institutes for biomedical research, horsham, uk ( ) university of michigan, usa obligatory for using oxygen in energy transfer pathways was the simultaneous co-evolution of enzymes that detoxify the reactive species formed as by-products. thus, we hypothesized that individuals with low aerobic function will have reduced anti-oxidant capacity and, therefore, be more susceptible to smoking-related lung diseases like copd. to test this hypothesis, we exposed high capacity runner (hcr) and low capacity runner (lcr) rats to months of whole-body smoke exposures.the animals, bred over successive generations on the same background strain for high or low running capacity, differ by over % (p< x - ) for exercise capacity, measured by running on a treadmill.after months of exposures, inflammatory cells in bronchoalveolar lavage fluid were increased in both the hcr-and lcr-smokeexposed(se) animals compared to air-exposed controls (p< . ); however there was a - -fold increase in the number of neutrophils and lymphocytes in the lcr-over the hcr-se group (p< . ).histopathology revealed there was greater inflammation and lung damage present in the lcr-versus hcr-se group (p< . ). metabonomic (metabolite profiling) analysis revealed that while peroxidation of lung lipids occurred for both se groups, oxidative damage to the lung surfactant layer was significantly more extensive for the lcr-se. systemic oxidative damage was also more apparent in the lcr-se group, with metabolic profiling suggesting a reduced capacity to regenerate muscle glutathione. the metabolic data suggest that repair processes maybe more effective in the hcrs. in summary, these data support the concept that aerobic capacity may be central to ones susceptibility to developing smoking-related lung disease. ( ), ap ligeiro-oliveira( ), jm ferreira-jr( ), sr almeida( ), w tavares de lima( ), shp farsky ( ) ( ) university of s¼o paulo, brazil ( ) regional integrated university of alto uruguai and missðes, brazil methods: male wistar rats were exposed to vehicle or hq ( mg/kg; ip.;daily, days, two-day interval every five days). on day , animals were ip sensitized with ovalbumin (oa). assays were performed on day . results: hq-exposed rats presented reduced number of leukocytes in the bronchoalveolar fluid and by impaired in vitro oa-induced tracheal contraction. the latter effect suggests reduction on mast cell degranulation, and it was corroborated by in vivo decreased mesenteric mast cell degranulation after topical application of oa. the oa-specificity response was confirmed by normal ability of mast cells to degranulate in both groups of animals after topical application of compound / . in fact, lower levels of circulating oa-anaphylactic ige antibodies were found in hq-exposed rats. this latter effect was not dependent on number or proliferation of lymphocytes, nevertheless reduced expressions of costimulatory molecules cd and cd on oa-activated lymphocytes indicated the interference of hq exposure on signaling of the humoral response during an allergic inflammation. contact information: ms sandra manoela dias macedo, regional integrated university of alto uruguai and missðes / university of s¼o p, department of clinical and toxicological analyses, s¼o paulo, brazil e-mail: smdmacedo@yahoo.com.br ( ) ( ) radboud university nijmegen, medical centre, nijmegen, the netherlands ( ) university hospital, zürich, switzerland toll-like receptors (tlr) are essential in the recognition of invading microorganisms. however, increasing evidence shows involvement of tlr in autoimmunity, such as rheumatoid arthritis (ra), as well. here we investigated whether synovial expression of tlr and tlr was associated with the expression of ifna, tnfa, il- b, il- , il- , and il- and studied in what way these receptors and cytokines were associated in vitro. using immunohistochemistry, we found that tlr / tlr expression in synovial tissue was associated with the presence of ifna, il- b and il- , but not tnfa, il- and il- . to investigate whether ifna, il- b and il- could induce tlr / tlr upregulation in vitro, we incubated separate lymphocyte populations with these cytokines and subsequently determined tlr / mrna expression. ifna incubation resulted in significant tlr /tlr upregulation, whereas il- b and il- did not. pre-incubation with ifna and subsequent stimulation of tlr /tlr significantly enhanced il- , tnfa and ifna/b production, indicating that the ifn-induced tlr upregulation was functional. low amounts of biologically active il- b were produced upon stimulation with atp, but not upon tlr / tlr stimulation, although mrna levels were high. interestingly, ifna-priming significantly increased the atp-induced il- b production. here, we demonstrated a dual role for ifna in vitro, which could explain the association between tlr and il- b / il- in synovial tissue. first, involvement in tlr /tlr regulation and second, involvement in atp-induced production of biologically active il- b. these results suggest involvement of anti-viral immune responses in ra and ifna as a key player in chronic inflammation. the pathogenesis of chronic joint inflammation remains unclear although the involvement of pathogen recognition receptors (ppr) has been suggested recently. here, we described the role of two members of the nacht-lrr (nlr) family, nod (nucleotide/ binding oligomerization domain) and nod in model of acute joint inflammation induced by intraarticular injection of tlr (toll-like receptor) agonist streptococcus pyogenes cell wall fragments. we found that nod deficiency resulted in reduced joint inflammation and protection against early cartilage damage. in contrast, nod gene deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage. to explore whether the different function of nod and nod occurs also in humans, we exposed pbmcs carrying either nod frameshift or nod frameshift mutation with scw fragments in vitro. both tnfa and il- b production was clearly impaired in pbmcs carrying the nod fs compared to pbmcs isolated from healthy controls. in line with the nod gene deficient mice, human pbmcs bearing the nod mutation produced enhanced levels of proinflammatory cytokines after h stimulation with scw fragments. these data indicated that the nlr family members nod and nod have a different function in controlling tlr -mediated pathways. we hypothesize that intracellular nod -nod interactions determine the cellular response to tlr triggers. whether lack of controlling tlr -driven pathways by nod signalling is involved in the pathogenesis of autoinflammatory or autoimmune disease, such as rheumatoid arthritis (ra), remains to be elucidated. leukocyte immunoglobulin-like receptors (lilrs) are a family of receptors with potential immune-regulatory function. activating and inhibitory receptors play a role in maintaining immunological equilibrium and an imbalance may lead to the onset of autoimmune diseases such as rheumatoid arthritis (ra). ra is a chronic inflammatory disease of joints caused by mediators (i.e. tnf-a) produced by activated leukocytes. we recently demonstrated expression of activating lilra in synovial tissue macrophages from ra patients. the aim of this study was to determine expression and function of lilra in monocytes and macrophages. peripheral blood mononuclear cells (pbmc) were prepared by standard density gradient separation and in vitro-derived macrophages were generated by differentiating thp- cells with vitamin-d . lilra expression was measured by flow cytometry before and after modulation with cytokines. differentiation to macrophages significantly up-regulated lilra expression (p= . ). treatment of macrophages with lps, tnf-a, il- b and ifn-g but not il- caused significant down-regulation of lilra (p< . ). function of lilra was assessed by cross-linking with plate-immobilised lilra -specific mab. soluble tnf-a was measured by elisa. activation of cells elicited tnf-a production in a dose-dependent manner while time-course analysis shows maximal production at h. correlation between lilra expression and response to cross-linking indicates that level of expression may relate directly to the degree of activation. decrease expression in response to acute-phase cytokines suggests controlled regulation during inflammation. in ra, abnormal regulation of lilra could potentially exacerbate inflammation by inducing uncontrolled production of proinflammatory cytokines. pharmacological blocking of lilra could potentially provide therapeutic benefit. ( ) ( ) university of valencia, spain ( ) northwick park institute for medical research, uk co-releasing molecules (co-rms) mimic the biological actions of co derived from heme oxygenase activity. in the present work we studied the effects of a water-soluble co-releasing molecule (corm- ) on an animal model of human rheumatoid arthritis. dba- /j mice were treated with corm- ( , or mg/kg/day, i.p.) from day to after collagen-induced arthritis (cia) and sacrificed on day . administration of corm- resulted in a significant improvement of the clinical profile of this disease since it markedly reduced joint swelling and redness. histological analysis of the joints in control arthritic mice indicated the presence of granulocytes and mononuclear cells, cartilage erosion, chondrocyte death and proteoglycan depletion. all these parameters were significantly reduced by corm- treatment with the most pronounced protective effect observed at mg/kg. the levels of pro-inflammatory mediators (pge , il- beta, tnfalpha, il- and il- ) in the hind paw homogenates were significantly inhibited by corm- . in addition, comp levels in serum, a marker of cartilage degradation, was reduced by the co-releasing agent. our studies show that therapeutic administration of corm- alleviates the clinical features of murine cia at the late phase of this response. the beneficial action of co liberated from corm- appears to be associated with a decrease in inflammatory cytokines and reduction of cell infiltration into the synovial tissues ultimately leading to a protective effect on the cartilage. aim: to setup a bovine model for cytokine-induced articular cartilage collagen degradation, and characterize the model using a variety of compounds targeting different disease mechanisms relevant to arthritis. methods: full thickness bovine articular cartilage punches were cultured with or without ng/ml il- a, tnf-a and oncostatin m. after three weeks the cartilage and culture medium were analyzed for weight changes, water content, dna content, glycosaminoglycans (gag), hydroxyproline (hyp), damaged collagen molecules, mmp activity, ctx-ii and comp. diclofenac, dexamethasone, pioglitazone, remicade, risedronate, galardin and a - were tested for their effect on cartilage degradation. results: exposure of articular cartilage to cytokines resulted in a decreased cartilage weight, increased proteoglycans degradation, increased collagen degradation, increased percentage of denatured collagen, increased water content and increased levels of active mmps (all p < . ). comp release during the first week of culture showed a trend towards up regulation during the first week of culture for all three donors, this was however not significant due to the small number of donors. most of the described processes were modulated by one or more of the drugs tested, indicating that this model for articular cartilage destruction is sensitive to treatment. discussion: stimulation of bovine articular cartilage explants with a cocktail of il- a, tnf-a and osm results in clear and consistent changes in the cartilage, highly reminiscent of cartilage destruction during arthritis. further research needs to establish whether the model is also sensitive to anabolic factors that potentially could repair the damage. toll-like receptors (tlrs) may contribute to the progression of rheumatoid arthritis through recognition of hostderived damage-associated ligands that have repeatedly been found in arthritic joints. involvement of tlr and tlr activation in the expression of arthritis was studied using interleukin- receptor antagonist deficient (il- ra-/-) mice, which spontaneously develop an autoimmune t-cell mediated arthritis. spontaneous onset of arthritis was dependent on tlr activation by microbial flora, as germ-free mice did not develop arthritis. after crossing with tlr knockouts, il- ra-/-tlr -/-mice developed more severe arthritis compared to il-ra-/-tlr +/+ littermates; whereas, tlr -/-il- ra-/-mice were protected against arthritis. to clarify the mechanism by which tlr and tlr differentially regulated the disease expression, we studied the role of these tlrs in il- production and th development, both important in il- ra-/-arthritis. wild type bone-marrow-derived dendritic cells (bmdcs) produced similar levels of il- upon stimulation with tlr and tlr ligands; however, il- ra-/-bmdcs produced less il- than wild type dcs upon tlr stimulation and more il- than wild type dcs upon tlr stimulation. furthermore, il- ra-/-t cells produced lower amounts of il- when cultured with tlr -activated apcs and higher amounts of il- when cultured with tlr -stimulated apcs, both in combination with cd stimulation. facs analysis of th (cd +/il- +) cells from both spleen and draining lymph nodes revealed % reduction in il- ra-/-tlr -/-mice compared to il- ra-/-tlr +/+ littermates. specific cd /cd stimulation of non-adherent splenocytes confirmed lower il- production in il- ra-/-tlr -/-. these findings suggest important roles for tlr and tlr in regulation of th development and expression of arthritis. prostaglandine (pge ) stimulates the transactivational activity of p through p map kinase-dependent ser phosphorylation (jbc ) .p controls cell-cycle progression, in part, by differential regulation of ap- proto-oncogenes (jun/fos).currently we studied pge control of cyclin d promoter activity with particular attention to the role of ap- oncogenes.pge induced a . fold increase in junb mrna expression (northern blot), a . -fold increase in junb promoter activity (luciferase assay), and increased ser junb phosphorylation in human synovial fibroblasts (hsf) (western blot).c-jun was strongly inhibited while jund, c-fos, fra / , and fosb expression were upregulated by pge .in cell-cycle experiments, transformation with a constitutively active ha-ras construct (ras g v) resulted in a . fold increase in cyclin-d promoter activity, cyclin-d synthesis, thr /tyr phosphorylation of erk / ( . fold) and ap- (c-jun)-dependent transactivity ( . fold); cyclin d /cdk - inhibitor p ink a synthesis was suppressed. addition of excess rass n dominant negative mutant construct to the plasmid mix abrogated the aforementioned processes.ectopic expression of c-jun, c-fos and especially jund expression constructs stimulated cyclin d promoter activity/protein synthesis, blocked p ink a synthesis; the latter effects were reversed by the addition of excess junb.pge exerted temporal and bi-phasic dose-dependent control of the cyclin d promoter activity, largely through differential ap- activation and promoted cell cycle arrest and apoptosis in hsf at high physiological concentrations.the results provide further insight into the biology of the cpla / cox/pges biosynthetic axis and highlight the complexity of pge action in terms of cell-cycle progression. di-glucopyranosylamine (diga) is an antikeratitic (roberts et al., , acvo conference, scottsdale) immunomodulatory pyranosyl disaccharide with parenteral anti-rheumatic activity (bolton et al., , inflammation res. (s ) s ) and unknown mechanism of action.interestingly, anti-tnf therapy is anti-keratitic.-diga hydrolyses to monoglucosylamine (mga) and glucose, which is prevented by n-acetylation (nacdi-ga).lider ( , pnas. : - ) showed that sulphated disaccharides are orally active, inhibit tnf synthesis and the dth reaction.we have investigated the anti-tnf and anti-rheumatic activity of the sulphated and free digas.human whole blood (hwb) was stimulated with pha ( mg/ml) to synthesise tnf.antigen induced arthritis (aia) was induced in methylated bovine serum albumin (mbsa) sensitised c bl/ mice challenged i.a. into the stifle joint.collagen arthritis (cia) induced in dba mice by sensitisation to bovine collagen, were boosted i.p. with collagen at day . hwb tnf synthesis was inhibited by diga, mga and nacdiga(ic < . mm). diga ( ml, mm) i.a. prevented hour aia (- . +/- . mm).diga at mg/kg reduced aia when administered i.v. (- . +/- . mm, p< . ) and i.p. (- . +/- . mm, p< . ), but is hydrolysed p.o. ( . +/- . mm ns). polysulphated diga (diga s) was unstable, but stabilised by n-acetylation (nacdi-ga s).tnf synthesis was potently inhibited by both nacdiga and nacdiga s (ic < . mm).nacdiga s ( mg/kg p.o.) inhibited aia (- . +/- . mm), and nacdiga s with lower degrees of sulphation (mw and kda) inhibited the development of mouse collagen induced arthritis as assessed by clinical score. sulphated diglucosylamines represent a new class of heparinoid which are potent inhibitors of tnf synthesis and possess oral anti-rheumatic activity. excessive no appears to play a key role in the pathogenesis of chronic inflammatory diseases. in this study we aimed to evaluate no synthesis in rheumatoid arthritis (ra) before and after therapy. it was performed on persons, divided into groups: a negative control group of healthy volunteers, a positive control group with ra, a group with ra and physiotherapy (phys), a group with ra and low doses of cimetidine (cim) + doxycycline (dox), a group with ra and combined treatment phys + cim + dox, and a group treated with usual doses of ibuprofen (ibu). serum nitrite/nitrate (griess) was measured in order to evaluate no synthesis. results: compared to the positive control group, in all the treated groups no synthesis decreased significantly. there was no significant difference between phys and cim+dox effect alone. the combined treatment, phys + cim + dox had a much better inhibitory effect on no synthesis. between the phys, cim + dox and phys + cim + dox groups and that treated with ibuprofen, there was no significant difference in reducing no synthesis. conclusions: ) in ra phys + cim + dox treatment was as efficient as ibuprofen in reducing no synthesis. ) the low doses of cim and dox may allow a longer treatment due to the lower side effects risk enhanced socs expression following exposure of murine macrophages to lps implicated socs in the control of lps-mediated signaling. socs regulates nfkb signaling in murine macrophages, blocking at the level of mal or ikba phosphorylation. we investigated the role of socs in regulating the production tnf by lps and pam csk -activated primary human monocytes. blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing socs (adv-socs ), control vector (adv-gfp) or left untreated. adv-socs monocytes were exposed to tlr and tlr ligands, lps ( ng/ml) or pam csk ( ng/ml). facs analysis demonstrated infection efficiencies of ae % and ae % (n= , mean ae sem) of monocytes expressing adv-gfp or adv-socs at moi . adv-socs blocked lps and pam csk induced tnf mrna and protein production in a dose-dependent manner. in contrast, il- and il- production by adv-socs -infected monocytes was not blocked. adv-socs also blocked lps and pam csk induced tnf production by macrophages isolated from synovial fluid. infection efficiencies of ae % or ae % were obtained. quantitative western blot analysis revealed that the classically defined nfkb pathway was not altered at the level of ikba or p activation. furthermore, the kinetics of lps and pam csk induced ikba phosphorylation and degradation in adv-socs monocytes remained unaffected (n= and donors, respectively). further, analysis of parallel mapk pathways demonstrated no block in p or erk mapk pathways. these data suggest that socs regulation of lps and pam csk -induced tnf production by human monocytes occurs downstream of tlrs, possibly at the level of transcription. recently, beta-nad+ has emerged as a novel extracellular player in the human urinary bladder. beta-nad+ is the natural substrate of cd which catalyzes the conversion of beta-nad+ to cadpr. under normal conditions in vivo, there is no or only very small quantities (submicromolar range) of extracellular beta-nad+ compared to intracellular levels ( - mm). during inflammation cell lysis may cause bursts of high local beta-nad+ levels. however, the effect of beta-nad+ on the human detrusor smooth muscle cells (hdsmc) was unknown. the effect of beta-nad+ on cultured (explant technique) hdsmc was determined by: ) measuring cytosolic free calcium ([ca +] i) in fura- loaded hdsmc using spectrofluorometry and ) force measurements in - mg detrusor strips. hdsmc responded to beta-nad+ ( - mm) with an immediate and transient increase in [ca +] i. the ca + transient was followed by one or two much slower and transient increases in [ca +] i, indicative of beta-nad+ enzymatic conversion into cadpr. the ca + responses persisted in the absence of extracellular calcium. the ca + responses to beta-nad+ were not affected by exposure of hdsmc to atp supporting the notion that the effects of beta-nad+ were not mediated via p x purinoceptors. furthermore, beta-nad+ caused a concentration-dependent detrusor muscle relaxation. this is the first study to report that extracellular beta-nad+ affect intracellular calcium homeostasis and force in hdsmc. these powerful actions of beta-nad+ suggest a role for beta-nad+/cadpr system as a novel extracellular player in the human detrusor during inflammation. aids remains a worldwide threat more than two decades after identification of hiv as the etiological agent. its wide dissemination can be partly attributed to its successful suppression of immunity resulting in disease progression and concomitant opportunistic infections including mycobacterial and cytomegalovirus infections. hiv trans-activator (tat) is one of the regulatory proteins that mediates hiv replication and dysregulates cellular functions such as apoptosis and cytokine expression. for example, tat induces tumor necrosis factor (tnf) and enhances gp -induced neurotoxicity. we recently showed that tat induces the overexpression of il- via cellular kinase pkr and activation of transcription factor ets- . in this study, we examined whether tat plays a role in perturbing interferon-& (ifn&) signal transduction. we showed that tat impaired ifn&-induced stat tyrosine phosphorylation, but had no effects on the serine residue of stat and jak kinases in primary human blood monocytes. furthermore, we found that the nuclear translocation of phospho-stat was abrogated by tat. the inhibition of phospho-stat led to the deformation of stat homodimers and subsequent stat-dna complex. to investigate the cellular consequences, we measured the expression of ifn&-stimulated genes including human leukocyte antigen (hla) and , oligoadenylate synthetase ( , oas), a key enzyme in the activation of latent ribonuclease l. the results showed that tat inhibited transcriptional activation of , oas and hla. taken together, we identified a new role for tat in which it impairs ifn& signal transduction and suppresses inflammation, thus crippling the immune system and contributing to hiv persistence, opportunistic infections and disease progression. caspase- belongs to the group of inflammatory caspases and is the activating enzyme for the pro-inflammatory cytokine interleukin- (il- ), a cytokine known to play an important role in the pathogenesis of psoriasis. the purpose of this study was to determine the expression of caspase- in psoriatic skin and the signaling mechanisms involved in stress induced activation of caspase- and il- . interestingly, increased caspase- activity in lesional compared with nonlesional psoriatic skin was seen as determined by western blotting. in vitro experiments in cultured human keratinocytes demonstrated anisomycin induced, p mapk dependent increased secretion of procaspase- and active caspase- . furthermore, anisomycin increased the mrna expression of il- through a p mapk dependent but caspase- independent mechanism, reaching a maximum level after hours of stimulation. finally, anisomycin caused a rapid ( hours) increase in the secretion of proil- and active il- . secretion of active il- was mediated through a p mapk/caspase- dependent mechanism, whereas secretion of proil- was mediated by a p mapk dependent but capsase- independent mechanism. these data demonstrate that the activity of caspase- is increased in psoriatic skin and that il- secretion is regulated by a p mapk/caspase- dependent mechanism, making caspase- a potential target in the treatment of psoriasis. prostaglandin e (pge ) regulates the stability of cyclooxygenase- (cox- ) mrna through adenylate/uridylate-rich elements (ares) in the untranslated region ( utr) by a positive autocrine/paracrine feed-forward loop. the principal objective of this study was to elucidate the molecular mechanisms involved in the pge dependent stabilization of cox- in human synovial fibroblasts (hsfs). transfection of well-known are binding proteins (aubps) demonstrated that tristetraprolin (ttp) potently destabilized a [luciferase-cox- utr] reporter fusion mrna ( ae . % decrease in luciferase activity vs. control). ttp protein levels in hsfs remained constant despite il- b-induced changes in ttp mrna levels, thus suggesting translational regulation of its expression. pge did not affect the transcription or translation of this gene in hsfs. western blot analysis of hsf ttp demonstrated the existence of a specific, covalent~ kda heterocomplex containing ttp (ttphcx). although ttphcxs exact composition and stoichiometry is yet to be defined, pge selectively regulated the amount of this heterocomplex in a time-dependent manner. furthermore, protein shuttling studies performed using real-time confocal microscopy revealed that pge can induce export of a small nuclear pool of ttp-gfp. finally, transfection of ttp into hsfs also influenced cox- gene transcription, thus enabling ttp to regulate cox- gene expression at both the transcriptional and post-transcriptional level. in conclusion, we have demonstrated that ttp is an rna binding protein capable of influencing cox- mrna stability and transcription and whose localization and interaction with other factors is regulated by pge . these data can provide important insight into deciphering the role of pge in fine-tuning physiological and pathophysiological gene regulation. ( ) ( ) chinese academy of sciences, shanghai, pr china ( ) ohio state university, usa mitogen-activated protein (map) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. map phosphatases (mkp)- is an archetypical member of the dual-specificity phosphatase family that deactivates map kinases. induction of mkp- has been implicated in attenuating the lipopolysaccharide (lps) and peptidoglycan (pgn) responses, but how the expression of the mkp- is regulated is still not fully understood. here, we show that inhibition of p map kinase by specific inhibitor sb or rna interference (rnai) markedly reduced the expression of mkp- in lps or pgn-treated macrophages, which is correlated with prolonged activation of p and jnk. depletion of mapkap kinase (mk ), a downstream substrate of p , by rnai also inhibited the expression of mkp- . the mrna level of mkp- is not affected by inhibition of p , but the expression of mkp- is inhibited by treatment of cycloheximide. thus, p mapk plays a critical role in mediating expression of mkp- at a posttranscriptional level. furthermore, inhibition of p by sb prevented the expression of mkp- in lpstolerized macrophages, restored the activation of map kinases after lps restimulation. these results indicate a critical role of p -mk -dependent induction of mkp- in innate immune responses. the i-kb kinase (ikk) complex regulates the activation of nf-kb a key transcription factor in inflammation and immunity. whilst ikka activity is necessary for proinflammatory and anti-apoptotic gene expression, ikka has distinct roles in lymphorganogenesis and b cell maturation. here we describe a role for ikka in cell mediated immunity (cmi). paw inflammation in methylated bsa-induced cmi was significantly reduced in transgenic mice expressing a mutant ikka protein that cannot be activated (ikka aa/aa ) compared to wild-type (wt). antigen-induced il- and ifng production by ikka aa/aa splenocytes and ikka aa/aa t cell:dc cocultures were also significantly reduced ex vivo. this could be normalised by using wt t cell: ikka aa/aa dendritic cell (dc) but not ikka aa/aa t cell:wt dc combinations. this suggests that reduced cmi in ikka aa/ aa mice is due to a defect in ikka aa/aa t cells not dcs. this is not due to a requirement of ikka in tcrmediated activation of t cells, since anti-cd /cd mediated activation of ikka aa/aa t cells was unaltered. however, lps-induced production of the important th cytokine il- is impaired in ikka aa/aa dcs. we are currently addressing the hypothesis that ikka activity may be required for the generation and maintenance of antigen-specific t cells in vivo. recently we described a role for ikkµ in the negative regulation of innate immunity and acute inflammation, which is in contrast to its role shown here in promoting adaptive immunity and antigen-driven inflammation. ikka may represent an alternative target for the treatment of autoimmune disease which would not compromise host defence. as a latent transcription factor, nf-kb translocates from cytoplasm into nucleus upon stimulations and mediates expression of genes important in immunity, inflammation and development. although extensive studies have been done regarding how nf-kb is triggered into nucleus, little is known about how it is regulated inside nucleus. by twohybrid approach, we identify a prefolding-like protein snip that is expressed predominantly and interacts specifically with nf-kb inside nucleus. we show that rnai knockdown of snip leads to impaired nucleus activity of nf-kb and dramatically attenuates expression of nf-kb dependent genes. this interference also sensitizes cells to apoptosis by tnf-a. furthermore, snip forms a dynamic complex with nf-kb and is recruited to the nf-kb enhancesome upon stimulation. interestingly, snip protein level correlates with constitutive nf-kb activity in human prostate cancer cell lines. the presence of nf-kb within nucleus of stimulated or constitutive active cells is significantly diminished without endogenous snip. our results reveal that snip is an integral component of nf-kb enhancesome and essential for its stability in nucleus, which uncovers a new mechanism of nf-kb regulation. bone remodeling is a tightly regulated process that couples resorption of old bone by osteoclasts and the deposition of new bone by osteoblasts. an imbalance between bone formation and bone resorption can result in various metabolic bone diseases, such as rheumatoid arthritis and osteoporosis. osteoclasts are terminally differentiated cells that arise from a haematopoietic stem cell lineage, which also gives rise to monocytes and macrophages. osteoclast differentiation and regulation of this process to maintain bone homeostasis are central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis. in vitro, osteoclast formation from bone marrow macrophages is induced by rankl (receptor activator of nf-kappa b ligand) in the presence of m-csf (macrophage colony stimulating factor). osteoclastogenesis is markedly enhanced in bone marrow macrophages from ifnar -/-and ifnar -/-mice and results in increased number of multinucleated cells positive for osteoclast marker, trap (tartrate-resistant acid phosphatase). consequently, the mutant mice develop osteoporotic phenotype, characterised by reduced bone density. these findings suggest that the ifn alpha/beta system is critical for the negative feedback regulation of osteoclastogenesis and that rankl signaling is essential for the induction of osteoclast differentiation. atp acting on p x receptors in macrophage is one of the main physiological signals that lead to the processing and release of the pro-inflammatory cytokine, interleukin- beta (il- b), their activation also leads to rapid opening of a membrane pore permeable to dyes such as ethidium. here we identify pannexin- , a recently described mammalian protein that functions as an hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin- is required for processing of caspase- and release of mature il- b induced by p x receptor activation. furthermore, maitotoxin and nigericin, two agents considered to evoke il- b release via the same mechanism were studied. maitotoxin evoked dye uptake whose kinetics were similar to a slow pannexin- -independent phase induced by p x receptor activation, and this was unaltered by pannexin- inhibition.nigericin did not induce dye uptake.inhibition of pannexin- blocked caspase- and il- b processing and release in response to this two stimuli.thus, while pannexin- is required for il- b release in response to maitotoxin, nigericin and atp, a mechanism distinct from pannexin- hemichannel activation must underlie the former two processes. introduction: saa is a classic acute-phase protein upregulated during inflammatory response. saa is active on leukocytes and modulates inflammation and immunity through the induction of cytokines, including the chemokine il- . here we verify the effect of saa on the mrna expression and release of mip- alpha, a chemokyne involved in the recruitment of dendritic cells. methods: peripheral blood mononuclear cells (pbmc) isolated from peripheral blood by density gradient were cultured in rpmi medium in the presence of saa. mip- alphaconcentration was determinated in the supernatant of cell cultures by elisa. mrnawas analyzed bythe ribonuclease protection assay (rpa). results: pbmc stimulated with saa ( ug/ml) induced the expression of mip- alpha mrna at , and hours. mip- alpha protein was found in the suppernatant of and hours cultures (p< , ) and the addition of sb (p inhibitor) and pd (erk / inhibitor) completely abolished the release of mip- alpha. conclusions: saa is an inducer of mip- alpha expression in pbmc and p and erk / are important pathway signaling to this effect. saa is one of the factors responsible by the recruitment of dendritic cells. the p pathway is activated in numerous inflammatory conditions, including ra, ibd asthma, acute coronary syndrome, and copd, and its activation helps drive the production of inflammatory mediators. inhibitors of p decrease mediator production and therefore can produce profound anti-inflammatory effects. arry- is a potent inhibitor of p enzyme (ic < nm) with a novel pharmacophore and physiochemical properties distinct from those of other p inhibitors, being very water soluble. it is extremely potent in human whole blood, blocking lps-stimulated tnf production with an ic < nm.in animal models of rheumatoid arthritis (cia and aia) the compound significantly normalized histologic endpoints, such as inflammation, bone resorption and cartilage damage (ed ~ mg/kg). a phase i single ascending dose clinical study was run in healthy volunteers. after an oral dose of , , , or mg), blood was drawn at various times, stimulated ex vivo with lps, and analyzed for cytokines and inflammatory mediatorsfj arry- was well-tolerated and drug exposure was proportional to dose. in ex vivo samples, there was both a time-and concentrationdependent inhibition of il , pge and tnffz with > % inhibition observed at the mg dose level. the plasma concentrations of drug peaked at~ ng/ml at the mg dose and cytokine inhibition was sustained for > hours, showing that low doses of arry- produced profound effects on clinical biomarkers. further evaluation of arry- in patients with inflammatory diseases is planned. introduction: we demonstrated that in vivo chronicle blockage of nos (l-name, mg/kg; oral route; days) impairs leukocyte-endothelial interactions and neutrophils migration into inflammatory focus. these effects may be depending, at least in part, on decreased expression of l-selectin on leukocytes and pecam- on endothelium. aimed to clarify the mechanisms involved on these inhibitory effects, we now investigated the role of l-name treatment on secretion of tnf and il- b; by circulating leukocytes and migrated peritoneal neutrophils. methods: male wistar rats were treated with l-name ( mg/kg; oral route; days) or sterile saline (control). circulating leukocytes were isolated from blood collected from abdominal aorta and migrated neutrophils were obtained hours after i.p. injection of oyster glycogen ( %; ml). no (griess reaction) and cytokines (elisa) were quantified in supernatants of x cultured cells before and hours after lps stimulation ( m;g/ml). results: levels of no, tnf and il- b; were reduced in circulating leukocytes from l-name-treated rats in both basal and lps stimulated conditions. on the other hand, only secretion of il- b; was impaired by migrated neutrophils. conclusions: results show that in vivo l-name treatment, which partially reduces no production, decreases the secretion of pro-inflammatory cytokines by circulating leukocytes. however, the same pattern of inhibition is not detected if neutrophils are in vivo primed. objectives: to investigate the ability and mechanism of ifn-g to suppress interleukin- (il- )-induced mmp- expression in articular chondrocytes. methods: human chondrocytes were treated with ifn-g or il- beta alone or in combination. mmp- mrna was analyzed by rt-pcr. mmp- protein, phospho-stat and p / mapk levels were measured by western blotting. mmp- promoter-luciferase, cmv-cbp/p plasmids and stat sirna were transfected by calcium phosphate method. ap- activity was monitored by elisa. stat -cbp/p interaction was studied by immunoprecipitation. results: ifn-gpotently suppressed il- -induced expression of mmp- and promoter activity. blockade with neutralizing ifn-gr antibody revealed that mmp- inhibition by ifn-¼ was mediated by the ifn-¼ receptor. ifn-beta-stimulated activation of stat was directly correlated with mmp- suppression. knockdown of stat gene by specific sirna or its inhibition with fludarabine partially restored the il- induction of mmp- expression and promoter activity. ifn-g did not alter activator protein (ap- ) binding ability but promoted physical interaction of stat and cbp/p co-activator. p overexpression reversed ifn-g inhibition of endogenous mmp- mrna expression and exogenous mmp- promoter activity. conclusions: ifn-g through its receptor activates stat , which binds with cbp/p co-activator, sequesters it from the cell system and thus inhibits transcriptional induction of mmp- gene in chondrocytes. ifn-g and its signaling pathways could be targeted therapeutically for ( ), p asmawidjaja ( ), r hendriks( ), erik lubberts ( ) ( ) erasmus medical center, department of rheumatology, rotterdam, the netherlands ( ) erasmus medical center, department of immunology, rotterdam, the netherlands the objective of this study was to identify the role of il- in th polarization in the prone autoimmune dba- mice with and without collagen-induced arthritis and to evaluate th specific cytokine and transcription factor expression. il- induced th cells in vitro from spleen cells of naïve and collagen-type ii (cii) immunized dba- mice. the percentage of th cells is markedly higher in cii-immunized versus naïve dba- mice. adding il- to tgf-beta/il- stimulated cd + t cells did not significantly increase the percentage of th cells. tgfbeta/il- in contrast to il- induced a relatively high percentage of il- +/ifn-gamma-cells and low il- -ifn-gamma+ cells. tgf-beta/il- did not increase il- receptor expression, which may explain why adding il- directly or two days after tgf-beta/il- did not result in an increase in the percentage of th cells. elevated expression of il- a and il- f as well as the th specific transcription factor rorgammat was found under il- as well as tgfbeta/il- conditions. interestingly, il- but not tgf-beta/il- is critical in the th cytokine il- expression in t cells from ciiimmunized dba- mice. these data show that il- was more pronounced in inducing il- +/ifn-gamma-(th ) cells under cii-immunized conditions. furthermore, il- did not markedly increase the percentage of th cells induced by tgf-beta/il- . however, il- is critical for the induction of il- expression, suggesting a unique role for il- in the induction of specific th cytokines ebi was initially discovered as a transcriptionally activated gene in epstein-barr virus-infected human b lymphocytes, and similar to p of il- . ebi protein has been shown to form heterodimers with p . p /ebi termed il- , can influence the function of multiple t cell subsets, including naive, effector, regulatory and memory t cells. however, previous studies showed that the overlapped expression of ebi and p is very limited. these data lead to the hypothesis that ebi may play a role independently from its association with p . thus, to define the function of ebi , we generated ebi transgenic (tg) mice expressing in multiple tissues. ebi tg mice exhibited no histologic abnormalities in various organs and normal numbers of naive and memory cd +, cd + t cells, b cells, nk cells and nkt cells. cd +t cells isolated from spleens of ebi tg mice, however, produced less ifn-g than cells from wt (wild type) control mice after in vitro stimulation with anti-cd and anti-cd antibodies. in vivo studies, delayed-type hypersensitivity (dth) and contact hypersensitivity (chs) responses were significantly reduced in ebi tg compared with wt mice. moreover, the chs responses in ebi tg mice were recovered with anti-ebi polyclonal antibody. notably, chs reaction in wt mice was increased by anti-ebi antibody. in contrast, anti-p antibody suppressed chs responses in wt mice. these data suggest that ebi acts in different from il- , and reduces th responses. ( ), o thaunat( ), x houard ( ), o meilhac ( ), g caligiuri( ), a nicoletti ( ) ( ) inserm u and university denis diderot-paris , chu xavier bichat, paris, france ( ) inserm umr s , universitØ pierre et marie curie-paris , centre de recherche des cordeliers, paris, france arteries are composed of three concentric tissue layers which exhibit different structures and properties. because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have been focused on the innermost layer (intima). in contrast, the role of the outermost tunica, the adventitia, has attracted relatively little attention and remains elusive. in the present review, we focus on involvement of the adventitia in the response to various types of arterial injury leading to vascular remodeling. several lines of evidence show that the initial insult and the early intimal response lead to the genesis of (neo-) mediators that are centrifugally conveyed by mass transport towards the adventitia. these mediators trigger local adventitial responses including angiogenesis, immuno-inflammation, and fibrosis. we propose that these three processes sequentially interact and that their net balance participates in producing each specific pathological condition. hence, an adventitial adaptive immune response predominates in chronic rejection. inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to proteolysis products in abdominal aortic aneurysm. centripetal adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. adventitial fibrosis mediated by low inflammation characterizes the response to mechanical stress and is responsible for constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. these adventitial events thus impact not only on the vessel wall biology but also on the surrounding tissue. atherosclerosis has many of the characteristics of an inflammatory disease, and thus would classically involve endothelial cox-derived prostaglandins such as pge and prostacyclin acting on ep and ip receptors, respectively.activation of vascular ip receptors is especially important in limiting the atherogenic properties of thromboxane a acting on tp receptors.more recently, expression of ghrelin receptors has been shown to be increased in atherosclerotic plaques, and ghrelin itself has anti-inflammatory properties in addition to its classical role as a hunger hormone.as well as the complex crosstalk between g-protein-coupled receptors (gpcrs), recent evidence indicates that many gpcrs exist constitutively as homodimeric complexes, and that the formation of heterodimers not only influences the classical cell signalling pathways used by these receptors, but also affects their subcellular distribution.we have found that ep -i, tp and ghrelin receptors readily form homodimers, but that co-transfection of hek cells with these receptors results in the formation of heterodimers with unpredictable effects on receptor distribution and cell signalling properties.since inflammatory conditions are thought to change the relative expression levels of gpcrs in the vasculature, and since varying the expression levels of gpcrs will affect their ability to form heterodimers, then one might predict that gpcr heterodimerization would indeed influence the reactivity of vascular tissue during inflammation. [this work was fully supported by grants from the research grants council of the hong kong special administrative region (cuhk / m and vascular inflammation leads to formation of leukotrienes through the -lipoxygenase pathway of arachidonic acid metabolism. leukotriene forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro-inflammatory actions within the vascular wall by means of cell surface receptors of the blt and cyslt receptor subtypes. recent mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis. leukotriene b (ltb ) is for example one of the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell-types of both hematopoietic and non-hematopoietic origin. initially identified on neutrophils, blt receptor activation is involved in monocyte chemotaxis and adhesion as well as in vascular smooth muscle cell migration and proliferation, providing examples of potential mechanisms in ltb -induced atherogenesis. targeting ltb -induced activation of vascular smooth muscle cells has beneficial effects in models of intimal hyperplasia and restenosis after vascular injury. furthermore, blt receptor expression has been demonstrated on t-cells, suggesting ltb as a potential link between innate and adaptive immunological reactions. taken together, the local formation of leukotrienes within the atherosclerotic lesion and the potent pro-inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role leukotrienes in this disease. further experimental and clinical studies are however needed to develop therapeutic strategies of treatments targeting leukotriene signaling in atherosclerosis. in normal physiological conditions, the prostanoid (prostaglandin (pg) and thromboxane (tx)) synthesis is dependent on the constitutive isoform of cyclooxygenase (cox- ). this synthesis and release happen few minutes after cell or tissue stimulation. in vascular preparations submitted to pro-inflammatory conditions for some hours, the inducible isoform of cyclooxygenase (cox- ) and other prostanoid synthases can be observed. as an illustration of the previous experimental results, there is an increased presence of cox- and the inducible enzyme responsible for pge synthesis (mpges- ) detected by immunocytochemistry in the carotid atherosclerotic plaque with strong inflammation. in vascular cells in culture, pgi is the major biological active prostanoid produced in the normal physiological conditions. however, when cox- is induced, pgi and pge are equally produced. the role of cox- , cox- and mpges- activities is also dependent on the expression of the various prostanoid receptors in the considered vessel. there is increasing evidence for the presence and a role of the ep receptor subtypes (ep , ep , ep or ep ) preferentially stimulated by pge in the vascular wall. for these reasons, we have characterized the receptors activated by pge in human mammary arteries. in these vessels incubated with a pro-inflammatory cytokine (interleukin- â) and lipopolysaccharides a reduced contractility to norepinephrine has been observed. this effect is abolished by treatment of the vascular preparations with a selective cox- inhibitor, suggesting that prostanoid synthesis and/or prostanoid receptors could be involved. rheumatoid arthritis is a syndrome which probably consists of a number of diseases for which the risk factors differ. two major processes were identified: the generation of the anti-citrullinated antigens immune response (highly sepcific for ra).we show that the different hla class ii alleles contribute to the development of anti-ccp-positive and anti-ccp negative ra.the se alleles do not independently contribute to the progression to ra, but rather contributed to the development of anti-ccp antibodies. next we determined the effect of smoking and observed that smoking only conferred risk to contract ra in the ccp-positive group and not in the anti-ccp negative group. for the risk factor ptpn (a gene that regulates treshold of lumphocyte activation) the allele c t only contributed to ccp-positive ra. in contrast to hla two other risk factors were found to be associated with both ccp-positive and ccp-negative ra. the risk factor in the fcrl-gene as has been identified in the japanese population was also tested in dutch ra cases and unrelated dutch controls. carrier analysis of the snp (rs ) revealed association of cc genotype with higher risk of developing ra as compared to tt & tc carriers (p = . and or = . ). in a meta-analysis of all studies comparing individuals, the or for the cc genotype to develop ra was . and the p-value < . . in conclusion, different steps in pathogenesis of the syndrome ra can be delineated this talk will focus on recent advances in understanding primary genetic factors predisposing to inflammatory bowel disease (crohns disease and ulcerative colitis). proven genes containing genetic variants predisposing to crohns disease include ibd / q , card /nod and il r. data is suggestive but not yet as convincing for many other genes. a common theme is of genetic variants influencing early innate immune responses to intestinal bacterial components, and subsequent adaptive immune responses, leading to intestinal inflammation. only for card /nod is there (partial) understanding of how genetic variation influences biological function to cause chronic disease. some mouse models (gene targeted) of card appear to show opposite effects to other models and human systems. in humans, card mutations impair responses to bacterial components (muramyl dipeptide) mainly at low dose sensing. it is likely this receptor system normally maintains intestinal crypt sterility and protection from invasive infection. pathogen-recognition receptors (prrs) are key components of immune systems and are involved in innate effector mechanisms and activation of adaptive immunity. since their discovery in vertebrates, toll-like receptors (tlrs) have become the focus of extensive research that has revealed their significance in the regulation of many facets of our immune system. recently a new family of intracellular prrs, the nod-like receptors (nlrs), which include both nods and nalps have been described. mutations within the nalp /cryopyrin/ cias gene are responsible for three autoinflammatory disorders: muckle-wells syndrome, familial cold autoinflammatory syndrome, and cinca/nomid. the nalp protein associates with asc and caspase- (thereby forming a molecular machine termed inflammasome that displays high proil- beta-processing activity. macrophages from muckle-wells patients spontaneously secrete active il- beta. increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with nalp -dependent autoinflammatory disorders. here we will emphasis on the ability of this protein complex to promote the development of autoinflammatory syndromes. allergic inflammation (ai) is a complex phenomenon initiated by allergen binding to ige sensitized mast cells and consequent mast cell activation. this causes the symptoms of the early phase of ai and the onset of the late phase characterized by the penetration in the inflamed tissue of inflammatory cells, notably the eosinophils. their subsequent activation is believed to cause tissue damage and to be the main responsible for the tissue remodeling, especially when the ai becomes a chronic process. we defined a novel functional unit that we termed the allergic synapse formed by mast celleosinophil couples. in the synapse these two old cellular players of ai have a cross talk via soluble mediators and receptor-ligand interactions. this results in mast celleosinophil functional synergism that consequently amplifies and prolongs the inflammatory response. in addition, mast cells and eosinophils are influenced and influence as in a sort of allergic niche the surrounding structural cells, i.e. fibroblasts and endothelial cells. we propose to view the allergic synapse/niche as a specialized effector unit worthy to be blocked for the treatment/prevention of allergic inflammation. ( ) ( ) erasmus mc, rotterdam, the netherlands ( ) department of immunology weizmann institute of science, rehovot, israel allergic asthma is one of the most common chronic diseases in western society, characterized by reversible airway obstruction, mucus hypersecretion and infiltration of the airway wall with th cells, eosinophils, and mast cells. if we are to devise new therapies for this disease, it is important to elucidate how th cells are activated and respond to intrinsically harmless allergens. dendritic cells (dcs) are the most important antigen presenting cells in the lung and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to aeroallergens. we have shown that intratracheal injection of ovalbumin (ova) pulsed dcs induces sensitization leading to eosinophilic airway inflammation upon ova aerosol challenge. we investigated the role of dcs in the secondary immune response in a murine asthma model. ova aerosol challenge in ova-dc sensitized mice, induced an almost fold increase in the number of airway dcs as well as an increase in eosinophils and t cells. to investigate the functional importance of dcs for the induction and maintenance of airway inflammation in response to allergen challenge, we conditionally knocked-out endogenous dcs in sensitised cd c-diphtheria toxin (dt) receptor (cd cdtr) transgenic mice by airway administration of dt h before ova aerosol ( x) challenge or during an ongoing inflammation (depletion after x ova aerosols continued with additional ova aerosols). numbers of balf eosinophils, th cytokine production by mediastinal lymph nodes and peribronchial and perivascular inflammatory infiltrates were dramatically decreased, illustrating an essential role for airway dcs during secondary challenge. karolinska institute, stockholm, sweden nk cells are innate lymphocytes with potent immunoregulatory functions. they are potent producers of several cytokines and chemokines, and also respond to similar molecules in the body and at inflammatory sites. even though traditionally best characterized for their role in anti-viral and anti-tumor immunity, they influence several other types of immune responses. for example, they are involved in, and affect, acute as well as chronic inflammatory responses. in the present talk, a general overview on our current knowledge of nk cell biology will be provided, with a special emphasis on the role of these cells in allergic inflammation. basophils are major effector cells in allergic reactions due to their ability to release substantial quantities of histamine and eicosanoids following activation of high affinity ige receptors (fc<epsilon>ri) with allergens. although these attributes are shared with their tissuefixed mast cell compatriots, basophils are unique in their ability to also rapidly elaborate th -type cytokines (e.g. il- and il- ), subsequently supporting ige synthesis and underlying atopy. importantly, these mediators are additionally secreted following primary exposure to certain parasites (e.g. s. mansoni) and immunoglobulin superantigens, suggesting a role for basophils in innate immunity and in assisting developing th -type adaptive immune responses. while we are beginning to understand the potential physiological functions of these cells regarding host defence, blocking their activity with respect to treating symptoms of allergic disease has remained an enigma. recent advances, however, have shed light upon the major intracellular signal transduction processes involved in fc<epsilon>ri activation and may lead to novel therapeutic strategies for inhibiting mediator secretions. an important discovery in this regard is the phosphatase ship, which downregulates pi -kinase signalling in both basophils and mast cells. recent data shows that ship expressions in basophils are reduced from donors with active allergic disease but that these levels may be increased, and the activity of basophils subsequently inhibited, by targeting receptors associated with ship recruitment (cd r, cd r). identifying the natural ligands for these inhibitory receptors may therefore pave the way for new therapies for the treatment of allergic inflammation. mitogenesis and proliferation of vsmc play an important role in atherogenesis. pro-inflammatory secretory phospholipases a (spla ) hydrolyse glycerophospholipids of hdl and ldl and release pro-inflammatory agents, lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.spla s lipolysis products localize in vascular wall in vicinity of vsmc.we have tested the impact of spla , hdl and ldl and of their hydrolysis products on mitogenesis and pge and ltb release from vsmc.mitogenesis was significantly enhanced by native hdl, and ldl, and by group v spla . spla hydrolysis of hdl and ldl enhanced mitogenic activity in order v>x>iia.the release of pge from vsmc was enhanced by group x spla s but not iia or v.the greatest effect was seen for hdl hydrolysed by group v and x spla .native ldl and its spla hydrolysis products enhanced the release of pge in order x>v>iia.the release of ltb from vsmc was markedly increased by native ldl and hdl, and hydrolysis products of group v and x, but not iia spla .migration of vsmc was significantly enhanced by spla iia and inhibited by hdl.this study demonstrates a complex interaction of hdl and ldl with pro-inflammatory spla s, which affects mitogenesis, eicosanoid release and migration of vsmc.study of biocompatible spla blockers in the therapy of atherosclerosis is indicated. contact information: professor waldemar pruzanski, university of toronto, department of medicine, toronto, ontario, canada e-mail: drwpruzanski@bellnet.ca ( ) ( ) ipmc-cnrs umr , valbonne, france ( ) university of washington, seattle, usa ( ) inserm umrs , paris, france ( ) university of naples, italy the superfamily of phospholipase a comprises at least intracellular enzymes and up to secreted pla s (spla s). elucidating the biological roles of each pla member is currently the most challenging issue in the pla field. the different spla s are not isoforms and are likely to function either as enzymes producing key lipid mediators (eicosanoids and lysophospholipids) or as ligands that bind to specific soluble or membrane-bound proteins (like cytokines). increasing evidence suggests that spla s iia, iii, v, and x are involved in inflammatory diseases including atherosclerosis. among spla s, the human group x (hgx) enzyme has the highest enzymatic activity towards phosphatidylcholine, the major phospholipid of cellular membranes and low density lipoproteins (ldl). on human alveolar macrophages, hgx spla can trigger secretion of tnf alpha, il and il in a non-enzymatic manner. on colorectal cancer cells, hgx spla stimulates cell proliferation, produces potent eicosanoids including pge , and activates the transcription of key genes involved in inflammation and cancer. the enzyme can also hydrolyze pc and platelet-activating factor (paf) of ldl particles very efficiently. finally, hgx spla is present in human atherosclerotic lesions and converts ldl into a proinflammatory particle that induces macrophage foam cell formation, as well as map kinase activation, arachidonic acid release, and expression of adhesion molecules in huvec cells. some other key molecular features of spla s including hgx will be presented. we have reported preferential release of polyunsaturated fatty acids during hydrolysis of lipoprotein phosphatidylcholine (ptdcho) by spla s, but the mechanism of this selectivity is not known. since both sphingomyelin (sm) and lysoptdcho inhibit the activity while increasing fatty acid specificity of other pla s, we have examined fatty acid release by spla siia, v and x in relation to relative increases in proportion of endogenous sm and lysoptdcho during lipoprotein digestion. the analyses were performed by normal phase liquid chromatography with on-line electrospray mass spectrometry (lc/esi-ms) and lc/collision induced dissociation (cid)/esi-ms using conventional preparations ofldl and hdl. the highest preference for arachidonate release from ldl by group x spla was observed when the residual sm/ pdcho molar ratio had reached . compared to a starting ratio of . .group v spla showed preferential release of linoleate at residual sm/ptdcho molar ratio . , while at intermediate ratios, both arachidonate and linoleate were released at more comparable ratios. the relative increases in lysoptdcho and sm during the digestion with spla iia were much more limited, and a preferential hydrolysis of polyunsaturated fatty acids was not observed. these results suggest a lipid phase separation as a likely basis for a differential hydrolysis of molecular species of ptdcho. the residual sm/ptdcho ratios reached during group v and x spla digestion are similar to those observed for lesional ldl, which promote release of ceramides by smase leading to ldl aggregation. the above findings support a potential role of sphingomyelins in atherogenesis. although sphingomyelin (sm) is one of the most abundant phospholipids in lipoproteins and cell membranes, its physiological significance is unclear. because of its localization in the outer surface of the cells, and its structural similarity to phosphatidylcholine (pc),we proposed that it competitively inhibits phospholipolysis of cell membranes by external phospholipases (pla). we showed that sm inhibits several lipolytic enzymes including secretory pla iia, v, and x, and hepatic and endothelial lipases, all of which hydrolyze pc. treatment of sm in the substrate with smase c not only relieved the inhibition but also activated the pla reaction further, suggesting that ceramide, the product of smase c, independently stimulates pla , possibly by disrupting the bilayer structure. smase d treatment, which produces ceramide phosphate, did not stimulate the spla . the fatty acid specificity of pla is significantly affected by sm. thus spla x exhibited enhanced specificity for the release of arachidonic acid ( : ) in presence of sm, due to a preferential inhibition of hydrolysis of other pc species. in contrast, sm inhibited the release of : by spla v. ceramide selectively stimulated the release of : by both enzymes. only the long chain ceramides (> carbons) were effective, while ceramide phosphate did not stimulate spla activity. sm-deficient cells released more : in response to spla -treatment than normal cells, and pretreatment of normal cells with smase c increased their susceptibility to spla attack. these studies show that sm and ceramide regulate the activity and specificity of pla, and consequently the inflammatory response. secretory phospholipase a (spla ) types iia, v, or x, have been associated with inflammatory diseases and tissue injury including atherosclerosis in humans and mice.given the link between spla and atherogenesis, a mouse model of atherosclerosis (apoe-/-) was used to study the effects of a- , an inhibitor of spla enzymes, on atherosclerosis and cholesterol levels over weeks of treatment. mice were fed with a high-fat, high cholesterol diet alone during the study ( % fat; . % cholesterol, . % casein) and were treated with vehicle or a- bid at mg/kg or mg/kg by oral gavage. total cholesterol was significantly decreased after one month of treatment and remained lower throughout the study.treatment with a- significantly reduced aortic atherosclerotic plaque formation in apoe-/-mice fed a high fat diet when compared to the untreated control by approximately %. in a different model that used angiotensin ii in conjunction with a high fat diet in a background of apoe-/-deficient mice for weeks, oral dosing of a- ( mg/kg bid) significantly reduced aortic atherosclerosis and aneurysm rate when compared to vehicle. these data suggest that a- is a potential novel therapeutic agent for the treatment of atherosclerosis. ( ), s doty( ), c antonescu ( ), c staniloae ( ) ( ) saint vincents hospital manhattan, new york, usa ( ) hospital for special surgery, new york, usa ( ) sloan-kettering institute for cancer research, new york, usa tnf-stimulated gene (tsg- ) is induced by tnf-a during inflammation and its secreted product tsg- glycoprotein is involved in immune-mediated inflammatory diseases and fertility. it regulates cox- and prostaglandin synthesis, and participates in extracellular matrix remodeling. considering the chronic inflammatory nature of atherosclerosis we hypothesized that tsg- is expressed in atherosclerotic plaques and investigated tsg- protein expression and cellular distribution on superficial femoral artery endarterectomy specimens from diabetic and non-diabetic patients with peripheral vascular disease. six histologically normal radial artery specimens were analyzed as control. paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-tsg- antibody. tsg- expression was consistently present in all atherectomy specimens but not in control specimens. a distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. patchy tsg- expression was noted in the extracellular matrix. within the inflammatory plaques from diabetic patients, tsg- stained the foamy macrophages. tsg- expression was also confirmed and quantified by qrt-pcr that showed a significant up-regulation of tsg- gene (more that fold induction compared to housekeeping genes). our study identifies for the first time the preferential expression of tsg- in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. tsg- is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation. ( ), r krohn ( ), h lue ( ), jl gregory( ), a zernecke ( ), rr koenen ( ), t kooistra ( ), p ghezzi( ), r kleemann ( ), r bucala( ), mj hickey ( ), c weber ( ) ( ) university hospital of the rwth aachen, germany ( ) centre for inflammatory diseases, monash university, melbourne, australia mediators, which cannot be classified into chemokine subfamilies but share functional patterns, e.g. signaling through chemokine receptors, constitute a group termed chemokine-like function (clf)-chemokines. the pleiotropic cytokine macrophage migration inhibitory factor (mif) plays a critical role in inflammatory diseases and atherogenesis. the underlying molecular mechanisms are poorly understood, but, interestingly, mif displays structural features resembling chemokines. we have identified the chemokine receptor cxcr as a functional receptor for mif. mif triggered galphai/integrin-dependent arrest and chemotaxis of monocytes specifically through cxcr , inducing rapid integrin activation. mif directly bound to cxcr with high affinity (kd of . nm). monocyte arrest mediated by mif in inflamed or atherosclerotic arteries involved cxcr as well as cd , a recently identified membrane receptor moiety for mif. accordingly, cxcr and cd were found to occur in a receptor complex. in vivo, mif deficiency impaired monocyte adhesion to the aortic/arterial wall in atherosclerosis-prone mice, as evidenced by intravital microscopy. thus, mif displays chemokine-like functions by acting as a non-cognate ligand of cxcr , serving as a regulator of inflammatory and atherogenic recruitment. these data harbor an intriguing novel therapeutic prospect by targeting mif in atherosclerosis and add a new dimension to mif and chemokine receptor biology. ( ), r toes ( ), h van bockel( ), paul quax ( , ) ( ) tno bioscienses, leiden, the netherlands ( ) department of vascular surgery, leiden university medical center, the netherlands ( ) department of rheumatoly, leiden university medical center, the netherlands the immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. here, we studied the role of lymphocytes in a murine model for artiogenenesis after acutehind limb ischemia. arteriogenesis was impaired in c bl/ mice depleted for natural killer (nk)-cells by anti-nk . antibodies and in nk-cell-deficient transgenic mice. arteriogenesis was, however, unaffected in jµ knockout mice that lack nk . + natural killer t (nkt)cells, indicating that nk-cells, rather than nkt-cells are involved in arteriogenesis. furthermore, arteriogenesis was impaired in c bl/ mice depleted for cd + tlymphocytes by anti-cd antibodies, and in major histocompatibility complex (mhc)-class-ii-deficient mice that lack mature peripheral cd + t-lymphocytes. this impairment was even more profound in anti-nk . treated mhc-class-ii-deficient mice that lack both nkand cd + t-lymphocytes. finally, collateral growth was severely reduced in balb/c as compared with c bl/ mice, two strains with different bias in immune responsiveness. correspondingly, fewer cd -positive lymphocytes accumulated around collaterals in balb/c mice. these data show that both nk-cells and cd + t-cells play an important role in arteriogenesis. moreover, our data hold promise for the development of novel clinical interventions as promoting lymphocyte activation might represent a powerful method to treat ischemic disease. post-interventional vascular remodeling in venous bypass grafts, seen as intimal hyperplasia (ih) and accelerated atherosclerosis, often causing graft failure. inflammation is an important trigger for these processe. complement is an important part of the immune system and participates in regulating inflammation. although involved in several other inflammatory diseases, the role of the complement cascade in vein graft remodeling is unknown. the involvement of the complement system in vein graft disease was studied here using a model in which caval veins are grafted in carotids arteries of hypercholesterolemic apoe leiden mice. in these veins ih and accelerated atherosclerotic lesions develop within days, consisting mainly of foamcells and smc. to study the functional role of complement in vein graft remodeling, cobra venom factor (cvf: u daily) was used to deplete complement starting one day prior to vein graft surgery. cvf-treatment reduced vein graft thickening by % (p= . ), when compared to saline treated controls (n= ).to confirm that the reduction by cvf was due to hampered complement function and not a direct effect of cvf, complement activation was blocked using crry-ig (inhibiting c convertases). crry-ig ( mg every other day) led to % decrease in vein graft thickening (p= . ) compared to controls receiving non-relevant control igg. these data prove that complement activation plays a major in intimal hyperplasia and accelerated atherosclerosis in vein grafts. ( ), m-c koutsing tine( ), p borgeat( ), h ong ( ), sylvie marleau ( ) ( ) universite de montreal, quebec, canada ( ) centre de recherche en rhumatologie et immunologie, canada we have previously shown that ep , a growth hormone-releasing peptide (ghrp) analogue binding selectively to the scavenger receptor cd , elicits a striking reduction in atherosclerosis development in apolipoprotein-deficient (apoe-/-), a condition associated with increased circulating numbers of primed/activated leukocytes. we investigated the effect of ghrp analogues on i/r-elicited remote lung injury in week-old apoe-/-mice fed a high fat high cholesterol (hfhc) diet from weeks of age. at weeks old, mice were treated daily with a s.c. injection of ep ( mg/kg) for days and were then subjected to unilateral hindlimb ischemia (by rubber band application) for minutes, followed by minutes reperfusion. our results show that ep significantly reduced leukocyte accumulation by % in the lungs, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice (n = - per group), as assessed by myeloperoxidase assay. this was associated with a % reduction of opsonized zymosan-elicited blood chemiluminescence. in contrast, neither blood chemiluminescence, nor leukocyte accumulation in the lungs were signicantly modulated in apoe-/-/cd -/-deficient mice, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice. we conclude that ep protects i/r-elicited circulating leukocyte priming/activation and remote lung injury, possibly through a cd -mediated pathway. glycogen synthase kinase beta (gsk- beta) is a serine/ threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. dysregulation of gsk- beta has been implicated in the pathogenesis of several diseases including sepsis. here we investigate the effects of two chemically distinct inhibitors of gsk- beta, tdzd- and sb , on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. male wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to mmhg for min) and subsequently resuscitated with shed blood for h. hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. treatment of rats with either tdzd- ( mg/kg, i.v.) or sb ( . mg/kg, i.v.) min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. the protection afforded by these compounds was confirmed by histological observations of lung, kidney and liver samples. in addition, tdzd- , but not sb , attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin . neither of the gsk- beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. thus, we propose that inhibition of gsk- beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. ( ), y ito ( ), h yoshimura ( ), h inoue ( ), n kurouzu ( ), h hara ( ), y mastui ( ), h kitasato ( ), s narumiya( ), c yokoyama ( ), m majima ( ) ( ) kitasato university school of medicine, japan ( ) kyoto university school of medicine, japan ( ) tokyo medical and dental university, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitors constitute the major determinant of revascularization. we hypothesized txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggregation. to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb ischemia. recovery from acute hind limb ischemia, as assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out mice (ipko) and thromboxane (tx) a receptor (tp) knock out mice (tpko) by using lase doppler. blood recovery in tpko significantly delayed compared to wt and ipko. immunohistochemical studies revealed that the number of cd positive cells in the ischemic quadriceps were less stained in tpko compared to wt and ipko.plasma sdf- and vegf concentration were significantly reduced in tpko mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. administration of selective tp agonist may open new therapeutic strategy in regenerative cardiovascular medicine. during renal ischemia/reperfusion (i/r) injury, apoptosis has been reported as a very important contributor to the final kidney damage. the determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. the aim of the study is to know the role of peroxynitrite as inductor of cytoskeleton derangement and apoptosis during the inflammatory process associated to renal ischemia-reperfusion. based in a rat kidney i/r model, by experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the i/r derived oxidative stress state, is able to provoke cytoskeleton derangement and apoptosis development. thus, the control in the peroxynitrite generation during the i/r could be an effective tool for the improvement of cytoskeleton damage and reduction apoptosis incidence in the renal i/r injury. metabolomics, the global profiling of metabolites, may inform about the multiple interacting processes involved in inflammatory disease. using nmr spectroscopy we analysed metabolite fingerprints in serum from early arthritis, and at a site of inflammation, in the posterior segment of the eye. serum from patients with synovitis of "t months duration whose outcome was determined at clinical follow-up was used. vitreous samples were from patients undergoing vitrectomy for vitreoretinal disorders. one dimensional h nmr spectra were acquired. principal components analysis (pca) of the processed data was conducted along with a supervised classification. with the arthritis serum there was a clear relationship between each samples score in the pca analysis and the level of crp. supervised classification of the initial samples was able to predict outcome, whether rheumatoid arthritis, other chronic arthritis or self-limiting arthritis, with high specificity and sensitivity. a similar approach using the eye fluids was able to give a clear discrimination between two pathologically similar conditions lens-induced and chronic uveitis. in this case differences were not due to a straightforward relationship with inflammatory markers (il- , ccl ), which did not correlate with pca in these samples. similarly, certain molecules, such as lactate, were associated with ocular disease, but not rheumatoid arthritis. these results suggest that underlying inflammatory processes may differ in these conditions or may reflect predisposing metabolic patterns in individual patients. h-nmr-based metabolomics may provide a useful measure of outcome in inflammatory diseases and give novel insights into the pathological processes involved. ( ), am artoli( ), a sequeira( ), c saldanha ( ) ( ) instituto de medicina molecular,faculdade de medicina de lisboa, portugal ( ) cemat, instituto superior tØcnico, universidade de tØcnica de lisboa, portugal the recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. the precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrest are still unknown. in this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behaviour of clusters of leukocytes. leukocyte-endothelial cell interactions in post-capillary venules of wistar rats cremaster muscle were monitorized by intravital microscopy. from these experiments the haemorheologic and haemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice boltzmann solver for shear thinning fluids. the dynamics of leukocyte clusters under non-newtonian blood flow with shear thinning viscosity was computed and discussed. in this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. we have also observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. we verified that the collective hydrodynamic behaviour of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. it was found that the lattice boltzmann solver used here is fully adaptive to the measured experimental parameters. this study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation. macrophages are essential for host defence, but when excessively and persistently activated, these cells contribute to the initiation and progression of inflammatory diseases such as rheumatoid arthritis. investigating the function of inflammatory genes in macrophages may identify novel therapeutic targets for inflammatory diseases. one family of transcripts that are highly expressed in activated macrophages are members of the schlafen (slfn) gene family; a recently identified family whose function is still unknown. this study examined the mrna expression of slfn in activated bone marrowderived macrophages in vitro, and in collagen-induced arthritis (cia) in vivo. real-time pcr expression analyses of bone marrow-derived macrophages stimulated with lipopolysaccharide (lps) over a time course, revealed differential expression of individual slfn family genes. in particular, slfn- , slfn- , and slfn- were maximally induced after hours. the maximal induction of slfn- and slfn- was observed after hours of lps treatment. individual members of the slfn family were also differentially expressed in cia, a model of rheumatoid arthritis. mrna levels of slfn- , slfn- , slfn- and slfn- were elevated in joints affected by cia. to investigate the role of slfn- , we have generated a transgenic mouse line, which over expresses slfn- specifically in cells of the mononuclear phagocyte system, by using a novel binary expression based on the c-fms promoter and gal . further characterisation of the slfn- over expressing mouse line will be used to assess the function of slfn- in macrophage biology and inflammation, and its potential as a therapeutic target. macrophages play an important role in resolving inflammation. it is known that the resolution of inflammation requires alternative activation of macrophages. but the precise events of phenotype switching in macrophages remain poorly understood. we show that lipocalin , lcn- , is able to provoke a switch in macrophage activation. in an in vitro co-culture model for renal epithelial cells and macrophages, we detected by sirna technique that the presence or absence of lcn- determines proliferation processes in damaged renal epithelial cells. the proliferative response was dependent on proinflammatory or anti-inflammatory environment. as lcn- is an acute phase protein synthesized during inflammation and unregulated in a number of pathological conditions, it may play an important role in survival and regeneration. we anticipate here that our results could be relevant for further research on the mechanisms of the phenotype switch induced by lcn- . ( ), y cao ( ), s adhikari ( ), m wallig ( ) ( ) national university of singapore, department of pharmacology, singapore ( ) university of illinois at urbana champaign, usa it has earlier been shown that the extent of apoptotic acinar cell death is inversely related to the severity of acute pancreatitis. our previous works have demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. the current study aims to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against acute pancreatitis by crambene. acute pancreatitis was induced in the mouse by administering hourly injections of caerulein ( mg/kg) for , and hours respectively. neutralizing monoclonal anti-il- antibody ( . mg/kg) was administered either with or without crambene ( mg/kg) hours before the first caerulein injection. rt-pcr, western blotting and immunostaining were performed to detect cd expression. apoptosis in pancreatic sections was visualized by tunel. severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (mpo), water content, and morphological examination. pancreatic levels of inflammatory mediators were examined by elisa. the protective effect of crambene is mediated by reducing production of pro-inflammatory cytokines such as mcp- , tnf-a and il- â and up-regulating anti-inflammatory mediators like il- . phagocytotic clearance in mouse acute pancreatitis may be essentially through macrophage surface receptor cd .the anti-inflammatory mediator il- plays an important role in crambene-induced protective action against acute pancreatitis. the release of anti-inflammatory mediator il- is downstream of phagocytosis. these results show that induction of pancreatic acinar cell apoptosis by crambene treatment protects mice against acute pancreatitis via induction of anti-inflammatory pathways. ( , ) ( ) northern ontario school of medicine, thunder bay, ontario, canada ( ) lakehead university, canada integrin receptors and their ligands are involved in adhesion and internalization of several human pathogens, including pseudomonas aeruginosa. we have recently established that beta integrins in lung epithelial cells (lec) provide co-stimulatory signals regulating inflammatory responses (ulanova et al, am j physiol, , : l -l ). we hypothesized that lec integrins serve as receptors to recognize pathogen-associated molecules and mediate the innate immune response to p. aeruginosa. to determine molecular mechanisms of integrin involvement in innate immunity, we used an in vitro model of p. aeruginosa infection of a cells. to investigate interactions of bacteria with lec, p. aeruginosa strain pak was chromosomally labeled with a green fluorescent protein gene using a mini-tn delivery system.using several fluorescence-based detection systems, we established that the natural beta integrin ligand, fibronectin, mediates bacterial adhesion to lec.p. aeruginosa infection caused rapid transcriptional upregulation of alphav and beta integrin expression followed by the increased cell surface protein expression. the surface expression of integrin beta increased shortly following bacterial exposure without alterations of mrna expression, suggesting rapid protein redistribution within the cells. the data indicate that p. aeruginosa are capable to modulate integrin gene/protein expression in lec, potentially using fibronectin to alleviate bacterial binding to beta integrins. upon their engagement, integrin receptors can initiate intracellular signaling involved in innate immune and inflammatory responses to the pathogen. integrin receptors in lec may represent significant therapeutic targets in pulmonary infection caused by p. aeruginosa. the purine nucleoside adenosine has a major modulatory impact on the inflammatory and immune systems. neutrophils, which are generally the first cells to migrate toward lesions and initiate host defense functions, are particularly responsive to the action of adenosine. through activation of the a a receptor (a ar) present on neutrophils, adenosine inhibits phagocytosis, generation of cytotoxic oxygen species, and adhesion. also, recent work showed that adenosine can transform the profile of lipid mediators generated by neutrophils, inhibiting leukotriene b formation while potentiating that of prostaglandin e through the up-regulation of the cyclooxygenase (cox)- pathway. moreover, our laboratory determined that a ar engagement can dramatically modulate the generation and secretion of neutrophil-derived cytokines/chemokines, including tnf-and mips. in mice lacking the a ar, migrated neutrophils expressed less cox- than their wild type counterpart while displaying higher mrna levels of tnf-and mip- . mononuclear cells from a ar knock out mice, which eventually replace neutrophils into the air pouch, also displayed a more pro-inflammatory phenotype than those from wild-type animals. signal transduction experiments, aiming to delineate the intracellular events leading to the modulation of neutrophil functions following a ar engagement, implicate pivotal metabolic pathways such as intracellular cyclic amp, p and pi- k. together, these results indicate that adenosine may have a profound and multi-pronged influence on the phenotype of neutrophils and present this cell as being pivotal in mediating adenosines anti-inflammatory effects. the newest developments regarding adenosines effects on neutrophil functions will be presented.this work is supported by the canadian institutes of health research (cihr). human skin serves not only as a physical barrier against infection, but also as a "chemical barrier" by constitutively and inducibly producing antimicrobial proteins (amps). to identify human skin amps, we analysed extracts of healthy persons stratum corneum by reversed phase-hplc and purified a novel kda amp that showed sequence similarity to mouse hornerin. suggesting that it originates from the human ortholog, we cloned it. human hornerin encodes a amino acid protein that contains a s domain, an ef-hand calciumbinding domain, a spacer sequence and two types of tandem repeats, suggesting that it represents a novel member of the fused s protein family. strongest constitutive hornerin mrna expression was seen in differentiated keratinocyte cultures. to follow the hypothesis, that hornerin fragments represent amps, we recombinantly expressed three hornerin peptides, rhrnr (tandem repeat unit b), rhrnr (tandem repeat unit a) and rhrnr (c-terminus) and subsequently analysed their antimicrobial activity using the microdilution assay system. the rhrnr peptide, containing the sequence motif found in the purified natural hornerin fragment isolated from stratum corneum, exhibited antimicrobial activity at low micromolar concentrations against escherichia coli, pseudomonas aeruginosa and candida albicans. the other peptides were found to be not or nearly not antimicrobially active. our results suggest that hornerin may have a yet unknown protective function in healthy human skin as part of the "chemical barrier" with preformed amps, which are generated from parts of the tandem repeats of a hornerin precursor molecule by a yet unknown cleavage mechanism. ( ), n lu( ), r jonsson( ), d gullberg ( ) ( ) department of biomedicine, university of bergen, norway ( ) the gade institute, university of bergen, norway a ß is the latest addition to the integrin family of heterodimeric receptors for the extracellular matrix. previously, it has been shown that this collagen receptor takes part in processes such as cell migration and matrix contraction. in this study we investigated the factors that regulate mouse integrin a ß expression. specifically, we have analyzed the influence of cell passage, growth factors and the -d microenvironment. using sv immortalized as well as primary fibroblasts, we show that a ß integrin is up-regulated when these cells are cultured within stressed collagen type i lattices. however, a ß is downregulated when the collagen gels are made under relaxed conditions, allowing cells contract the lattice diameter. we also show here that a is upregulated by tgf-a on planar substrates. these findings suggest that mechanical tension and tgf-a are important factors in the regulation of a ß that need to be to taken into consideration when evaluating the role of a ß in wound healing and fibrotic disorders. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada the objective of this study was to investigate the effects of par deficiency in various models of colonic inflammation in order to elucidate the role of endogenous par in the process of inflammation in the gut.colonic inflammation in c bl wildtype and par -/-mice was induced by treatment with . % dss (in drinking water) or tnbs ( mg or mg in ul of % ethanol, single intracolonic injection) or pre-sensitizing mice with % oxazolone (in olive oil) applied to the skin of the abdomen, and days later, a single intracolonic injection of % oxazolone (dissolved in % ethanol).intravital microscopy was performed, days (tnbs/dss) or days (oxazolone) after induction of colitis on the colonic venules to assess changes in leukocyte rolling, adhesion and vessel diameter.lastly, various parameters of inflammation were assessed following the intravital microscopy.par -/-mice showed significantly lower leukocyte adherence and vessel dilation compared to the wildtype mice in dss, and tnbs challenge. in all three challenges, mpo activity, macroscopic damage score and bowel thickness were significantly higher in wild-type mice, compared to par -/-.our evidences indicate that deficiency in par attenuates inflammatory responses in the experimental models of colitis associated with either th (tnbs/dss) or th (oxazolone) cytokine profile.therefore, par deficiency in the gut exerts antiinflammatory properties that are independent of th or th cytokine profile.the present study further highlights par as a potential target for inflammatory bowel diseases. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada in a previous study, inflammatory responses induced by three different models of colitis (tnbs/dss/oxazolone) were significantly attenuated in mice deficient for par (par -/-). among the inflammatory parameters observed, infiltration of granulocytes to the colon was consistently reduced by par deficiency. aim of this study was to assess the effects of par deficiency (via par -/-mice) on the recruitment of leukocyte in colonic venules. in anaesthetized animals, leukocyte rolling/ adherence and vasodilation were induced, by topical administration of fmlp ( mm) or paf ( nm) or by intraperitoneal injection of tnf-a; ( . mg -given hours before the intravital microscopy). using intravital microscopy, we evaluated the ability of various leukocyte stimuli to induce leukocyte trafficking and vasodilation in colonic venules of par -/-versus par +/+ mice. fmlp and paf as well as tnf-a; induced significant vasodilation and an increase in rolling/adhesion of leukocytes in mouse colonic venules. par -/-mice showed significantly lower leukocyte rolling compared to the wildtype mice in response to fmlp topical administration. leukocyte adherence induced by fmlp and tnf-a; was significantly lower in par -/-mice compared to wild types as well. no difference was observed between par -/-and wildtype for leukocyte rolling/adherence-induced by paf. the lack of functional par attenuated leukocyte trafficking in response to fmlp and tnf-a; but not to paf. the involvement of par activation in mouse colon leukocyte trafficking highlights par as an important mediator of inflammatory cell recruitment and thereby a potential target for the treatment of inflammatory bowel diseases. ( ), kk hansen( ), k chapman( ), n vergnolle ( ), ep diamandis ( ), md hollenberg ( ) ( ) advanced center for detection of cancer, mount sinai hospital, university of toronto, toronto, on, canada ( ) proteinases and inflammation network, university of calgary, calgary, ab, canada kallikreins (klks) are secreted serine proteinases identified in many cancers and multiple sclerosis lesions. we have recently shown that klks can activate proteinaseactivated receptors (pars), a family of g-protein coupled receptors associated with inflammation. we hypothesized that like trypsin, kallikreins can trigger inflammation via the pars. we studied the ability of klks and to activate pars , and in vitro and to cause oedema in a mouse model of paw inflammation in vivo. we found that klk is able to activate both of pars and and to prevent thrombin from activating par . on the other hand, klk was a specific activator of par . kallikrein administration in vivo resulted in a paw oedema response comparable in magnitude and time to that generated by trypsin. the oedema was accompanied by a decreased threshold of mechanical and thermal nociception. our data demonstrate that by activating pars and and by inactivating par , kallikreins, like klks and , may play a role in regulating the inflammatory response and perception of pain. ( ), d park ( ), b short( ), n brouard( ), p simmons( ), s graves ( ), j hamilton ( ) ( ) melbourne university, melbourne, victoria, ( ) peter maccallum cancer institute. melbourne, australia mouse mesenchymal stem cell enriched populations can be isolated from bone tissue by employing lineage immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cyto-kines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. using are-driven and nf-kb-targeted reporter genes, transfection of the nf-kb p subunit and nrf into hepg or other cells, as well as sirna technique to knockdown endogenous p in cells, we found that nf-kb p subunit repressed the anti-inflammatory and anticarcinogenetic nrf -are pathway at transcriptional level. in p -overexpressed cells, the are-dependent expression of heme oxygenase- was strongly repressed. in the cells where nf-kb and nrf were simultaneously activated, p unidirectionally antagonized nrf transcriptional activity. the p -mediated are inhibition was independent of the transcriptional and dna-binding activities of p . co-transfection and rna interference experiments revealed two mechanisms which coordinate the p -mediated repression of are: ( ) p selectively deprives creb binding protein (cbp) from nrf , but not mafk, by competitive interaction with the ch -kix domain of cbp, resulting in inactivation of nrf transactivation domain and concomitant abrogation of the nrf -stimulated coactivator activity of cbp; ( ) p promotes recruitment of histone deacetylases (hdac ) to are by enhancing the interaction of hdac with either cbp or mafk, leading to inactivation of cbp and deacetylation of mafk. this study may establish a novel pro-inflammatory and pro-carcinogenic model for the transrepression of the are-dependent gene expression by p subunit. since various inflammatory and tumor tissues constitutively overexpresses p in their nuclei, the finding in this study implies a strong repression of are-dependent gene expression must take place in those tissues. in this regard, the findings in this study may help to explain why oxidative stresses and toxic insults usually occur in those pathological loci. dendritic cells (dc) play a pivotal role in the induction of immune response and tolerance. it is less known that dc accumulate in atherosclerotic arteries, where they might activate t-cells and contribute to the progression of disease. the serine protease thrombin is the main effector protease of the coagulation cascade. thrombin is also generated at sites of vascular injury and during inflammation. hence, thrombin generation is observed within atherosclerotic and other inflammatory lesions including rheumatoid arthitis. thrombin activates various cells via protease-activated receptors (pars). immature dc do not express pars. upon maturation with lps, tnfalpha, or cd l, only lps-matured dc expressed par and par on their surface. stimulation of dc with thrombin, par -or par -activating peptides elicited actin polymerization and concentration-dependent chemotactic responses in lps-, but not in tnf-alphamatured dc. the thrombin-induced migration was a true chemotaxis as assessed by checkerboard analysis. stimulation of pars with thrombin or respective receptoractivating peptides led to activation of erk / and rho kinase i (rock-i) as well as subsequent phosphorylation of the regulatory myosin light chain (mlc ). the erk / -and rock-i-mediated phosphorylation of mlc was indispensable for the par-mediated chemotaxis as shown by use of pharmacological inhibitors of rock, erk and mlc kinases. in addition, thrombin significantly increased the ability of mature dc to activate proliferation of naive t-lymphocytes in mixed leukocyte reactions. in conclusion, our work demonstrates expression of functionally active thrombin receptors on lps-matured dc. we identified thrombin as a potent chemoattractant for mature dc, acting via rho/ erk-signaling pathways. data concerning the role of circulating modified low density lipoproteins (modldl) in atherogenesis and other pathologies are scarce. one reason for this is the lack of suitable radiolabeling methods for direct assessment of metabolic pathways of modldl in vivo. we report a novel approach for specific labeling of human native ldl (nldl) and modldl (iron-, hypochloriteand myeloperoxidase-oxidized, nitrated, glycated, and homocysteinylated ldl) with the positron emitter fluorine- ( f) by either nh -reactive n-succinimidyl- -[ f]fluorobenzoate or sh-reactive n-[ -( -[ f]fluorobenzylidene)-aminooxyhexyl]maleimide (radiochemical yields, - %; specific radioactivity, - gbq/ mmol). radiolabeling itself caused neither additional oxidative structural modifications of ldl lipids and proteins nor adverse alterations of their biological activity and functionality in vitro. the approach was evaluated with respect to binding and uptake of f-nldl and f-modldl in cells overexpressing various lipoproteinrecognizing receptors. the metabolic fate of f-nldl and f-modldl in vivo was delineated by dynamic small animal pet studies in rats and mice. the in vivo distribution and kinetics of nldl and modldl correlated well with the anatomical localization and functional expression of ldl receptors, scavenger receptors, and receptors for advanced glycation end products. the study shows that ldl modification, depending on type and extent of modification, in part or fully blocks binding to the ldl receptor, and reroutes the modldl to tissuespecific disease associated pathways. in this line, flabeling of modldl and the use of small animal pet provide a valuable tool for imaging and functional characterization of these pathways and specific sites of pathologic processes, including inflammatory processes, in animal models in vivo. the p mapk signaling pathway, which regulates the activity of different transcriptions factors including nuclear factor-Þb (nf-Þb), is activated in lesional psoriatic skin. the purpose of the present study was to investigate the effect of fumaric acid esters on the p mapk and the down stream kinases msk and in cultured human keratinocytes. cell cultures were incubated with dimethylfumarate (dmf), methylhydrogenfumarate (mhf) or fumaric acid (fa) and then stimulated with il- b before kinase activation was determined by western blotting. a significant inhibition of both msk and activations was seen after pre-incubation with dmf and stimulation with il- b whereas mhf and fa had no effect. also, dmf decreased phosphorylation of nf-kb / p (ser ), which is known to be transactivated by msk . furthermore, incubation with dmf before stimulation with il- b resulted in a significant decrease in nf-kb binding to the il- kb and the il- kb binding sites as well as a subsequent decrease in il- and il- mrna expression. our results suggest that dmf specifically inhibits msk and activations and subsequently inhibits nf-kb induced gene-transcriptions which are believed to be important in the pathogenesis of psoriasis. these effects of dmf explain the anti-psoriatic effect of fumaric acid esters. a humanized model of psoriasis was successfully established by transplanting non-lesional skin biopsies from psoriasis patients onto bg-nu-xid mice lacking b, t and nk cells. in this system, a psoriatic process is triggered by intradermal injection of activated autologous peripheral blood lymphocytes. inflammation is associated with the expression of activation markers and inflammatory medi-ators such as tnf-alpha, hla-dr and cd a and this results in increased proliferation and differentiation of keratinocytes, demonstrated by increased expression of ki- and ck- . epidermal hyperplasia is a typical readout in this model. in a series of studies, this model was found to be sensitive too a wide range of compounds, including inhibitors of tnf-alpha, antibodies directed against growth factors, mmp-inhibitors, calcipotriol, metothrexate, betamethasone and cyclosporine a.in addition, we showed that inhibition of fatty acid oxidation had an anti-psoriatic effect in this model (caspary et al. brit j dermatol ; , - ) . employing lesional skin it was demonstrated that inhibition can also be performed in a therapeutic setting.due to its humanized nature this model represents a powerful tool for the identification or validation of compounds with potential for the treatment of psoriasis. kristian otkjaer ( ), e hasselager( ), j clausen( ), l iversen ( ), k kragballe ( ) ( ) aarhus university hospital, denmark ( ) novo nordisk a/s, denmark interleukin- (il- ) is assumed to be a key cytokine in the pathogenesis of psoriasis. increased levels of il- are present in lesional psoriatic skin compared with nonlesional skin where it is barely detectable. whether il- is derived from antigen-presenting cells or keratinocytes remains unsolved. the aim of the present study was, therefore, to characterize il- expression in non-lesional psoriatic skin ex vivo. mm punch biopsies from nonlesional psoriatic skin were collected. biopsies were transferred to cacl enriched keratinocyte basal media and cultured with vehicle or il- beta ( ng/ml) for , , , , , and hours, respectively. the samples were analyzed by in situ hybridisation, qrt-pcr, immunofluorescent staining and elisa. incubation with il- beta rapidly induced il- mrna expression in the biopsies. the highest level of il- mrna was detected after hours and in situ hybridisation revealed that basal as well as suprabasal keratinocytes throughout the epidermis were the only cellular source of il- mrna. increased levels of il- protein were detected in the supernatant of the il- beta stimulated biopsies. immunofluorescent staining of the biopsies showed no il- protein in the keratinocytes, whereas the il- protein was present in epidermal cd a positive dendritic cells. our data emphasize the keratinocyte as the cellular source of il- expression in human skin. interestingly, immunofluorescent staining of our cultured biopsies showed il- protein in epidermal dendritic cells whereas no il- was detected in the keratinocytes. this indicates that epidermal dendritic cells are the target for keratinocytederived il- . one response of epidermal keratinocytes to inflammatory stress is the induction of matrix metalloproteinases (mmps) that participate in tissue remodeling. excessive proteolytic activity is associated with chronic wounds and tissue damage during persistent inflammation. calcitriol, the hormonally active form of vitamin d, is known to have beneficial effects during cutaneous inflammation. we hypothesized that one way in which calcitriol exerts its effect on inflamed skin is by attenuation of damages caused by excessive mmp proteolytic activity. our experimental model consists of hacat keratinocytes cultured with tnf to simulate an inflammatory state. pro-mmp- was quantified by gelatin zymography and mrna by real-time pcr. the levels and activation of signaling proteins were determined by immunoblotting. the increase in pro-mmp- activity and mrna levels induced by tnf was inhibited by~ % following h treatment with calcitriol. using specific inhibitors we established that the induction of mmp- was dependent upon the erk pathway, while p -mapk and pkc inhibited, and jun-kinase, pi- -kinase and src did not affect it. levels of c-fos, a component of ap- transcription complex known to mediate mmp- induction, were elevated by tnf and further increased by calcitriol. the induction of mmp- by tnf was abolished by inhibition of the egfr tyrosine kinase attesting to the requirement for egfr trans-activation. calcitriol also inhibited the induction of mmp- by egf. we conclude that calcitriol inhibits the induction of mmp- gene expression by tnf in keratinocytes by affecting an event downstream to the convergence of the egfr and the tnf signaling pathways. ( ), p verzaal ( ), t lagerweij ( ), c persoon-deen ( ), l havekes ( ), a oranje ( ) ( ) tno pharma, department of inflammatory and degenerative diseases, leiden, the netherlands ( ) erasmus medical center, department dermatology and venereology, rotterdam, the netherlands mice with transgenic overexpression of human apolipoprotein c in liver and skin display a strongly disturbed lipid metabolism. moreover, these mice show a loss of skin-barrier function evident from increased trans epidermal water loss. these mice develop symptoms of atopic dermatitis, i.e. scaling, lichenification, papules, excoriation and pruritus. both hyperplasia of epidermis and dermis are observed. histological analysis shows increased numbers of cd + t cells, eosinophils, mast cells and ige-positive cells in the dermis. serum levels of ige are increased as well. cytokine profiling of draining lymph nodes is in favor of a th -mediated disease. development of atopic dermatis in this model was found to be sensitive to topical treatment with triamcinoloneacetonide, fluticasone-proprionate and tacrolimus. moreover, oral treatment with dexamethasone successfully inhibits the development of disease in this model. impairment of the skin barrier is most likely the underlying cause of the development of atopic dermatitis in this model.this model is useful for identifying new therapeutic strategies and obtaining new insight into the pathogenesis of atopic dermatitis. topical immunosupppressants such as elidel and protopic are highly efficacious therapeutics for the treatment of atopic dermatitis and other dermatological conditions.-when delivered topically, these calcinuerin inhibitors offer several advantages over topical steroids; however, these marketed drugs have received a controversial "black box warning" because of a potential cancer risk. we speculated that systemic exposure of these drugs over long term use may contribute to the cancer risk.accordingly, we have designed and discovered a series of "soft" cyclosporin a (csa) derivatives as potentially safer alternatives.in general, soft drugs are engineered, via medicinal chemistry, to be effective upon local delivery but upon systemic exposure they are rapidly inactivated by metabolic pathways.in this way, exposure of active drug to distal organs is greatly minimized resulting in a significant enhancement in therapeutic index.the results or our drug discovery efforts around soft csa derivatives will be presented. ( ), y sawanobori ( ), u bang-olsen ( ), c vestergaard( ), c grønhøj-larsen ( ) background: a strain of japanese fancy-mice, nc/nga, serves as a model for atopic dermatitis. under specific pathogen-free conditions, the mice remain healthy, but when kept under non-sterile conditions, they exhibit pruritic lesions like atopic dermatitis. scratching behaviour of the mice precedes the development of dermatitis, and a correlation between registered scratching counts and expression of il- mrna has been shown. also, transgenic mice over-expressing il- exhibit increased scratching behaviour and develop severe dermatitis. consequently we decided to explore the therapeutic effect of an anti il- antibody on scratching behaviour and dermatitis in nc/nga mice. methods: prior to clinical manifestation of dermatitis, we commenced treatment of nc/nga mice with il- ratanti-mouse mg/kg intraperitoneally every fifth day for seven weeks. clinical dermatitis, scratching behaviour and weight gain, was assessed throughout the intervention period. serum analysis for ige and il- as well as histopathological and immunohistochemistry analysis on skin biopsies were also performed at end-point. results: taken over the entire intervention period, treatment with anti il- antibody in nc/nga mice from age seven weeks did not meet the primary end points, which were scratch, dermatitis and body weight. however, post hoc analysis revealed a significant reduc-tion of scratch by the anti il- antibody treatment in the time interval day - . our results suggest an anti pruritic role for il- antibody in an atopic dermatitis-like animal model. anti il- antibody is therefore a new therapeutic opportunity for the treatment of pruritus in atopic dermatitis and perhaps other pruritic diseases. ( ), p ferro ( ), hm asnagli ( ), v ardissone ( ), t ruckle ( ), f altruda ( ), ch ladel ( ) ( ) rbm merck serono/university of torino, italy ( ) merck serono pharmaceutical research institute, geneva, switzerland ( ) university of torino, dipartimento di genetica, biologia, biochimica, italy class-i phosphoinositide -kinases (pi ks) play a critical role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells, such as granulocytes and lymphocytes. since pi k-g plays a pivotal role in mediating leukocyte chemotaxis and activation, as well as mast cell degranulation, the pharmacological blockade of pi k-g might offer an innovative rationale-based therapeutic strategy for inflammatory skin disorders. in our study the inhibitory properties of a selective pi k-g inhibitor as on inflammation was applied to murine models modeling skin diseases like psoriasis and dermatitis. two mouse models were used: the first, irritant contact dermatitis (icd), is an innate inflammatory skin condition arising from the release of pro-inflammatory cytokines in response to haptens, usually chemicals. the second, contact hypersensitivity (chs) is a t-cell dependent model, modeling in part t-cell-mediated skin diseases such as psoriasis. we demonstrated the therapeutic effect of pi kg inhibition and subsequent inhibition of chemotaxis in models of skin diseases and showed that a selective pi k-g inhibitor can excert an important therapeutic efficacy (dose-dependent) in models of innate immunity (icd) -effective dose , mg/kg p.o. once -as well as in t-cell mediated skin pathology (chs)effective dose mg/kg p.o. bid. we conclude that the mechanism of action related to inhibition of pi k-g are demonstrable after oral administration of selective inhibitors like as in models of acute and chronic skin inflammation and are mediated by modulation of innate and acquired immunity. introduction: high mobility group box (hmgb ) has recently been identified as a late mediator of endotoxin lethality. we newly developed an extra corporeal hmgb absorber. the purpose of this study was to test the hypothesis that hmgb removal could prevent or reduce endotoxin induced lethality or tissue injury of rats. methods: all experiments were conducted in accordance with the institutional care and use committee.male wistar rats were randomly allocated into three groups; hmgb absorber group (group i),hmgb nonabsorber group (group ii), and vacant column group (group iii).we applied these columns for each groups at hours after lps injection. the rats were sacrificed hours after lps injection for pulmonary histology. we statistically analyzed survival rate with kaplan-meier and the levels of hmgb with anova. results: survival rate was % in the group i at hours after lps injection, as compared with % in the group ii and % in the group iii. the pulmonary histology in both group ii and group iii showed acute inflammatory injuries, whereas group i showed less inflammatory changes.the level of hmgb in the group i was significantly lower than those of group ii and iii. discussions:these results demonstratethat specific absorption of endogenous hmgb therapeutically reverses lethality of established sepsis indicating that hmgb inhibitors and absorber can be treated in a clinically relevant therapeutic window that is significantly wider than for other known cytokines. contact information: dr hideo iwasaka, oita university, anesthesiology and intensive care unit, yufu city, japan e-mail: hiwasaka@med.oita-u.ac.jp ( ) ( ) department of pharmacology, national university of singapore, singapore ( ) dso national laboratories, singapore hydrogen sulfide (h s) is increasingly recognized as a proinflammatory mediator in various inflammatory conditions. in this study, we have investigated the role of h s in regulating expression of some endothelial adhesion molecules and migration of leukocytes to inflamed sites in sepsis. male swiss mice were subjected to cecal ligation and puncture (clp) induced sepsis and treated with saline, dl-propargylglycine (pag, mg/kg i.p.), an inhibitor of h s formation or sodium hydrogen sulfide (nahs) ( mg/kg, i.p.), a h s donor. pag was administered either hour before or hour after induction of sepsis while nahs was given at the time of clp. using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of pag significantly reduced the leukocyte rolling and adherence in mesenteric venules coupled with decreased mrna and protein levels of adhesion molecules (icam- , p-selectin and e-selectin) in lung and liver. in contrast, injection of nahs significantly upregulated leukocyte rolling and attachment as well as tissue levels of adhesion molecules in sepsis. on the other hand, normal mice were given nahs ( mg/kg i.p.) to induce lung inflammation with or without pretreatment of nf-x×b inhibitor, bay - . h s treatment enhanced the pulmonary level of adhesion molecules and neutrophil infiltration in lung. these alterations were reversed by pretreatment with bay - . moreover, expression of cxcr in neutrophils obtained from h s treated mice was significantly upregulated leading to an obvious elevation in mip- directed migration of neutrophils. therefore, h s act as an important endogenous regulator of leukocyte trafficking during inflammatory response. transient receptor potential vanilloid (trpv ) is primarily found on sensory nerves. we have demonstrated its pro-inflammatory potential in arthritis and now present evidence that it is protective in an endotoxininduced model of sepsis. selective trpv antagonists are not available for use in the mouse in vivo, thus established trpv knockout (-/-) mice were used. c bl wt and trpv -/-mice were matched for age and sex and injected intraperitoneally (i.p.) with lipopolysaccharide (lps). the response was monitored for h. blood pressure, measured before and at intervals after lps in conscious mice via a tail cuff was reduced in both wt and trpv -/-mice, with trpv -/-mice showing an enhanced drop at h. in a separate group temperature, a proposed pre-mortality marker was also reduced by h, again with a significantly increased drop in trpv ko. furthermore higher levels of two inflammatory markers tnfa and nitrite (as an indicator of no) were measured in peritoneal lavage and higher levels measured intrpv -/-as compared with wt samples. finally aspartate aminotransferase (ast) levels also enhanced in trpv -/-versus wt mice, although markers for kidney and pancreatic damage were similar in both genotypes. we conclude that trpv plays a protective role in sepsis. trpv is known to be present on nonneuronal (e.g. vascular components) and their relative involvement in sepsis is unknown. ( ), da souza-junior( ), l de paula ( ), mc jamur ( ), c oliver ( ), sg ramos ( ), cl silva ( ), lucia helena faccioli ( ) ( h rv) . infected balb/c mice developed an acute pulmonary inflammation and higher levels of tnf-a, il- , kc, mcp- and mip- were detected in the lungs by day . in vivo degranulation of mast cells by c / led to a reduction of the inflammatory reaction associated to a marked decline proinflammatory cytokine and chemokine levels in the lungs. the magnitude of cellular immune response was also partially impaired in infected mice and treated with c / . histologically, the exacerbated granulomatous inflammation shown in the lung parenchyma of infected mice was attenuated in infected mice and treated with c / . of interest, the number of mycobacterial bacilli recovered from the lungs was log higher after treatment of infected mice with c / . these findings suggest that mcs participate in host defense against m. tuberculosis infection through of the modulation of cytokines and chemokines, which are important for the recruitment and activation of inflammatory cells. ( ), ms chadfield ( ), db sørensen ( ), h offenberg ( ), m bisgaard ( ), he jensen ( ) ( ) department of veterinary pathobiology, faculty of life sciences, university of copenhagen, denmark ( ) novo nordisk a/s, cell biology, gentofte, denmark introduction: pasteurella multocida is an important cause of pneumonia in several animal species and may spread systemically. the aim of this study was to evaluate initial inflammatory reactions and the inocula effect due to strains of p. multocida of different origin in an aerogenous murine model. materials and methods: female balb/ c-j mice (app. g, taconic, denmark) were infected intranasally with two clinical isolates of p. multocida of avian (vp ) and porcine (p ) origin, at three different levels of inocula concentration. after euthanasia, specimens of lung and liver tissue were collected for bacteriological and histopathological evaluation. furthermore, lung tissue samples were taken for measurement of expression of metalloproteinase mmp- and metalloproteinase inhibitor timp- . results: all mice infected with the avian strain were euthanized after hours. viable counts recovered from lung and liver tissue were high, and histopathology revealed pronounced acute bronchopneumonia. in the liver, disseminated necrosis with formation of microabscess was also seen. on the contrary, a dose response was observed with the porcine strain with regard to recovery of viable counts and development of lesions was apparent after , and h. furthermore, differences were seen in the nature of the lesions caused by the two strains. there was a difference in expression of mmp- and timp- between infected and noninfected mice. the model proved suitable for the evaluation of pulmonary inflammatory reactions between the two different host-derived strains as demonstrated through viable counts, histopathology and expression of mmp- and timp- . ( ), r molinaro ( ), a franÅa ( ), m bozza ( ), f cunha( ), s kunkel ( ) ( ) universidade do rio de janeiro, brazil ( ) universidade de s¼o paulo, brazil ( ) university of michigan, usa introduction and objectives: studies reveal that regulatory t (treg) cells control immune responses; therefore these responses must be controlled to enable the effective protection against infections and cancer. ccr knockout mice (ccr -/-) are more resistant to lps shock. so, our aim is to study the mechanisms involved in the resistance of ccr -/-subjected to severe sepsis by cecal ligation and puncture (clp) and how tregs modulate this effect. results: c /bl mice were subjected to clp model, whereby the cecum was partially ligated and puncture nine times with a g needle. sham-operated mice were used as control. mice subjected to clp and sham surgery were treated with antibiotic since h after and until days. ccr -/-mice subjected to clp presented an increase in survival rate ( %) compared with wildtype mice ( %), and a marked improvement in the innate response concern to neutrophil migration to peritoneum and lung, bacteria load and cytokine levels compared to wild-type mice. besides, tregs from ccr -/-clp mice did not inhibit proliferation of t effector cells as observed for treg from wild clp mice, at a proportional ratio of teffector:treg. interesting, treg from ccr -/-clp mice did not inhibit neutrophil migration to bal when co-injected with fungal challenge as secondary infection, while the treg from wild clp mice did, as expected. conclusions: these results suggest that treg cells from ccr -/-mice did not present suppressive response and it could be an important factor in their survival. inflammation and oxidative stress are known to be one of the important causes that are responsible for many diseases. inflammation has been associated with diseases like cancer, diabetes and many other. proinflammatory cytokine (tnf -µ and il- â) and no are considered as pivotal mediators in inflammatory conditions like rheumatoid arthritis, sepsis and cancer etc. thus inhibition of pro-inflammatory cytokines and no production are important targets for treatment of inflammatory disorders. nowadays due to the emerging side effects of cox inhibitors, these targets have been paid more attention for the treatment of these conditions. some medicinal plants such as curcuma longa ( ), commiphora mukul ( ) in humoral memory, antibodies secreted into serum and other body fluids protect an individual against repeated challenges of previously encountered pathogens. antibody-secreting plasmacells are mostly considered to be shortlived, terminally differentiated b lymphocytes, eliminated after a few days or weeks by apoptosis. however, in secondary lymphoid organs and in the bone marrow, plasma cells can survive for months and years, without dna-synthesis and refractory to signals from antigen or antigen-antibody complexes. the lifetime of these longlived plasmacells depends on an intrinsic competence to survive in the distinct environment of those organs, which defines a specific survival niche. the niche provides survival signals like il- , cxcl and tnf. within a functional niche, the lifetime of a plasma cell is apparently not limited intrinsically. the number of niches in the body has to be limited, in order to maintain physiological concentrations of serum immunoglobulins. thus recruitment of new plasmacells to the pool of old memory plasma cells has to be competetive. this competition is probably controlled by a simple molecular mechanism, namely the dual functionality of chemokines like cxcl , which attract newly generated plasmablasts to a survival niche and at the same time are a survival signal for the plasma cell. plasmablasts and plasma cells express cxcr , the receptor for cxcl . while plasmablasts migrate in response to cxcl , plasma cells depend on it for survival in the niche, but are no longer migratory. thus once disloged from their niche, they will die. plasmablasts newly generated upon systemic secondary immunization, upon concommittant stimulation with interferon-gamma, can also express cxcr , the receptor for the interferon-gamma-induced chemokines cxcl , and , which may lure the plasmablasts into inflamed tissue. the switch in the potential to migrate provides also an efficient means to eliminate plasma cells of the peak of an immune response, which as plasmablasts had migrated to the tissue inflamed in that pathogenic challenge. inflamed tissue contains survival niches for plasma cells. in the inflamed tissue, plasma cells provide high local antibody concentrations while the tissue is inflamed. upon resolution of the inflammation the plasma cells will be dislodged and die. longlived plasma cells provide longlasting antibody titers (protective memory) and leave memory b cells a role in reactive memory, generating memoryplasmacells in secondary challenges, and if serum titers are not sufficient to protect. in chronic inflammation, this mechanism can contribute to pathogenesis. thus in the nzb/w model of lupus, longlived autoreactive plasma cells are generated early in pathogenesis, which survive in bone marrow and spleen. later, in established disease, autoreactive plasma cells are shortlived and continuously generated. they do not compete with the longlived plasma cells, and both populations coexist as prominent populations. interestingly, longlived plasmacells are resistant to therapeutic immunosuppression, while the generation of shortlived plasma cells is blocked. this may be the reason for the failure to cure antibodymediated immunopathology, e.g. in autoimmunity and allergy, by conventional immunosuppression, ( ), h lee ( ) c a is a potent inflammatory mediator produced during complement activation. unregulated c a signalling through its receptor (c ar) on neutrophils and other leukocytes is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. considerable effort has gone into development of c ar antagonists for human therapy. we took neutrophils from genetically modified human c ar knock-in mice in which the mouse c ar coding region was replaced with human c ar sequences and immunized wild-type mice to generate high affinity antagonist monoclonal antibodies (mabs) to human c ar. these mabs inhibit c a-induced neutrophil migration and calcium-flux, and bind to a region of the nd extracellular loop of c ar loop that seems to be critical for receptor activity. this study investigated the effectiveness of these mabs in the k/bxn serum-transfer model of inflammatory arthritis. human c ar knock-in mice were given - mg/kg mab intraperitoneally, before or after inflammatory arthritis developed. mice treated with anti-c ar mab one day before serum transfer did not develop swelling or clinical signs of arthritis in contrast to controls. histopathology of the joints in anti-c ar mab-treated mice revealed a complete block of the massive influx of leukocytes and cartilage erosion seen in controls. furthermore, and most significantly, a single mg/kg dose of anti-c ar mab given days after initiation of disease completely reversed inflammation. in the collagen-induced arthritis (cia) model, injection of anti-c ar mab after development of inflammation also reversed inflammation to baseline. these potent new antibodies to human c ar are in preclinical development. the cytokine macrophage migration inhibitory factor (mif) participates in fundamental events in innate and adaptive immunity. the profile of activities of mif in vivo and in vitro is strongly suggestive of a role for mif in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (ra), asthma, and sepsis. mif also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of mif is in fact induced by glucocorticoids. thus, mif functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. therapeutic mif antagonist may therefore provide a specific means of steroid sparing. since mif are highly conserved among different species, it is hard to develop high affinity antibodies due to immune tolerance.we developed a proprietary technique to break the immune tolerance and selected high affinity mouse monoclonal antibodies against mif.the antibody can neutralize mif activity in cell based assays, and is very effective in a lps induced mouse sepsis model. using this antibody as a tool, we are studying the function of mif in comparison with the function of lps.we found that lps induced inos expression and no secretion are dependent on the secretion of mif.we also found that although both lps and mif induce g arrest in macrophage cell line raw . , their functions are independent to each other. structure-based small molecule drug design. an effective agent would be the first orally active cytokine antagonist. methods: collagen-induced arthritis (cia) was induced in dba- mice by immunisation with bovine type ii collagen/adjuvant on day and . cor , synthesized on the basis of computer modeling of mif protein x-ray crystallographic data, was administered by daily oral gavage from day . etanercept ( mg/kg ip q d) was used as a positive control. the mek-erk pathway is activated in numerous inflammatory conditions, including ra, ibd and copd. arry- is a potent (ic = nm), selective, atp-uncompetitive mek / inhibitor.arry- is highly efficacious in cia and aia rat models, with ed s of and mg/kg, respectively, equal to or better than standard agents. addition of arry- to methotrexate, etanercept, ibuprofen or dexamethasone regimens in these models results in improved efficacy that is at least additive, if not synergistic. in tpa-stimulated human whole blood, this compound inhibited tnf, il- and il- production (ic s of , and nm, respectively). in contrast, inhibition of perk required nm to achieve a % reduction, demonstrating that inhibition of pro-inflammatory processes is very sensitive to perk inhibition.in clinical studies, healthy volunteers were administered a single oral dose of , , , or mg); blood was drawn at various times after dosing and stimulated ex vivo with tpa. arry- was well-tolerated and drug exposure was dose-proportional. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpainduced il and tnffz with > % inhibition observed at plasma concentrations of and ng/ml, respectively. similar inhibition of perk required ng/ml. a multiple ascending dose clinical study has confirmed the pharmacokinetics and pharmacodynamics of arry- and helped define tolerability. clinical evaluation of arry- in combination with methotrexate in patients with rheumatoid arthritis is on-going. p (a mitogen-activated protein kinase) has been shown to play a key role in the release of cytokines such as tnfand il- a from monocytes in signaling cascades that are initiated due to extra cellular stress stimuli. inhibition of p activity is expected to regulate the levels of tnf-a and il- b thereby alleviating the effects of inflammation in ra. a new class of p inhibitors based on the naphthyridininone scaffold have been discovered. x-ray crystallography and site directed mutagenesis studies were critical tools that aided the evolution of the naphthyridinone lead class starting from a pyrido-pyrimidinone template. this presentation will discuss the derivation of key benchmark pre-clinical candidates in these novel scaffold classes (shown below) as influenced by structural biology studies, mutagenesis data and molecular modelling. efficacy studies in animal models for benchmark compounds will also be presented. ( ), h aaes ( ), w-h boehncke( ), j pfeffer( ), t skak-nielsen( ), i teige ( ), k abell ( ), ph kvist ( ), e ottosen ( ), tk petersen ( ), lars svensson ( ) ( ) discovery, leo pharma, industriparken, ballerup, denmark ( ) department of dermatology, johann wolfgang goethe-university, frankfurt am main, germany p map kinase plays an important role in mediating an inflammatory response in mammalian cells. as a consequence of activation, several inflammatory mediators are released including il- b] and tnfa. both cytokines have a central role in the pathogenesis of inflammatory conditions such as psoriasis. approximately % of psoriasis patients develop psoriatic arthritis. leo is a member of newly developed class of selective p map kinase inhibitors. the compound was tested orally in in vivo models relevant for psoriasis and psoriatic arthritis. the in vivo models selected include the cia arthritis model, the human psoriasis xenograft scid mouse model, the uvb-induced dermatitis model, the lps induced tnfa model and a local gvh model. treatment with leo led to an amelioration of the ongoing inflammation in all investigated in vivo models. in the cia model, a clear dose response effect was observed on the developing arthritis in both rats and mice ( % reduction in mice and % in rats at mg/kg p.o.). in the humanised psoriasis model, leo at a dose of mg/kg, had an effect on both the hyperplastic epidermis (epidermal thickness reduced by %) and on the infiltrating inflammatory cells. the anti-inflammatory effect of leo was even close to the effect of systemically delivered steroids in both models. we believe that the new highly selective class of p map kinase inhibitors has a strong potential as an orally delivered therapy for systemic inflammation diseases such as psoriasis and arthritis. slx- is a potent, selective, orally bioavailable inhibitor of the rho-kinase rock- . its ic for rock- and rock- inhibition is nm and > mm respectively. the ability of slx- to inhibit septic liver injury was investigated in c bl/ j mice challenged with lipopolysaccharide (lps) and d-galactosamine (d-gal). mice were given lps ( mg/mouse) and d-gal ( mg/ mouse) i.p and . hours later were sacrificed for analysis of liver injury. mice challenged with lps/d-gal had a > fold increase in serum alt and ast levels. this increase was reduced by > % in mice pretreated with slx- either orally ( mg/kg, and hrs prechallenge) or i.p. ( - mg/kg, min pre-challenge). slx- inhibited the increase in hepatic levels of tnffalpha produced by lps/d-gal by > %. to assess the kinetics of slx- s benefit, slx- ( mg/kg i.p.) was given min prior to, or or min after the lps/ d-gal. slx- was effective at inhibiting the rise in alt and ast levels at all time points suggesting that inhibiting rock- even after the initiation of the lps/d-gal driven cascade protects against septic liver injury. in a survival study, out of mice given lps/d-gal were dead by hrs whereas in mice given slx- ( mg/kg orally) animal died at hours and the remaining mice were alive hrs later. these results show that specific inhibitors of rock- may have therapeutic utility in the treatment of sepsis and subsequent liver injury. theta through three key hydrogen bond mediated interactions.they potently inhibit protein kinase c activity in vitro as demonstrated by inhibition of il- secretion in human purified t-cells stimulated with anti-cd and anti-cd or whole blood seb challenge.the pkc-theta inhibitors are orally bioavailable and demonstrate immunosuppressive activity in a mouse model of human delayed-type hypersensitivity responses. ( ), j zhang ( ), k henley ( ), m white ( ), d hilton ( ), b kile ( ) ( to investigate pathogenesis of rheumatoid arthritis, we used mice transgenic for the uniquely human fcgam-mariia in inducible and passively transferred models of arthritis. transgenic mice developed severe ra-like disease in both model systems, indicating that the transgene played a major role in arthritis pathogenesis. disease could be reduced by the administration of either specific monoclonal antibodies to fcgammariia or small chemical entities (sce) designed to bind to the fcgam-mariia dimer. to investigate the cause of this enhanced sensitivity to auto-immune stimuli, the phagocytic capacity of transgenic mice compared to c bl/ control mice was examined using phagocytosis of fluorescent beads coated with ova or ova/anti-ova immune complex or opsonised sheep red blood cells. in both assays macrophages from fcgammariia transgenic mice showed significantly increased phagocytosis comapred with cells from control mice at hours.this difference diminished over time andwas only seen where particles were opsonised. treatment of macrophages with specific fcgammariia blocking monoclonal antibody fragments . f(ab) or iv. f(ab) reduced phagocytosis to background levels . macrophages from transgenic mice also showed significantly greater production of inflammatory cytokines tnf-alpha and il- beta when stimulated in vitro with heat aggregated immunoglobulin (hagg). moreover, this response was also blocked by specific fcgammariia monoclonal antibody fragments or sce. thus, expression of the fcgammariia transgene in these mice leads to increased uptake of and reactivity to immune complexes, resulting in enhancement of inflammatory sequelae in the form of increased th cytokine secretion andamplification of the pro-inflammatory response leading to arthritic disease. harald burkhardt ( ), u hüffmeier ( ), i kçnig ( ), j lacorz ( ), a reis ( ), k reich ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander-unisversity of erlangen-nuremberg, germany results: whereas the earlier described strong association of allele tnf*- a with psoriasis could be confirmed, our study revealed that this association was completely dependent on carrying the psors risk allele. for psa, but not psoriasis vulgaris without joint involvement strong association with the allele tnf*- t was detected (or= . , % ci . - . ; pcorr= . ) also in patients negative for the psors risk allele. our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. while the previously reported association between tnf*- a and psoriasis seems to primarily reflect ld with psors , tnf*- t may represent a risk factor for psa independent of psors . ( ), n modi ( ), m stanford ( ), e kondeatis ( ), r vaughan ( ), f fortune ( ), w madanat ( ), c kanawati( ), p murray ( ) methods: dna was obtained from patients with bd, from the uk and from the middle east (me), and controls individuals, from the uk and from me. dna was prepared by proteinase k digestion, and salt extraction and - and snp were detected by a pcr-ssp. results: there was no significant difference in expression of - c/t or a/g when all bd patients were compared to all control individuals, (p= . and . , respectively). as we have previously shown differences in snp expression in different patient groups we tested the uk and me patients separately. however, there was no significant difference in either snp in uk bd patients, (p= . , p= . ) or me bd patients (p= . , p= . ) when compared to the appropriate controls. ( ), da brown ( ), h johnen ( ), mr qiu ( ), t kuffner ( ), pgm curmi ( ), l brown ( ), m mazzanti ( ), sn breit ( ) ( introduction: cerebral palsy (cp) is a nonprogressive motor disorder caused by white matter damage in the developing brain. it is often accompanied with neurocognitive and sensory disabilities. the cause and pathogenesis of cp is multifactorial and continues to be poorly understood. chorioamnionitis, clinically silent or manifest, has been reported to be a risk factor for cp both in term and preterm infants. il- gene is single copy gene located on chromosome q . - in humans. interleukin- is synthesized by a variety of immune (t cells, eosinophils and dendritic cells) and non-immune (fibroblasts, epithelial and neuronal) cells. it is also detected in organ-specific secretions in a number of inflammatory processes. amniotic fluid interleukin- concentrations decreased with advancing gestational age. but, women with preterm labor and women with chorioamnionitis have higher interleukin amniotic fluid concentrations than those who delivered at term or those with sterile amniotic fluid. the aim of our study was to estimate allelic frequency for regulatory il - snp in the children with the cp. methods: dnas obtained from peripheral blood of cp patients and unrelated healthy volunteers were genotyped for the il - snp pcr-rflp method. results and conclusions: il - genotype fncc was more common in the population with cerebral palsy in the comparison to healthy volunteers. the significance of the association between il fngene polymorphisms and cerebral palsy has to be investigated in the future studies. ( ), d delbro ( ), e hansson ( ) ( ) kalmar university, sweden ( ) gçteborg university, sweden background: acetylcholine (ach) is a major signalling molecule, binding partly at nicotinic receptors (nachrs; a family of ions channels with nicotine as a selective ligand). one subtype, the alpha nachrs, has antiinflammatory effects by way of down-regulation of tnfalpha release from macrophages. the alpha nachrs have been demonstrated in neuronal as well nonneuronal tissues, e.g. astrocytes and microglia. aim. in rat astrocytes in primary culture, and in astrocytes cocultivated with primary microvessel cultures study: . the expression of alpha nachrs and alpha nachrs by immunofluorescence and western blot. . intracellular ca +-transients spread within the astroglial networks after stimulation with nicotine. . pro-inflammatory cytokines, il- b, il- , and tnfalpha, released from microglial cells after stimulation with nicotine. results: alpha nachrs expression was more evident in astrocytes co-cultivated with endothelial cells, suggesting that endothelial cells release factors, which increase the maturity of astrocytes. the ca + transient evoked by nicotine was also more pronounced in the co-cultured astrocytes. these ca + responses were blocked by the alpha nachr antagonist alpha-bungarotoxin. the release of pro-inflammatory cytokines was down-regulated after stimulation by nicotine. conclusion: alpha nachrs appear to be involved in some of the effects of nicotine administration to rat astrocytes. an anti-inflammatory action of cholinergic nerves on the astrocytes via nachrs seems probable, which may have therapeutic implications. university, department of natural sciences, kalmar, sweden e-mail: ann.pettersson@hik.se gedeon richter plc., budapest, hungary tramadol, an atypical opioid analgesic, is increasingly used for the treatment of osteoarthritis because it does not produce typical side effects of nsaids. review of clinical data show that it produces symptom relief and improves function, but these benefits are small and adverse events often cause participants to stop taking the medication (cohran database ). efficacy of tramadol in animal models of inflammatory pain is well established; however complex characterization of the drug regarding analgesic and side effects is missing in rats. our aim was to assess oral efficacy of tramadol at side effect free doses in rats. anti-hyperalgesic effect was determined in complete freunds adjuvant (cfa) induced thermal hyperalgesia test (th) and in model of cfa-induced knee joint arthritis. effect on inflammation was characterized in carrageenan induced paw edema test (et). for the characterization of side effect accelerating rotarod assay (arr) was used. significant impairment in arr was noted at mg/kg dose, and above. in the th test weak % effect was seen at side effect free dose ( mg/kg). in the arthritis model b.i.d. mg/kg dose of tramadol given on days - after cfa caused a maximum % effect on weight bearing incapacitance (day ). however, its effect seemed to diminish ( % on day ) upon repeated treatment. in et tramadol had significant anti-inflammatory effect at but not at mg/kg dose. these results show that efficacy of tramadol in rat inflammatory pain models is limited by its side effects, in accordance with clinical data. chronic relapsing experimental allergic encephalomyelitis (cr eae) is a disease that bears striking similarities to the human condition multiple sclerosis (ms). in particular, cr eae and ms have major inflammatory events in the central nervous system (cns) that culminate in demyelination and disruption of axonal function.one group of mediators involved in the progressive cns inflammation are the prostaglandins (pgs).pg generation is regulated in experimental non-immune conditions of the cns by n-methyl-d-aspartate (nmda) receptor activation.nmda receptor-mediated events are also evident in eae and may therefore have the potential to influence pg production.the study was designed to profile pge and pgd in cns tissues during the development of cr eae and to examine the role of the nmda receptor in pg production through the use of the specific antagonist mk- .biozzi mice were inoculated for cr eae and cns tissues were sampled during the course of disease.enzyme immunoassay of processed samples revealed pge and pgd levels within normal limits during the acute and subsequent remission phases of cr eae.in contrast, dramatic changes in pg concentrations were observed with a relapse of symptoms and a remission of disease.mk- was therapeutically administered to cr eae-diseased mice at the onset of relapse and changes in cns pg levels were recorded.the relapse phase of cr eae, but not the acute stage of disease, is characterised by an increase in cns pg production that may be influenced by nmda receptor activation. livia l camargo( ), lm yshii ( ) ( ), sk costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college, london, uk ( ) butantan institute, s¼o paulo, brazil objectives: the neuropeptide substance p (sp) released by capsaicin-sensitive nerves (csn) plays a pivotal role in neurogenic inflammation. despite the prevalence of arthritis, the contribution of sp to the progression of arthritis has not been established. this study investigated the effect of csn ablation and sr , a sp antagonist, on knee joint inflammation and pain induced by intraarticular (i.a.) injection of kaolin ( %, h time course) in female wistar rats. the kaolin-injected knee (ipsilateral -ipsi) of vehicle-treated rats exhibited a significant, timedependent oedema as compared to the contralateral knee. in addition, increased pain score and high levels of myeloperoxidase (mpo, marker of neutrophil accumulation) and both pro-inflammatory (il- b and il- , but not tnfa) and anti-inflammatory (il- ) cytokines was detected in the ipsi synovial fluid of these animals. both destruction of knee joint csn fibres by neonatal capsaicin treatment and i.a. injection of rats with sr ( nmol/cavity) significantly attenuated the kaolin-induced pain score and knee oedema, suggesting that kaolin is acting, at least partially, via a neuronal mechanism. in contrast, the same treatment caused increased mpo activity and cytokine concentrations measured h post kaolin injection. conclusions: peripheral release of sp after kaolin injection acts to increase pain generation, oedema formation and inflammatory cell influx. however, chronic tachykininergic depletion by capsaicin treatment up-regulates the production of pro-inflammatory cytokines that are important in triggering cell influx in the synovial cavity. ( ) ( ) university of s¼o paulo, s¼o paulo, brazil ( ) butantan institute, s¼o paulo, brazil objectives: previously, intra-tracheal (i.tr.) injection of dep or , -naphthoquinone ( , -nq) evoked plasma extravasation and cell influx in rat airways. we now investigated whether simultaneous injection of these pollutants had a synergistic inflammatory action. we also determined the ability of dep and , -nq-induced airway inflammation to evoke changes in the rat isolated thoracic aorta (rta) and corpus cavernosum (rcc) reactivity using organ bath assays. results: capsaicin-or vehicle-treated male wistar rats received i.tr. injection of dep ( mg/kg) and , -nq ( nmol/kg). after min, dep and , -nq produced a potent (additive) plasma extravasation in the trachea and bronchi, but not lung, compared with each compound alone. in capsaicin-treated rats or treated with tachykinin antagonists, the response was inhibited, suggesting an important role for c-fibres, primarily tachykinins. increased mpo and cytokine levels were detected in bronchi of capsaicin-treated rats following h treatment with pollutants. this treatment contributes to augment the ach ( - - - m)-induced relaxation in the rta but not in the rcc. in capsaicin-treated rats, the rta response to the pollutants was not affected but it was capable of evoking a marked relaxation in rcc in animals challenged with pollutants. conclusions: , -nq exacerbates dep-induced plasma extravasation and mpo activity in the airways, indicating a neurogenic mechanism through tachykinins. the exposure to dep and , -nq affects the endotheliumdependent response in rta without interfering with rcc. neuropeptides are unlikely to affect the pollutantsinduced changes in the rta. acknowledgements: capes, cnpq, fapesp. we thank ma alves for technical assistance. trans-resveratrol (rv) is a naturally occurring polyphenolic compound present in certain foods that has anticancer and anti-inflammatory properties. the purpose of this study was to determine the effect of rv on the production of proinflammatory cytokines and reactive oxygen species stimulated by lipopolysaccharides (lps) in glial cells. rt-pcr showed that rv ( , , mm) dose-dependently inhibited ng/ml lps induced tnfa, il- b, il- , mcp- and inducible nitric oxide synthase mrna expression. rv also inhibited lps-induced production of these cytokines (elisa), nitric oxide and reactive oxygen species in a dose-dependent manner. western blot analyses showed that resveratrol could inhibit lps-stimulated phosphorylation of erk / and jnk but not p . nf-kb reporter assay showed rv could inhibit nf-kb activation by lps in microglia and astrocytes. these results suggest that rv may inhibit lps-induced microglial and astrocyte activation through erk / , jnk and nf-kb signaling pathways. therefore, rv is a natural product with therapeutical potential against disease conditions in cns that involve an overproduction of proinflammatory cytokines and reactive oxygen species. ( ), cs patil( ), sv padi ( ), vp singh ( ) ( ) university institute of pharmaceutical sciences, panjab university, chandigarh, india ( ) pharmacology r & d, panacea biotec ltd. lalru, punjab, india persistent stimulation of nociceptors and c-fibers by tissue injury causes hyperalgesia and allodynia by sensitization of nociceptors and facilitation of synaptic transmission in the spinal cord. the important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (no). the aim of the present study was to test the sensitivity of pde inhibitor sildenafil in chronic constriction injury (cci) model, a rat model of neuropathic pain. sciatic nerve injury is associated with development of hyperalgesia days after the nerve ligation. sildenafil ( and fÝ g/rat, i.t.) produced a significant decrease in pain threshold, which in lower dose did not alter the nociceptive threshold. the hyperalgesic effect of sildenafil was blocked by l-name and methylene blue (mb), which on per se treatment showed antinociceptive effect in nerve ligated rats. the results from the present study indicated that the major activation of no cgmp pathway in the chronic constriction injury model of neuropathic pain. the aggravation of hyperalgesic response might be due to the increased cgmp levels resulting in pkg-i activation and its upregulation. glycine transporter (glyt) and glyt are expressed in glia and neurons, respectively. glyt make clearance of glycine released from glycinergic neuron in synapse and thus terminates the neurotransmission and also regulates over-stimulation by glycine spilled over to nmda receptors, while glyt supplies glycine into synaptic vesicles in glycinergic neurons. therefore, glyt inhibitors could modulate inhibitory glycinergic or excitatory glutamatergic neurotransmission. the present study examined the effects of glyt inhibitors on pain in animal models. inhibitors of glyt (sarcosine and org ) and glyt (alx and org ) by intrathecal (i.t.) injection reduced formalin-induced nociceptive behaviors. glyt inhibitors reduced allodynia score and reversed the reduction of paw withdrawal threshold in complete freund adjuvant (cfa)-induced inflammation mice but the antiallodynia effects appeared after latent period. on the other hand, glyt inhibitors produced antiallodynia effects immediately after the i.t. injection in cfa-treated mice. these inhibitors produced the similar antiallodynia effects in the partial sciatic nerve ligationinjury or streptozotocin-induced diabetic neuropathic pain models, either by i.t. injection or i.v. injection.pretreatment of specific antagonists of glycine site of the nmda receptors disappeared the latent periods of glyt inhibitors and potentiated the antiallodynia effect. glycine receptor antagonist, strychnine by i.t. injected reversed the antiallodynia effect of i.v. injected glyt inhibitors. these results suggest that both glyt and glyt inhibitors by enforcing glycinergic inhibitory neurotransmission in spinal cord produce potent antinociceptive effect and may be novel candidate for medicament of pain control. the tumour microenvironment in particular tumour associated macrophages (tams) play a role in determining tumour outcome. despite strong causative links between inflammation and human gastric cancer progression, little is known of the role of tams in this disease. we have utilized our mouse model of gastric tumourigenesis, the gp ff mouse to assess the effect of the gp ff mutation on macrophage function and ascertain the role of macrophages in tumor formation. this mouse has a knockin mutation in the il- family cytokine receptor gp preventing shp /ras/erk signaling and leading to constitutive stat transcriptional activation. tumour development is inflammation dependent, there is a requirement for il- , and development is inhibited by nsaid treatment or microbial eradication, however an adaptive immune response is s inflamm. res., supplement ( ) posters dispensable for tumorigenesis. in gastric antrum the gp ff mutation results, decreased erk / activation and constitutive phosphorylation of stat , abnormal signaling is replicated in macrophages. antral stat activation is unaffected by depletion of il- . however in macrophages an absence of il- results in higher stat activation demonstrating the anti-stimulatory role of il- on macrophages. the gp ff mutation results in macrophages with decreased il- and increased inos mrna expression reflecting a more basally activated phenotype. manipulations of the gp ff mouse that reduce tumor size (eg. antibiotic or nsaid treatment or stat hemizygosity) coincidently result in reduced macrophage infiltration of antral mucosa. macrophages of gp ff mice display aberrant gp signaling potentially resulting in exaggerated response to stimuli. the key difference between mutant gastric mucosa and macrophages are changes in transcription of target genes. ( ), y riffo-vasquez( ), s brain( ), s costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college london, uk objectives: we have previously shown that simultaneous intra-tracheal injection of diesel exhaust particles (dep) and , -naphthoquinone ( , -nq) caused a potent inflammation in rat airways partially dependent on neurogenic-mediated mechanism. this study investigates the mechanism of action of thee pollutants using inflammatory assays and histopathological approach in the lung and trachea of wild type (wt) and trpv knockout (ko) mice exposed to dep and , -nq. dep ( mg/kg) and , -nq ( nmol/kg)-induced airway inflammation was assessed via i-labelled albumin h post pollutants injection. mpo assay and histopathology were performed h after treatment. staining of lung and trachea specimens with h&e provided a profile of cell infiltration in both wt and ko animals. results: injection of dep and , -nq evoked a potent plasma extravasation into the trachea and lung of wt, but not trpv ko, suggesting these pollutants act via a trpv -mediated mechanism. in contrast, mpo activity in the airways of trpv ko mice was exacerbated compared to wt mice data. likewise, the histopathology revealed high numbers of leukocytes and macrophages infiltrated into the lungs and trachea of trpv ko mice compared to wt. conclusions: inhibition of increased microvascular permeability in the airways of trpv ko mice treated with pollutants suggests that these receptors are the predominant mechanism involved in the inflammation. however, neutrophils/macrophages accumulate more in trpv ko, indicating that the lack of trpv receptors up-regulates production of inflammatory cells in response to pollutants, thus supporting a protective role for trpv receptors. acknowledgements: capes, cnpq, fapesp. ( ), n sato( , ), y endo ( ), s sugawara ( ) ( ) division of oral immunology, department of oral biology, tohoku university graduate school of dentistry, sendai, japan ( ) division of fixed prosthodontics, department of restorative dentistry, tohoku university graduate school of dentistry, sendai, japan biotin is a water-soluble vitamin of the b complex and functions as a cofactor of carboxylases.biotin deficiency causes alopecia and scaly erythematous dermatitis.moreover, serum biotin levels are significantly lower in atopic dermatitis patients than in healthy subjects, indicating that biotin deficiency is involved in inflammatory diseases.however, immunological effects of biotin on allergic inflammation remain unclear.in this study, we investigated the effects of biotin-deficiency on metal allergy using nickel (ni)-allergy model mouse and a murine macrophage cell line j . . female balb/c mice ( weeks old) received a basal diet or a biotin-deficient diet for weeks.ten days after sensitization with intraperitoneal injection of lps and nicl , the mice were challenged with intradermal injection of nicl into the pinnas.allergic inflammation was measured by ear swelling.the ethical board for nonhuman species of the tohoku university graduate school of medicine approved the experimental procedure followed in this study.j . cells were cultured in biotin-sufficient or deficient medium for weeks. ear swelling was significantly higher in biotin-deficient mice than biotin-sufficient mice.il- beta productions by splenocytes were significantly higher in biotin-deficient mice than in biotinsufficient mice.moreover, biotin-deficient j . cells produced il- beta significantly higher than biotinsufficient j . cells.to investigate the therapeutic effects of biotin-supplementation, biotin-deficient mice received biotin contained-water for weeks.ear swelling was significantly lower in biotin-supplement mice than biotin-deficient mice.these results indicated that biotindeficiency deteriorates allergic inflammation.the augmentation of il- beta production is probably involved in those deteriorations. one of the most important targets of cytokine action is the blood vessels, which undergo some structural and functional changes that result in activation of endothelium. applying the elisa technique, levels of il- , icam- and e-selectin were studied in patients with acute pancreatitis. mediators levels were studied in arterial, venous and pancreatic ascites samples. according the atlanta criterion the mild pancreatitis was established in patients and severe -in patients. the highest levels of il- were noted in ascites and lowest in arterial samples. the highest concentration of adhesion molecules was in venous samples and lowest in ascites. it was a clear correlation between levels of il- and adhesion molecules and severity of pancreatitis. during first week the levels of il- gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased starting from the third day. icam- levels gradually increased during first three day with the following decrease after this term. the highest levels of e-selectin were noted at the time of admission. the clear correlation between il- and adhesion molecules was noted in both groups of patients. besides that, the clear strong correlation was observed between il- and quantity of circulating granulocytes and between e-selectin and hematocrite in patients with necrotizing pancreatitis. our study confirms the importance of activation of endothelium as a part of the systemic inflammatory response in patients with acute pancreatitis. subsequently eos infiltrate the tissues, are activated, and release mediators inducing the late phase response. this is characterized by tissue damage and repair, and in chronic reactions, tissue remodeling and fibrosis. mc/eos cross-talk by physical and non-physical contact is an essential feature of late-phase and chronic allergic reactions. we have previously described an mc/eos interaction facilitated by soluble mediators and shown to enhance allergic inflammation. still, pathways that mediate mc/eos cross-talk in allergy are not fully characterized. methods: human cord-blood derived mc were cocultured with peripheral blood eos, and activated with anti-ige. mc/eos couples in co-culture and in human nasal polyp tissue sections were specifically stained and counted using microscopy. expression of surface molecules was analyzed by facs. mc activation was measured by chromogenic assays for â-hexosaminidase. relevant surface molecules were neutralized using antibodies, to assess interference with couple formation and activation. results: mc and eos physically interact, forming welldefined couples in-vitro and in-vivo. in the presence of eos, mc are more releasable under baseline or igeactivating conditions. this effect is partially mediated by cd and dnam- on mc, and b and nectin- on eos. we describe a novel physical interaction between mc and eos that we name "the allergic synapse". this synapse may upregulate allergic reactions, thus serving as a target for therapeutic intervention in allergic and inflammatory diseases. methods: mc were obtained from cord blood mononuclear cells ( - weeks with scf, interleukin- and prostaglandin e , cbmc). cbmc were activated, after their culture for days with myeloma ige ( mg/ml), by rabbit anti-human ige antibodies ( mg/ml), for , and h at c. activation was measured by b-hexosaminidase release, determined by enzymatic-colorimetric assay. the expression of flip, mcl- , bcl- , bcl-xl, bak and bax was assessed by immunoblot analysis. results: two anti-apoptotic proteins were found to be upregulated: flip, which is involved in the extrinsic apoptotic pathway and mcl- that is mainly implicated in the intrinsic apoptotic pathway. in contrast, the expression of two other anti-apoptotic proteins that we have examined (i.e. bcl- and bcl-xl) was not altered. likewise, the expression of pro-apoptotic proteins from the bcl- family (i.e. bak and bax) was either undetectable or unchanged. conclusions: our findings reveal that ige-dependent activation of human mc mainly induces the selective increase in the expression of two pro-survival molecules. this may be one of the mechanisms that underlay mc hyperplasia in the chronic allergic inflammation. ( ), c armishaw( ), z yang ( ), h cai ( ), p alewood( ), c geczy ( ) ( ) university of new south wales, faculty of medicne, sydney, australia ( ) university of queensland, institute for molecular biosciences, st lucia, australia purpose: human s a , s a and s a are closely related proteins associated with inflammation. s a is expressed in the human genome, but not in the mouse. s a is a potent monocyte chemoattractant and mast cell (mc) activator. mouse (m) s a and human (h) s a share % structural identity, but the hinge domains are more divergent. the ms a hinge (ms a - ) is a potent chemoattractant for leukocytes whereas this sequence in hs a (hs a - ) is inactive. methods: s a hinge domain and its alamine scan mutants were synthesized and their activities were tested by using thp cells or murine mc in vitro and mouse footpad injection. results: s a hinge domain (s a - ) was chemotactic for monocytes and mc and provoked mc degranulation in vitro and oedema, and leukocyte recruitment in vivo. in contrast to s a , the hinge domain only weakly induced cytokine production (il , il ) by macrophages. residues essential for oedema were hydrophobic in nature (leu , isoleu , isoleu and isoleu ). n and i were essential for responses provoked by - m s a - whereas mutation of k , l , n , i , d , k and i significantly reduced migration with - m s a - . conclusions: s a and ms a may be functional chemattractant equivalents; s a may have arisen by duplication of human s a . isoleucine residues in s a hinge domain are essential for its proinflammatory properties. ( ), g kiriakopoulou ( ), e tsimara( ), a voultsou( ), k zarkadis ( ) ( ) general hospital of zakynthos, greece ( ) medical centre of katastari, zakynthos, greece schistosome is a disease which pests in tropical countries. the endemic areas are south america, far and middle east, and africa. our aim is to present an incident which concerning a parasitic infection, not endemic in greece. patient: man years old who immigrated from pakistan (at the side of the aparkenar river) in greece, a year ago. he turned out with anlage, headache and weight loss. he is a swimmer. he mentioned also a fever before migrating to greece. clinically: largely-scaled paleness, stomach -mild and diffused sensitivity, with also lightly increased enteric sounds and a small degree of hepatomegaly, when palpated deeply. laboratory examination: leucocytes . eo . %, hb . g/dl, ht . %, mcv , fl, mch . pg, thrombocytes . , blood sedimentation mm, fe mg/ml, ferritin . ng/ml. normal biochemical examinations. u/s: livers size lightly increased . mm, hepatoportal vein with normal amplitude. parasitology of feces: in the immediate confection with lugol of the second sample, there were observed: big scattering oval ovules ( - mm) with a thin wall and quills on the side, as well as three imago worms (> mm): schistosoma mansoni. treatment: praziquantel was given. recheck in a months time: blood examinationleucosites . , eo . %, hb , g/dl, ht . %. parasitology of feces is negative. in the diagnosis of anemia combined with fever, to patients who are immigrants, schistosomiasis posters inflamm. res., supplement ( ) should be taken into account, especially when sideropenic anemia is accompanied with intense eosinophile, because the disease is mostly a reaction of retardate supersensitivity. bronchial asthma is a chronic airway inflammatory disease caused by immune cells such as t lymphocytes and eosinophils. recently, high-sensitivity crp (hs-crp) has become available for detecting small changes in crp levels within the normal range, allowing for assessment of subclinical inflammation. this study was undertaken to investigate the relationship between hs-crp and bronchial asthma. we collected blood samples from patients with bronchial asthma with or without attacks and measured serum eosinophil cationic protein (ecp) and pulmonary function as well as serum hs-crp. serum crp levels in patients without attacks (average . mg/ l; p < . ) and with attacks (average . mg/l; p < . ) were significantly higher than those of normal controls (average . mg/l). serum hs-crp levels were inversely correlated with fev . % in asthmatic patients (r = - . , p < . ). in conclusion, these results indicate that serum hs-crp as well as ecp may be related to the state of asthma exacerbation and allergic inflammation. objectives: the pshr assay can be used to test the biologic activity of allergens since it mimics the effector phase of a type i hypersensitivity reaction. in this study we tested methods for removing ige-antibodies (stripping) from donor basophils. moreover a method was developed for improving the antigen specificity profile in pshr. proof-of-concept was provided using absorption with complete allergen extracts. methods: buffy coats were screened to exclude reactivity against relevant allergens. subsequently pbmcs were purified and basophils were stripped with either lactate or a phosphate buffer. cells were passively sensitized with patient sera and challenged with allergen extracts in various concentrations. released histamine was measured spectrofluorometrically (hr-test, reflab). cutoff was % hr. for absorption experiments patient sera (from a peanut allergic and a codfish allergic patient) were incubated with streptavidin-coated sepharose beads coupled with biotinylated allergens (peanut, and bsa as control). after centrifugation the supernatant was used to passively sensitize stripped basophils. hr was measured as described above. results: stripping experiments using the two buffers only partially removed surface ige but passive sensitization of stripped basophils was equally effective enabling determinations of sub-nanogram quantities of peanut allergen. absorption experiments showed that it was possible to specifically remove peanut specific ige from patient serum. removal of specific ige reactivity to peanut extract was verified by western blotting. conclusions: using peanut extract as a model it was demonstrated that in pshr antigen specificity can be modified. ( ), sk bk( ), v sharma( ), rp bhandari ( ) ( ) nepal medical college teaching hospital, nepal ( ) all india institute of medical sciences, new delhi, background: nepal has one of the highest maternalmortality rates in the world. this study was to evaluate the incidence, disease pattern, and risk-factors for thromboembolism in pregnant nepalese women. methods: women with thromboembolic diseases were identified and their case records retrieved and reviewed s inflamm. res., supplement ( ) posters from january to december . demographiccharacteristics were compared between women with and without thromboembolism. the total number of deliveries over the study period was , , giving an incidence of . per deliveries. there were two cases of pulmonary-embolism and one resulted in a maternal-death. the others had deep-vein-thrombosis of which over % were limited to calf veins only. the ultrasound-examinations requested for suspected deep-venous-thrombosis before and after the event of maternal-death were . and . per deliveries (p <. );the corresponding cases of deepvenous-thrombosis diagnosed were . and . per deliveries, respectively (p <. ). the majority ( %) of cases were diagnosed in the postpartum period, mainly after cesarean-delivery. women with venous-thromboembolism were older, had higher bmi, and a higherincidence of preeclampsia. there were approximately twice as many postpartum as antepartum events. bloodgroup a, multiple-pregnancy, caesarean-section, cardiacdisease, delivery at gestational-age of < weeks, a bmi of , or more and maternal-age of or over were all found to increase incidence of venous-thromboembolism. the long-standing belief that thromboembolism is rare among nepalese is at least partly because of undiagnosis most of these events are deep-veinthromboses occurring in the postpartum period and it is very essential for primary prevention developing country like nepal. ( ), ch ladel ( ), t ruckle( ), c rommel( ), r cirillo ( ) ( ) rbm-merck serono, colleretto giacosa, italy ( ) serono pharmacological research institute, geneva, switzerland rheumatoid arthritis (ra) is a severe articular disease. massive leukocyte activation and infiltration into joints result in cartilage and bone destruction. blockade of pi k signalling pathway has been demonstrated to be curative in a murine model of ra, collagen-induced arthritis (cia). in this study we explored the molecular mechanisms by which pi k signalling inhibition resulted in clinical amelioration of disease symptoms. as , a novel isoform non-selective, yet specific class-i-pi k inhibitor, administered at mg/kg twice-a-day for days, to mice showing signs of arthritis, paw swelling and inflamed digits, induced a significant amelioration of disease course. at the end of treatment, post-arthritic paws were removed and phosphrylation levels of akt (p-akt), downstream target in pi k-mediated signal, were determined by semi-quantitative immunohistochemistry, also immunophenotyping of circulating cells by flowcytometry was conducted. akt phosporylation resulted to be significantly enhanced by disease induction and as was able to decrease its levels down to values comparable with naïve animals. controls and as treated mice were bled before treatment, after two days of treatment and at treatments end. no changes in cellular composition (morphology and hematology parameters) between experimental groups were observed. t cell number was not affected, however a significant decrease of natural-killer, memory and regulatory t cells was observed after as administration. finally, a non-significant, moderate reduction of bcell number was also observed. these data demonstrate that efficacy of as in arthritis models is mediated by direct modulation of the target resulting in a mixed anti-inflammatory (via pi kg) and immunosuppressive (via pi kd/a/b) activity. ( ) ( ) institute of biomedical science, university of sao paulo, brazil ( ) ibilce -sao paulo state university, brazil epidemiologic data suggest that female sex hormones are involved in the pathophysiology of allergic asthma. we investigated in rats the immunomodulatory potential of estradiol and progesterone on the expression of allergic asthma. seven days after being ovariectomized (ovx), groups of rats were sensitized with ovalbumin (oa). fourteen days after sensitization, the animals were oachallenged and used day thereafter. allergic,shamoperated animals were used as controls. some ovx animals were treated with estradiol ( ìg/kg) or progesterone ( ìg/kg) h before being challenged. mast cell degranulation was quantified in samples of isolated, oa-challenged bronchi. the airway reactivity of inner bronchi to methacholine and the functional activity of cells we analysed. ovx caused reduction of the allergic lung inflammation and bronchial hyperresponsiveness with regard to intact female rats. estradiol reverted the reduced cellular recruitment into lungs, whereas progesterone reduced the pulmonary inflammatory response and reverted the bronchial hyperresponsiveness. cultured bal and bone marrow cells from allergic rats increased posters inflamm. res., supplement ( ) s the release of il- and reduced that of il- and tnf. the release of il- by bone marrow cells was significantly reduced. these effects were reverted by estradiol, and progesterone reduced il- and increased il- production in bal and increased that of il- and tnf in bone marrow cells. bronchial mast cell degranulation upon direct contact with oa in ovx rats was less than in controls. it is suggested that female sex hormones can modulate the allergic lung inflammation in rats by acting on cellular migration/activity and airway responsiveness. objectives: to study the participation of fsh on modulation of e-and l-selectin, icam- and mac- expression in ovariectomized (ovx) rats made allergic. methods: female rats were sensitized (oa/alumen) after (ovx- ) or days (ovx- ) of ovx or sham-operated (sh). fourteen days thereafter animals were challenged (oa, %; aerosol) and sacrificed h after. bronchoalveolar lavage (bal) was collected and flow citometry analyses oficam- , mac- and l-selectin expression was studied. in parallel, lungs were frozen and sections were analysedfor e-selectin expression by immunohistochemistry. results: at day , e-selectin expression increased(ovx- = , ae , ) and at day , decreased (ovx- = , ae , ) as compared to respective controls (sh- = , ae , ; sh- = , ae , ). estrogen treatment reverted this profile in both groups (ovx- +e= , ae , ; ovx- +e= , ae , ). mean fluorescence intensity of bal cells showed increase of mac- expression (ovx- = ae , vs sh- = , ae , ), icam- (ovx- = , ae , vs sh- = , ae , ) and l-selectin (ovx- = , ae , vs sh- = , ae , ) at day i.e., ovx- group. on the other hand, we observed a decrease in icam- (ovx- = , ae , vs sh- = ae , ) and mac- expression (ovx- = , ae , vs sh- = , ae , ) was seen in ovx- group. conclusions: oscillation of hormone levels during immunization with oa increased (ovx- ) and decreased (ovx- ) expression of adhesion molecules. estradiol treatment reverted this effect. this results suggest that fsh modulates the ali in rats by acting on cell ( ), s lim ( ), y lin ( ), bp leung ( ), c thiemermann ( ), wsf wong ( ) ( ) national university of singapore ( ) the william harvey research institute, london, uk glycogen synthase kinase b (gsk- b) is known to regulate various cellular functions including inflammatory responses. we hypothesized that inhibition of gsk- b may have anti-inflammatory effects in a mouse asthma model. balb/c mice were sensitized with ovalbumin (ova) and challenged with aerosolized ova. tdzd- , a non-atp competitive gsk- b inhibitor, was administrated by i.v. injection one hour before ova challenge. tdzd- significantly reduced the ova-induced eosinophilia in a dose-dependent manner and inhibited the levels of il- , il- and eotaxin in bronchoalveolar lavage (bal) fluid. tdzd- also suppressed the mrna levels of icam- , vcam- and chitinase proteins in the lung. histological studies revealed that tdzd- substantially reduced the inflammatory cell infiltration and mucus secretion in the lung tissue. tdzd- also decreased ova-specific ige level in the serum. in addition, ova-induced increase in airway resistance and reduction in dynamic compliance were attenuated by tdzd- . our findings suggest that inhibition of gsk- b may have therapeutic potential for the treatment of allergic airway inflammation. ( ), a yildirim ( ), f ercan ( ), n gedik ( ), m yuksel( ), inci alican ( ) ( materials and methods: sprague-dawley rats ( - g) were exposed to oc (burn group) or oc water bath (control group) for s under ether anesthesia. adm ( ng/kg; s.c) was administered min before burn and all rats were decapitated h after the burn insult. trunk blood was collected for the measurement of tnf-alpha level and the lung, ileum and kidney samples were stored for microscopic scoring and for determination of lipid peroxidation (lp), myeloperoxidase (mpo) activity and formation of reactive oxygen metabolites (roms) using chemiluminescence assay. results: burn resulted in severe morphologic damage in tested tissues. lp increased in lung and kidney (p< . - . ) and mpo activity showed a marked increase in all tested tissues (p< . ) of the burn group. adm reversed these parameters effectively (p< . - . ). luminol chemiluminescence levels showed increases in both ileum and lung (p< . - . ) whereas lucigenin chemiluminescence levels increased in ileum and kidney (p< . ) of the burn group. adm treatment was also beneficial in reducing chemiluminescence levels near to controls (p< . ). adm reduced plasma tnf-alpha level (p< . ) which showed a significant increase in burned animals compared to controls (p< . ). conclusions: adm is beneficial in remote organ damage following burn insult via decreasing neutrophil infiltration, rom generation, lp, and the release of proinflammatory cytokine tnf-alpha. kalpana panday ( ), sd joshi ( ), kr reddy ( ) ( we determined the crystal structure of human hematopoietic prostaglandin (pg) d synthase (h-pgds) as the quaternary complex with glutathione (gsh), mg +, and an inhibitor, hql- , with anti-inflammatory activities in vivo, at . resolution. hql- was found to reside within the catalytic cleft between trp and gsh in the quaternary complex. hql- inhibited h-pgds competitively against the substrate pgh as well as noncompetitively with respect to gsh. surface plasmon resonance analysis revealed that hql- bound to h-pgds with an affinity that was -fold higher in the presence of gsh and mg + than in their absence. hql- inhibited selectively pgd production by human h-pgds-expressing megakaryocytes but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between h-pgds and cyclooxygenase. orally administered hql- inhibited antigen-induced production of pgd , and airway inflammation in mice without affecting the production of pge and pgf f¿. knowledge about this quaternary structure should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit pgd production specifically. introduction: it has been proved that high levels of mechanical ventilation produce lung injury as well as local inflammation. this study was designed to evaluate how the generation of inflammatory mediators by an over-stretched lung affects the non-hyperventilated lung. methods: male wistar rats ( - g) were anesthetized and paralyzed, and the two lungs were independently intubated. differential ventilation was applied for h ( breath/min). one lung was subjected to hyper-ventilation ( ml/kg/lung) and the other was ventilated with a normal volume ( ml/kg/lung). in a control group, both lungs were ventilated with a normal volume. after sacrifice, samples of lung, plasma and liver were collected. the expression of the pro-inflammatory chemokine mip- was evaluated by rt-pcr and the edema was assessed by the ratio between the wet and dry weight of the lung. systemic inflammation was estimated in liver by measuring the expression of tnfa by rt-pcr as well as its levels in plasma. the hiper-ventilated lung showed an increase in the ratio between the wet and dry weight and in mip- expression compared to the normal ventilated lung. no differences were found in edema, neither expression of mip- between the normally ventilated lung and control lungs. no significant changes were observed in liver expression and plasma levels of tnfa as a consequence of unilateral lung hyper-ventilation. the over-straining caused to the hyperventilated lung leaded to a local inflammatory response without systemic effects. the normal ventilated lung is not affected by the inflammatory process triggered on the over-strained lung. ( ), j-y gillon( ), v lagente ( ), e boichot ( ) ( ) university of rennes , rennes, france ( ) serono international s.a., geneva, switzerland macrophage elastase (mmp- ) is a metalloproteinase involved not only in emphysema but also in the inflammatory process associated with copd (chronic obstructive pulmonary disease). the mechanism of action of mmp- in the development of pulmonary inflammatory process is still unknown. in the present study, we investigated the effect of recombinant human mmp- (rhmmp ) on il- /cxcl release from alveolar epithelial cell line a and we explored the underlying mechanisms. a cells were stimulated with rhmmp- ( . - - . - u/ml) during hours and il- /cxcl level in supernatant was determined by elisa. involvement of map (mitogen activated protein)-kinases was studied by western-blotting and also using chemical inhibitors. nfkb activation was examined with the transam nfkb p kit. we observed that mmp- elicited il- /cxcl release in a dose-dependent manner. this production could be prevented by a pretreatment for hour with a selective mmp- inhibitor (as - mm) or with the nonselective inhibitor batimastat ( - mm) . the il- /cxcl production was also inhibited by actinomycin d ( mg/ml), erk / inhibitors (u mm and pd mm) and nfkb inhibitors including bay - ( mm) and nfkb activation inhibitor ( nm), whereas p kinase inhibitor (sb ) had no effect. stimulation with mmp- was rapidly followed by a phosphorylation of erk / ( min) and an nfkb nuclear translocation and activation ( h). the nfkb activation was not inhibited by a treatment with u . these data suggest that alveolar epithelium is a target of mmp- since it upregulates gene expres-s inflamm. res., supplement ( ) posters sion and release of il- /cxcl , via erk / and nfkb activation, but these two pathways appears to be distinct. agents which are associated with lung inflammation, such as cigarette smoke and lipopolysaccharide (lps), induce the production of pro-inflammatory chemokines in lung epithelial cells in vitro, and the induction of interleukin (il)- , in particular, is often used a measure of relative toxicity.in this study we have compared mrna expression and mediator release in nci-h human lung epithelial cells exposed to lung toxicants, namely: cigarette smoke total particulate matter (tpm), lps, bleomycin, diesel exhaust particles, residual oil fly ash (rofa), carbon black and vanadyl sulphate.polystyrene, poly(methyl-methacrylate) and the tpm vehicle, dimethyl-sulphoxide were used as negative controls.confluent monolayers of h cells were exposed to serial dilutions of test agents in serum-free medium for hours.the conditioned medium was then removed and assayed for a range of pro-inflammatory cytokines and other selected mediators by luminex technology.the levels of gene expression of il- , matrix-metalloprotease- (mmp- ), the gel-forming mucin muc ac, heparinbinding epidermal growth factor-like growth factor (hb-egf) and the cytochrome p s cyp a and cyp b were determined by quantitative-polymerase chain reaction.all of the toxicants induced similar responses whereas the negative controls were largely ineffective.-such a panel of biomarkers may enable an in vitro assessment of the potential to cause lung inflammation.-moreover the use of several biomarkers could give a more accurate picture of toxicity than the determination of il- alone, particularly in the case of agents such as tpm, where the conventional vehicle is found to have some biological activity. respiratory tract infections are a major public health issue. prevention in high risk populations relies mainly on vaccination. vaccination is highly recommended in decrease absenteeism. immunomodulating drugs are important tools in the treatment of infectious diseases. immunomodulatory agents are probably contributive in decreasing exacerbation rates. the authors present different classes of immunomodulators that are currently in use. the vaccine, created from a bacterial protein, reeducates the immune system to stop inflammation. by preventing infections, vaccines prevent the development of a strong t helper (th ) response. the challenge is now to inform about new possibilities of an optimal prevention respiratory tract infections. deoxypodophyllotoxin (dpt) is a medicinal herbal product that is isolated from anthriscus sylvestri. that inhibits cyclooxygenase- (cox- ) and cox- dependent phases of prostaglandin d (pgd ) generation in bone marrow-derived mast cells (bmmc) in a concentration-dependent manner with ic values of . mm and . mm, respectively. this compound inhibited cox- and -dependent conversion of the exogenous arachidonic acid to pgd in a dose-dependent manner with an ic values of . mm and . mm, respectively. however, dpt did not inhibit cox- protein expression up to a concentration of mm in the bmmc, indicating that dpt directly inhibits cox- activity. furthermore, this compound consistently inhibited the production of leukotriene c (ltc ) in a dose dependent manner, with an ic value of . mm. these posters inflamm. res., supplement ( ) s results clearly demonstrate that dpt has a dual cox- / -lox inhibitory activity in vitro. therefore, this compound might provide the basis for novel anti-inflammatory drugs. in order to determine anti-allergic and antiasthmatic activity of dpt in vivo, we used rat pca model which was activated by anti-dinirophenyl ige and ovalbumin/alum induced mouse asthmatic animal model, respectively. as a result, dpt strongly inhibited pca reaction as well as it reduced infiltrated eosinophil numbers in bronchoalveolar lavage fluid. furthermore, dpt decreased the mrna levels of the th cytokines in a murine asthmatic model. in addition, northern blot analysis showed that dpt also reduced both the eotaxin and arginase i mrna levels in a dose dependent manner. these results suggest that dpt may be beneficial in regulating various inflammatory diseases. an imbalance of proteases and anti-proteases in the lung has been implicated in the pathogenesis of chronic obstructive pulmonary disease (copd), a smokingrelated disorder associated with accelerated lung function decline.in particular, the activity of matrix metalloproteases (mmps) have been implicated in driving both the inflammation and parenchymal destruction observed in copd patients.here, we tested whether a broad spectrum mmp inhibitor, pkf- , could block the inflammation induced by an acute exposure to cigarette smoke in strains of mice, balb/c and c /bl .animals were administered the compound ( - mg/kg) either per os (p.o.) or intranasally (i.n.) hour before and hours after exposure to smoke on three consecutive days.bronchoalveolar lavage (bal) was performed and lungs were flash frozen for inflammatory marker analysis.pkf- dose-dependently reduced lung neutrophilia in balb/c mice when dosed either p.o. (~ % at mg/kg; p < . )or i.n. (~ % at mg/kg; p < . ).however, the compound had no clear effect on bal neutrophil infiltration in c /bl mice by either route of administration.interestingly, in both strains bal macrophages dose-dependently trended towards an increase when the compound was dosed p.o. and decreased when dosed locally (p < . ). examination of lung tissue cytokine levels revealed that while smoke-exposure increases il- beta, kc, and mip- , pkf- had little effect on these cytokines.these data suggests the ability of broad spectrum mmp inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, while its ability to limit macrophage infiltration may be route dependent. to investigate the role of seh in the regulation of the pulmonary inflammatory response, we have used seh deficient mice in a locally administered lps model. male seh deficient mice (ko) and their wildtype (wt) littermates were exposed to inhaled lps.four hours later they were sacrificed and bal was performed. differential counts and cytokine levels in bal were evaluated. results: lps induced a significant increase in total cell number and neutrophil number in bal in the seh deficient mice and in wt mice;no significant differences between the groups were seen (table ) cytokine analysis showed significant increases in tnfalpha, il- , kc, gm-csf, mcp- , il- beta and rantes in the lps-exposed wt mice. no significant differences were seen between lps exposed wt and ko mice except for a significant increase in tnfa in ko mice. our results show that seh has no pivotal role for the regulation of the acute inflammatory response to lung administered lps. fam.liliaceae. based on literature data which signaled the presence of steroidic saponins in the rhyzomes, isolation, identification and quantitative determination of these compounds were done. the antiinflammatory activity was tested in non-immune chronic inflammation model:the cotton-pellets granuloma test in rats, and in an immune chronic inflammation model:arthritis test induced by freunds adjuvant in rats. in the first test, the antiinflammatory effect of steroidic saponins mg/kg is weak, statistically insignificant. in the arthritis test, steroidic saponins mg/kg proved an antiinfalammatory activity, influencing especially the primary response, but also the secondary one, in the last part of the experiment (after days). in both tests, the suprarenal glands weight was modified. objectives: dendritic cells (dcs) are professional antigen presenting cells. many types of dc with subtle difference in phenotype have been reported in several organs. existence of dc was reported in synovial tissue of rheumatoid arthritis (ra), however, the details in ra dc still remains unclear. in this study, we generated a new lineage of dc with gmcsf (+tnfa) and investigated their functions. furthermore the ability of osteoclastgenesis was examined. methods: monocyte-derived dcs or macrophage were generated in ( ) tnfa + gm-csf ( ) gm-csf ( ) il- + gm-csf ( ) mcsf. the phenotypes of these cells were analyzed by morphological examination and flow cytometry (facs calibur). cell proliferation was examined by wst- assay. dc functions were assessed in antigen presenting ability (mlr assay), cytokine production (elisa), and endocytosis (fitc-dextran uptake). concerning osteoclastgenesis, monocyte-derived dcs were incubated with rankl and mcsf. trap stain was performed and the resorption ability was assessed on osteologic cell culture system and dentine slices. results: these cells were dendritic-like and their surface markers were cd a low cd b + cd c + cd + cd + cd low hla-dr + dc-sign low and different from conventional dc or macrophage. they had an antigen presenting ability to induce na*ve cd + t cell proliferation, il- production and endocytosis. in the presence of rankl and mcsf, they differentiated multinuclear trap-positive cells with bone resorption ability, which was strengthened by tnfa. we generated a new lineage of dc with gm-csf (+ tnfa). the dc seemed to play a pivotal role in inflammatory arthritis under tnf immunity. the clinical effectiveness of rituximab and other b cellattenuating rheumatoid arthritis (ra) therapies has increased interest in understanding the role of b cells in ra pathogenesis.the possible mechanisms underlying the effectiveness of rituximab were investigated by performing biosimulation research in the entelos ra physiolab platform, a mathematical model of the joint of an ra patient.the platform dynamically integrates the contributions of immune cells (t cells, b cells, and macrophages), resident cells (fibroblast-like synoviocytes and chondrocytes) and mediators to the joint inflammation and structural damage observed in ra.the b cell lifecycle is represented in the platform, as well as effector functions such as antigen presentation, mediator and autoantibody production, and immune complex formation.the dynamics of these b cell properties were calibrated to reproduce reported experimental behaviors and clinical outputs from ra patients (e.g., acr score and radiographic progression rates).an assessment of the contribution of individual b cell functions to clinical outcome suggests that plasma cell-derived immune complexes are key modulators of inflammation in ra patients. in contrast, proinflammatory cytokine production by b cells contributes minimally to synovial hyperplasia, but plays a role in the progression of structural damage.immune complex formation leads to monocyte activation, increased mediator production by macrophages and an increase in antigen availability to t cells.biosimulation research in the ra physiolab platform is advancing our understanding of the mechanisms underlying effective b cell-targeting ra therapies, and may guide the development of improved second-generation therapeutic approaches. methods: the hmscs were obtained at the operation.the mononuclear cells were extracted and the colony forming assay were performed after weeks. the hmscs were cultured and in passage ,the cell surface antigens of both groups were analyzed by flow cytometory.in passage , in control group, the cells were cultured with beta glycerophosphate(bgp) and in osteogenic group, the cells were cultured with bgp and ascorbic acid(aa) and dexamethasone(dex).after weeks, alp staining and activity were measured in each group.after weeks, alizarin red s assays were performed.rna was extracted from the cells cultured with bgp and aa and dex for weeks and weeks and the gene expressions of bone formation markers were examined by real time pcr. the mann-whitney test was used for the statistical analyses. the colony forming assays showed no significant differences in oa and ra. in flow cytometory, the cell surface antigens in oa and ra were almost same.in alp activity,there were no significant differences in oa and ra.in alizarin red s, there were significant differences.in real time pcr,the gene expressions showed no significant differences in oa and ra. conclusions: the hmscs of ra will be able to use for regenerative therapy. silje vermedal høgh ( ), hm lindegaard ( ), gl sorensen ( ), a høj ( ), c bendixen ( ), p junker ( ), u holmskov ( ) ( cytosolic phospholipase a (cpla ) plays a crucial role in eicosanoid production, by releasing an arachidonic acid from membrane phospholipids. in addition, cpla regulates the phagocyte nadph oxidase-releasing superoxides and that is the only isozyme responsible for the production of eicosanoid in phagocytic cells. collageninduced arthritis (cia) in mice is an experimental model of auto-immune diesease with several clinical and histological features resembling rheumatoid arthritis in humans. previous studies show that cpla -deficient mice are resistant to cia. thus we aimed to study whether cpla is up-regulated during the development of cia and to detect its exact location in the inflamed joints. immunoblot analysis revealed an increase in the level of joint cpla protein during the development of the disease which correlates with the severity of the inflammation, as examined by paw thickness. immunohistochemistry with specific anti-cpla antibodies revealed low positive cpla protein levels in skeletal muscles, sebaceous glands and skin (epidermis, dermis) tissues of healthy paws. in the joints of the cia mice, large amounts of inflammatory infiltrate containing cpla were detected. in addition, robust cpla protein expression in the skeletal muscles surrounding the joints and strong cpla positive staining in sebaceous glands were detected. the high correlation between the severity of inflammation and the elevated cpla protein, due to an inflammatory infiltrate and increased cpla expression, suggests an important role of cpla in the development of arthritis. rheumatoid arthritis (ra) is the complex disease depending on environmental as well as genetic factors. in spite of the large research efforts we know in fact only small number of genes involved in this disease. animal models are useful tools for better understanding of the pathogenic mechanisms and genes leading to the disease process. the aim of the current project is to identify the genes and their functional role importance for arthritis. two loci associated with arthritis were identified using a cross between the b .q (intermediate susceptible) and nod.q strains (resistant to arthritis). one on chromosome a disease protective locus (cia ) and another on chromosome a disease-promoting locus (cia ). nod.q allele at cia promotes arthritis whereas cia , has a protective effect in contrast to the b .q allele on chromosome . a promising candidate gene in cia locus is complement factor (c ) as the nod.q allele produced defective c protein. cia locus contains several genes of potential importance for disease such as fc riib, fc riii, fc riv ncf , fh. the results of cia (collagen induced arthritis) and caia (collagen antibody induced arthritis) experiments using the subcongenic mice generated that contains fc r region showed a significant difference in incidence and severity of arthritis. the disease is controlled in a recessive pattern. the fragment devoid of fc r region seems to be protective. the subcongenic for the cia locus has been generated recently which will be tested for cia and caia susceptibility. the results from these experiments will be discussed in detail. angela pizzolla, ka gelderman, r holmdahl ncf is a component of the nadph oxidase complex, which produces reactive oxygen species (ros) upon activation into phagosomes and extracellularly. polymorphisms in the ncf gene that impair the capacity to produce ros, enhance susceptibility of both mice and rats to arthritis. activation of autoreactive t cells drives arthritis development but neither ncf expression nor oxidative burst have been detected in t cells. we hypothesize that antigen presenting cells influence t cell activity by producing ros during antigen presentation. we aimed to clarify the role of ros produced by dendritic cells (dc) on t cell activation. dc were grown from bone marrow from ncf mutated and wildtype mice. we could show that ncf mutated dc proliferated and differentiated better, had higher expression levels of costimulatory molecules and mhc ii upon stimulation as compared to ncf wildtype dc. in addition, ncf mutated dc induced higher levels of il- production by hybridoma t cells. to analyze the role of ncf in dc on arthritis, mice were developed expressing functional ncf restricted to dc (b .qdcn). these mice are characterized for burst, ncf expression and ability to present antigen. we published that immunization with myelin oligodendrocyte glycoprotein (mog) protein resulted in higher disease severity than immunization with mog peptide in ncf deficient mice. this suggests that ncf plays a role in the uptake and processing of posters inflamm. res., supplement ( ) s antigens, probably by dc. this will be further investigated with the b .qdcn mice using in vitro assays as well as in vivo models for arthritis and multiple sclerosis. purpose: macrophage migration inhibitory factor (mif) is a pro-inflammatory cytokine involved in both innate and adaptive immune responses. it is expressed in human ra synovial tissue and its suppression inhibits t or b cell dependent animal models of ra. we investigated the role of mif in k/bxn serum transfer arthritis. methods: arthritis was induced by injection of k/bxn serum on days and in littermate wt and mif-/-mice. arthritis was scored clinically, ankle thickness was measured using microcallipers, and joints collected for histology. sections were scored for synovitis, synovial fluid exudate, cartilage degradation and bone damage. results: wt mice exhibited arthritis as early as day and % incidence was observed on day . mif -/-mice exhibited delayed arthritis, with onset on day and % incidence on day . mif -/-mice exhibited significantly reduced disease severity as measured by clinical disease score ( methods: osteoarthritis was induced by bilateral transection of the medial meniscus in dunkin-hartley guinea pigs using minimal invasive surgery to avoid cartilage damage due to inflammation and/or intra-articular bleeding. results: the first signs of osteoarthritis development were macroscopically observed four weeks after meniscal transection. twelve weeks after surgery the lesions were still restricted to the medial side of the joint and did not reach into the subchondral bone. cartilage destruction due to meniscal transection was also histologically detected. however, biomarkers for cartilage destruction (ctx-ii, hp/lp ratio, comp) were not increased. treatments aiming at different processes in osteoarthritis, such as bone destruction (risedronate), inflammation (pioglitazone and anakinra), and cartilage destruction (galardin) were not effective in this model. the early degenerative changes in this transection model are probably too mild to be measured in the systemic circulation using classic biomarkers. further research into new biomarkers is needed to detect and monitor the early stages of osteoarthritis. the ineffectiveness of the compounds tested in this model underscores the urgent need for new strategies to treat the disease. the meniscal transection model might prove to be useful tool for identifying new biomarkers and treatments. livia l camargo ( ), a denadai-souza ( ), lm yshii ( ), a schenka ( ), ma barreto ( ), d boletini-santos ( ), c lima ( ), v rioli ( ), mn muscar ( ), e ferro ( ), sk costa ( ) ( methods: aia was induced via intraarticular (i.a.) injection of methylated bovine serum albumin in immunized male rats. knee oedema and pain score were assessed daily in controls and animals treated with hemopressin ( or ìg/day; i.a.). histopathological changes, cell number and cytokines in the synovial fluid of aia rats were determined at day . results: aia rats developed a severe mono-arthritis characterized by a joint oedema and pain. at day , there was marked cellular infiltration, hyperplasia, pannus formation and destruction of bone and cartilage, but pro-inflammatory cytokines were undetectable by elisa. both doses of hemopressin significantly reduced the knee oedema, but only mg of hemopressin attenuated the pain score. acute joint inflammation was significantly reduced by hemopressin, but failed to significantly affect chronic histopathological signs (hyperplasia, pannus etc.). conclusions: hemopressin has potential for treating acute signs of aia by reducing synovial plasma protein extravasation, alleviating pain and reducing acute joint histopathological changes, thus providing an alternative strategy for treatment of oedema and pain in arthritis. calcitriol, the hormonal metabolite of vitamin d and it synthetic analogs exert an anti-inflammatory action on psoriatic skin lesions while eliciting mild inflammation on healthy skin. the map kinase erk plays an important role in the induction of chemokines, cytokines and adhesion molecules in keratinocytes that maintain the epidermal inflammatory response.we hypothesized that the dual effect of calcitriol may be partially attributed to differential effects on erk activation in the presence or absence of inflammatory mediators. our experimental model was immortalized non-tumorigenic human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators. inflammation was mimicked by exposure to tnf. level and activation of signaling molecules were determined by immunoblotting. by using the specific egf receptor (egfr) tyrosine kinase inhibitor, ag , we established that tnf activates erk in an egfr-dependent and egfrindependent modes. the egfr-dependent activation resulted in the induction of the transcription factor, c-fos, while the egfr-independent activation was of a shorter duration and did not affect c-fos expression. treatment with calcitriol alone increased erk activation and c-fos induction. pretreatment with calcitriol enhanced egfr-dependent erk activation and tyrosine phosphorylation of the egfr, but completely abolished egfr-independent tnf-induced erk activation. pretreatment with calcitriol increased the rate of de-phosphorylation of activated erk, accounting for the inhibition of egfr-independent erk activation by tnf. it is possible that effects on the erk cascade underlie the dual action of calcitriol and its synthetic analogs on cutaneous inflammation. christina barja-fidalgo ( ), r saldanha-gama ( ), ja moraes ( ), r zingali ( ), c marcienkewicz ( ) ( ) universidade do estado do rio de janeiro, rio de janeiro, brazil ( ) universidade federal do rio de janeiro, rio de janeiro, brazil ( ) temple university, philadelphia, usa neutrophils adhere on vascular endothelium and directly migrate toward inflamed tissue to exert their primary defense function. integrin are receptors that drive cell adhesion and motility and interfere with cell activation, functions and survival.acting as both anchoring molecules and signaling receptor, transducing signals outsidein and inside-out, integrins are potential targets for therapeutic and diagnostic opportunities. disintegrins are a family of cystein-rich low-molecular weight peptides that usually contain an rgd sequence, a cell attachment site of ecm and cell surface proteins recognized by integrins. they are considered selective and competitive antagonists of integrins, being potent inhibitors of platelet aggregation and cell-cell/cell-ecm interactions. we reported that rgd-disintegrins, selectively interact with integrin amb , a b and/or avb ) on human neutrophils, interfering with cell functions through the activation of integrin-coupled intracellular signaling pathways. recently showed that, a selective ligand of a /a b integrins, vlo , induces neutrophil chemotaxis, cytoskeleton mobilization and potently inhibiting neutrophil spontaneous apoptosis. these effects are mediated vlo interaction with a b integrin, activating the focal adhesion cascade. vlo effects on the delay of neutrophil is modulated by pi k, erk- mapk and nf-kb pathways that seems to interfere with the balance between anti-and pro-apoptotic bcl- family members and with mitochondrial membrane potential. data emphasize mechanistic details of the role of a b integrins interactions on human neutrophils and support the use of disintegrins as prototypes to develop logical combinations of drugs to optimize or minimize the susceptibility of a selected target cell population to apoptosis during therapeutic interventions. (faperj, cnpq, capes, ifs-sweeden) ( ), h serezani ( ), m peters-golden( ), sonia jancar ( ) '( ) university of s¼o paulo, brazil ( ) university of michigan, usa it is been shown that leukotriene (lt) b and cysteinyl lt (ltc , ltd and lte ) enhances fcr-mediated phagocytosis in alveolar macrophages (am), dependent on protein kinase c (pkc). in contrast, ltb but not cyslt effects are exclusive on syk activation. in the present study we investigated the role of specific pkc isoforms and its upstream and downstream targets involved in lt-enhanced fcr-mediated phagocytosis. to this purpose, ams were pretreated or not with inhibitors of pkc-d (rottlerin - um), pkc-a (ro- - - nm), pi k (ly - um and wortmannin- nm), erk / (pd - um), cpla (aacocf - um), p mapk (sb - um) and ca++ (bapta/am- um), before stimulation with ltb or ltd and addition of igg-opsonized red blood cells for min. activation (phosphorylation) of signaling molecules by lts were analyzed by western blot. our results demonstrate that ltb -enhanced phagocytosis is dependent on pkc-a, while ltd effects are mediated by pkc-d. cell proliferation and differentiation, adhesion, cell activation and apoptosis. while galectin- mainly acts as an anti-inflammatory and pro-apoptotic molecule, galectin- is known as a strong pro-inflammatory and anti-apoptotic signal. we have recently recognized galectin- as a new molecular target of immunomodulatory drugs in monocyte/macrophage-like cells. in this study we investigated the effects of immunomodulatory drugs (aspirin, indomethacin, hydrocortisone and dexamethasone), applied in therapeutic ranges on the expression of galectin- at gene and protein level in monocytic thp- cells. we have also tested the effects of these drugs on both galectins in the cells activated by lipopolysaccharide from e. coli (lps). the targeted mrna level was evaluated using quantitative rt-pcr technique and the expression of both galectins in cell homogenates was determined by western-immunoblot analysis. the results showed that immunomodulatory drugs affected the expression of galectin- on both, gene and protein level, and that the effects were dependent on drug type and applied concentration as well as time of the exposure. the modulatory effects of the applied drugs on galectin- and - expressions were also observed in the cells activated by lps. these findings represent important step in the understanding of the effects of immunomodulatory drugs on galectin- and- expressions, as well as the role of these lectins in the physiology of monocytes. introduction: pancreatitis-associated protein (pap) has been recently described as an endogenous mechanism involved in the regulation of inflammation. in the present study, we show some of the molecular mechanisms implicated in the intracellular signaling pathways modulated by pap. the pancreatic human cell line panc was incubated with pap ( ng/ml) and/or tnfa ( ng/ml). total rna was obtained and the expression of tnfa was examined by rt-pcr. in addition, the effect of pap on nfkb activation was measured by inmunofluorescence in cells. western blot analysis was used to determine the expression of nfkb mediators: phosphorylated ikk, ikba and p . results: we observed that pap administration to cells prevented nfkb translocation to the nucleus as well as the tnfa-induced tnfa gene expression. when tnfastimulated cells were treated with cycloheximide in order to block protein synthesis, the induction of tnfa gene expression was completely restored. on the other hand, pap had no effect on ikk phosphorylation or ikba degradation. conclusions:in this study we have provided evidence that pap modulates the inflammatory response by blocking nfkb translocation to the nucleus. this pap-induced nfkb inhibition requires a jak/stat-dependent de novo protein synthesis. objectives: this study investigated the inhibitory mechanism of hyaluronan (ha) on lipopolysaccharide (lps)stimulated production of proinflammatory cytokines in u macrophages. methods: ha was added to u macrophage cultures in the presence of lps, with or without pretreatment with anti-intercellular adhesion molecule- (icam- ) antibody. secreted levels of tumor necrosis factor a (tnfa), interleukin (il)- b, and il- were determined by enzyme-linked immunosorbent assay. the phosphorylation of nuclear factor (nf)-kb, ikba, and mitogenactivated protein kinases (mapks) was analyzed by immunoblotting. results: lps stimulated production of tnfa, il- b, and il- . in contrast to kda ha, kda ha at mg/ ml inhibited lps-induced cytokine production. anti-icam- antibody blocked the effects of ha on the lps actions on u cells. lps activated nf-kb and mapk pathways, whereas ha down-regulated p nf-kb and ikba phosphorylation by lps without affecting mapks. inhibition studies revealed the requirement of nf-kb for lps-stimulated cytokine production. anti-icam- antibody reversed the inhibitory effects of ha on phosphorylation of p nf-kb and ikba. conclusions: ha of intrinsic molecular weight suppresses lps-stimulated production of proinflammatory cytokines via icam- through down-regulation of nf-kb and ikb. exogenous ha into arthritic joints could act as an anti-nf-kb agent by the mechanism demonstrated in the present study. the principal eicosanoid product of endothelial cox- is prostacyclin (pgi ), which is a potent vascular patency factor. induction of endothelial cox- under hypoxic conditions is well documented. this response, along with associated pgi release, is likely an important protective homeostatic response. in order to explore the role of candidate signalling agents in cox- expression in response to hypoxia, studies were undertaken using luciferase reporter constructs of the cox- promoter region in huvec. cox- induction under hypoxic conditions was confirmed with the wild type construct. strategic mutations of transcription factor binding sites showed that sites for hypoxia inducible factors (hifs) were more important for cox- expression than those for nfkb. furthermore, expression of cox- was increased under normoxic conditions by transfection of huvec with normoxia-stable hif mutants. emsa showed hif binding in nuclear extracts from untransfected hypoxic huvec. under these hypoxic conditions, increased release of pgi but not vaso-occlusive thromboxane a was seen. thus the putative protective induction of cox- in endothelial cells in response to hypoxia involves signalling by hifs. crescentic glomerulonephritis is characterized by crescent formation and rapid progress to renal failure, where the predominance of th immune response plays a crucial role. however, the therapeutic efficacy of the regulation of th -predominant immune responses remains to be investigated. therefore, the effects of a th selective inhibitor tak- were investigated in a model of crescentic glomerulonephritis in wky rats. methods: tak- was administered orally, starting at the time of induction of glomerulonephritis. in group , the drug was administered daily for the initial days. tak- was administered on day only in group , and from day to in group . in each group, nephritic rats were killed on days and . results: in group , glomerular damage, including crescent formation, was improved on day , with reductions in the numbers of cd , cd and ed- positive cells, as well as in urinary protein excretion. protein and transcript levels of th cytokines in the diseased kidneys were markedly decreased by tak- treatment. renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. elevated levels of serum creatinine showed concomitant improvement. in group , in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. treatment only on the first day in group , partially rescued renal dysfunction. conclusions: these results suggest that the initial inhibition of th immune response has an appealing therapeutic potential for crescentic glomerulonephritis. parasitic nematodes and their hosts are now known to produce a wide range of galectins. whilst host galectins have been shown to modulate the recruitment and effector function of inflammatory cells including mast cells, neutrophils and eosinophils, the role of secreted parasitic galectins is less well defined. studies at moredun have demonstrated that both the endoparasitic helminth, haemonchus contortus, and the ectoparasitic mite, psoroptes ovis, produce galectin-like factors which, in vitro, directly influence the migration and survival of eosinophils from their natural sheep host. excretory-secretory extracts from both parasites contained potent chemokinetic activity and were also able to promote eosinophil survival in the presence of dexamethasone. separation by affinity chromatography, as well as specific sugar inhibition and mass spectrometric profiling, revealed the active components to be galectins. in the case of h. contortus, there was homolgy with known est sequences, which allowed subsequent cloning and expression studies to be undertaken. a functional in vivo role for these parasitederived galectins awaits confirmation, but the possibility is raised that they could directly influence the host immune response following infection. this may have particular significance in mite infections in which exudates from the associated eosinophilc lesion appear s inflamm. res., supplement ( ) posters to provide the primary nutrient source for their survival. moreover, the observation that two very different parasites may have evolved similar mechanisms for manipulating the host inflammatory response to their benefit, raises the further possibility that parasite galectins may provide potentially novel therapeutic targets. the aim of the study was to investigate the time course of cytokine gene expression in liver and lungs of mice with lipopolysaccharide (lps)-induced septic shock and to assess the effect of three different immunomodulatory agents on cytokine mrna levels in these tissues. male cd- mice were injected intraperitoneally with mg/kg lps alone or concomitantly with an intravenous dose of pentoxifylline ( mg/kg), lisofylline ( mg/kg) or prednisolone ( mg/kg). the tissues were harvested , , , , and h following lps administration and stored at - c. relative quantification of tumor necrosis factoralpha (tnf-alpha), interleukin- beta (il- beta), interleukin- (il- ), and interferon-gamma (ifn-gamma) mrna levels was performed by real-time rt-pcr. the highest levels of cytokine mrna were observed at or h after lps administration, whereas the expression of tnf-alpha and il- beta in lungs and il- in liver reached the peak values at h and then decreased gradually. in addition, the lps effects on cytokine mrna were more pronounced in liver when compared to lungs. all administered compounds inhibited the lpsinduced tnf-alpha mrna expression (by up to approximately %), whereas lisofylline significantly increased ifn-gamma mrna levels in both tissues at most investigated time points. for other cytokines, the observed differences did not reached statistical significance. in conclusion, with the exception of ifn-gamma, the time course of cytokine mrnas differed considerably depending on the type of tissue. in the murine model of lps-induced septic shock, only tnf-alpha gene expression was suppressed by all compounds under investigation. maria sanz( ), m losada ( ), c company ( ), c lope-gines( ), l piqueras( ), j cortijo ( ) the migration of leukocytes into inflamed tissues involves a cascade of molecular events finely regulated by cell adhesion molecules and chemokines. fractalkine/ cx cl (fr) is a membrane-bound chemokine that functions as a mononuclear leukocyte chemoattractant and as an adhesion molecule. clinical studies and animal disease models have shown that fr is also involved in the pathogenesis atherosclerosis. we have demonstrated that angiotensin-ii (aii) has proinflammatory actions inducing the initial attachment of mononuclear cells to the arteriolar endothelium. in the present study we have investigated whether aii can cause the synthesis and expression of fr on human umbilical arterial endothelial cells (huaecs). huaecs were stimulated with ang-ii microm or with tnfalpha ( ng/ml) for , and h. fr was determined in the culture supernatants by conventional sandwich elisa. fr was only detected after h and h stimulation with tnfalphafnwhereas aii was unable to provoke the cleavage of the chemokine. semiquantitative rt-pcr analysis of huaecs showed increased fr mrna expression in aii-stimulated cells for and h. these effects were caused by the interaction of aii with its at receptor since they were abolished by losartan (at receptor antagonist). tnfalpha also increased fr mrna. immunohistochemical analysis of the cultured endothelial cells showed a clear expression of fr in huaecs stimulated with aii or tnfalpha for and h. these results suggest that fr could be a key chemokine in the selective adhesion of mononuclear leukocytes to the arterial endothelium elicited by aii. the lipophilic yeast malassezia is an exacerbating factor in atopic dermatitis (ad).among organisms of the malassezia species, m. globosa and m. restricta are particularly dominant on the skin of ad patients. our previous study has demonstrated that human keratinocytes responded to the two malassezia species with different th -type cytokine profiles, i.e. m. globosa induced il- , il- , and il- secretion from the keratinocytes, whereas m. restricta induced il- secretion.these findings suggest that m. globosa and m. restricta play a synergistic role in triggering or exacerbating ad by stimulating the th immune response. pattern recognition receptors (prrs) of human keratinocytes play an important role in the induction of inflammatory and innate immune responses. in this study, we assessed the role of prrs for cytokine production by human keratinocytes in response to malassezia species. human keratinocytes were pretreated with various anti-prr monoclonal antibodies (mabs) and stimulated with m. globosa or m. restricta. cytokine secretion from keratinocytes was measured by using fast quant elisa kit. exposure of human keratinocytes to m. globosa and m. restricta resulted in enhanced secretion of il- and il- , respectively. the m. globosainduced increase in il- secretion was inhibited by mabs against cd and cd . in case of m. restricta, mabs against toll-like receptor (tlr ) and cd suppressed significantly il- secretion from keratinocytes. these findings suggest that the distinct prrs interactions with fungal pathogen-associated molecular patterns (pamps) are key factors in differential cytokine secretion from keratinocytes stimulated with malassezia species. atopic dermatitis (ad) is a chronic, relapsing inflammatory skin disease associated with allergy. mdc (macrophage-derived chemokine/ccl ) and tarc (thymus and activation-regulated chemokine/ccl ) are th type cytokines, and it has been reported that serum mdc and tarc levels are associated with ad disease. in present study, we investigated the effect of prunus yedoensis matsum barks on the inflammatory chemokines (mdc and tarc) and jak-stat pathway in hacat keratinocytes. as a result, etoac fraction and e sub-fraction inhibited the mrna expression and protein level of mdc and tarc in a dose-dependent manner. also, e sub-fraction showed inhibitory activity on the stat protein level. these results suggest that p. yedoensis may have an anti-atopic activity by suppressing the inflammatory chemokines (mdc & tarc). the il- family now consists of members, most of which have assigned functions.there are members of the il- receptor family (including the decoy receptor type ii il- r).many of the il- family members possess neither a signal peptide nor an apparent prodomain, but nevertheless manage to exit the cell.the il- family members il- f , f and f signal through a complex of the il- r family member rp in association with il- r acp to activate common inflammatory pathways.the specific activity is is low, on the order of - ug/ml ec .we have found that removal of a few n-terminal amino acids from il- f , f , f and f can increase their bioactivity approximately -fold. the location of the n-terminus leading to increased specific activity is quite specific; removal of one more or one fewer amino acid eliminates the effect.in addition, n-terminally truncated il- f is capable of antagonizing signaling via il- rrp , but full-length f is inactive. ( ) university of ulsan, japan kyoto university, japan inflammation plays a pathogenic role in the development of obesity-related complications such as type ii diabetes and atherosclerosis. tumor necrosis factor alpha (tnfa) is closely associated with the enhanced inflammatory responses in obesity and the obesity-related pathologies. tr (hvem/tnfrsf ), which is a member of the tnf receptor superfamily and the receptor for lymphotoxins-related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells (light/tnfsf ), is a potent mediator of inflammatory responses. the purpose of this study is to examine the hypothesis that obesity-induced inflammatory responses can be attenuated by inhibiting tr pathway. c bl/ tr knockout mice and their wild-type control were fed a high-fat diet for weeks and the obesity phenotypes were determined in the obese tr knockout mice and the control. the obese tr mice fed a high-fat diet elicited the attenuation of body weight gain and insulin resistance relative to wild-type control mice. expression levels of inflammatory genes significantly decreased in the adipose tissue of the obese tr knockout mice compared with those of the control. our results demonstrate that obesity-induced inflammatory responses and insulin resistance can be attenuated in obese tr knockout mice fed a high-fat diet. objectives: the present study was undertaken to investigate the role of insulin on allergic airway inflammation. methods: diabetic male wistar rats (alloxan, mg/kg, i.v.) and controls were sensitized with ova ( ìg) and al(oh) ( mg, s.c.) days after alloxan or saline injection. the animals were challenged days later by the intratracheal instillation of ova ( mg/ . ml). the following analyses were performed h thereafter: (a) total and differential cell counts in bronchoalveolar lavage (bal) fluid; (b) quantification of tnf-alpha and il- beta in the bal by elisa; and (c) immunohistochemistry for p-and e-selectins on lung vessels. results: compared to the control animals, diabetic rats exhibited reduced number of neutrophils ( %) and mononuclear cells ( %); reduced levels of tnf-alpha ( %) and il- beta ( %), and reduced p-selectin expression ( %) in response to ova challenge. these abnormalities were corrected after treatment of diabetic rats with a single dose of nph insulin ( iu, s.c.), h before ova challenge. although we did not find differences in e-selectin expression between diabetic rats and controls, expression of this molecule was amplified by insulin. conclusions: data presented show that insulin controls neutrophils migration during allergic airway inflammatory possibly by modulation of tnf-alpha and il- beta production and selectin expression. supported by fapesp and pronex. hormonally active vitamin d derivatives are beneficial in the treatment of cutaneous inflammatory disorders, particularly in psoriasis. their anti-inflammatory effect is usually attributed to inhibition of the activity of infiltrating immune cells.we examined whether vitamin d also interferes with the pro-inflammatory action of the keratinocytes themselves. human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators were exposed to tnf to simulate an inflammatory challenge and their response was monitored by assessing mrna levels of the cytokine tnf, the chemokine il- and the adhesion molecule icam- by real-time pcr. icam and il- were induced rapidly peaking after h, their mrna levels increased again from h to reach a plateau between h to h after exposure to tnf, whereas tnf mrna levels increased steadily between h and h. h pretreatment with calcitriol, the hormonal form of vitamin d, inhibited induction of il- but did not affect that of icam- or tnf h following exposure while calcitriol markedly inhibited the induction of all pro-inflammatory genes h after the tnf challenge.calcitriol inhibits the activation of jun kinase (jnk) and p by tnf. this action was mimicked by the posters inflamm. res., supplement ( ) s jnk inhibitor sp and the p inhibitor sb .the combination of the two inhibitors fully reproduced the time and gene dependent modulatory effect of calcitriol. we conclude that vitamin d attenuates the active contribution of keratinocytes to cutaneous inflammation and that this modulatory effect is explained by inhibition of the jnk and p cascades. ( ), cm lotufo ( ), p borelli ( ), zs ferreira ( ), rp markus ( ), shp farsky ( ) ( ) department of clinical and toxicological analyses, school of pharmaceutical sciences, university of s¼o paulo, brazil ( ) department of physiology, bioscience institute,university of s¼o paulo, braziil introduction: we showed that endogenous glucocorticoids (eg) control neutrophil mobilizations from the bone marrow and peripheral compartment by modulating the expression of l-selectin on segmented cells. aims: we evaluated the role of eg on endothelial cells (ec) and the molecular mechanisms responsible for hormonal actions in neutrophils and ec. methods: neutrophils were collected from blood, segmented leukocytes from femoral bone marrow and ec were cultured from testis vessels. cells were obtained from adrenalectomized (adx), ru -treated, shamoperated, vehicle-treated and non-manipulated (nm) wistar rats. results: circulating neutrophils and segmented cells from ru -treated rats presented elevated and decreased expressions of l-selectin vs cells from control animals, respectively. the effects were not dependent on alterations of l-selectin mrna levels. ec from adx animals presented more ability to adhere neutrophils from nm rats and enhanced mrna levels and membrane expressions of icam- , vcam- and pecam- . participation of the glucocorticoid cytosolic receptor(gcr) on these effects was shown by similar results in cells from ru treated rats. nfkappab translocation in neutrophils was equivalent in all groups of animals, but it was enhanced in ec from adx or ru -treated rats. conclusions: data show the participation of the gcr on events involved in neutrophil mobilizations, but nfkappab transcription is only involved on ec. ( ), y naito( ), t okuda ( ), k mizushima ( ), t okayama ( ), i hirata ( ), h tsuboi ( ), t suzuki ( ), o handa ( ) background: despite the inhalation of co at high concentrations had been considered as a toxic gas, the inhalation of co at low concentration has recently been shown the cytoprotective and anti-inflammatory effect against various animal models. however, it is unclear whether the direct exposure of co to the intestinal inflamed mucosa is effective or not. in this study, we investigated the therapeutic efficacy of the rectal co administration for rat colitis model. acute colitis was induced with trinitrobenzene sulfonic acid (tnbs) in male wistar rats. co( ppm- ml) was intrarectally administrated twice a day after the induction of colitis. rats were sacrificed at days after the administration of tnbs. the distal colon was removed, and the ulcer lesions were measured. thiobarbituric acid (tba)-reactive substances and tissueassociated myeloperoxidase (mpo) activity were measured in the colonic mucosa as indices of lipid peroxidation and neutrophil infiltration, respectively. moreover, we evaluated the expressions of cinc- mrna/protein and p-p mapk protein. the intracolonic administration of co ameliorated tnbs-induced colonic ulceration. the increases in tba-reactive substances and mpo activity after tnbs administration were both significantly inhibited by treatment with co. moreover, the rectal administration of co significantly inhibited the increased expression of cinc- mrna/protein and p-p mapk protein after the induction of tnbs-induced colitis. the rectal administration of co protected from the intestinal inflammation in rats. based on these data, the beneficial effects of co on the intestinal mucosal injury may be attributed to its anti-inflammatory properties. alessandra gambero ( ), m maróstica ( ), m saad( ), j pedrazzoli jr ( ) ( ) s¼o francisco university medical school, brazil ( ) state university of campinas, brazil recent studies have shown that adipocytes produce and secrete several bioactive molecules like adipocytokines. the adipose tissue can also present short and long-term changes during inflammation and infectious pathologies. in this study, the alterations of mesenteric and perinodal mesenteric adipose tissue during experimental colitis induced by repeated intracolonic tnbs instillations were evaluated. the adipocyte size was measured after collagenase digestion. the basal lipolysis (glycerol release) and adipocytokine production was monitored after short time culture of adipose tissue. the colitis animals showed higher mesenteric fat mass ( . +- . and . +- . % of body weight for colitis and control, respectively; p< . ) in despite of the lower body weight. the mesenteric adipocytes from colitis animals presented reduced diameter ( . +- . and . +- . um for colitis and control, respectively; p< . ), higher basal lipolysis ( . +- . and . +- . ug.mg- for colitis and control, respectively; p< . ) and tnf-alpha production ( . +- . and . +- . ng.mg- for colitis and control, respectively; p< . ). perinodal mesenteric adipocytes presented normal diameters, higher basal lipolysis ( . +- . and . . +- . ug.mg- for colitis and control, respectively; p< . ), increased tnfalpha ( . +- . and . +- . ng.ml- for colitis and control, respectively; p< . ), leptin ( . +- . and . +- . pg.ml- for colitis and control, respectively; p< . ) and adiponectin production ( +- and +- ng.ml- for colitis and control, respectively; p< . ). the mesenteric adipose tissue was modified during the experimental inflammation, but some alterations were site specific. perinodal adipose tissue retained the ability to produce anti-inflammatory and pro-inflammatory cytokines, while mesenteric adipose tissue only the pro-inflammatory one. this work was financially supported by fapesp. inflammatory bowel disease (ibd) is a group of chronic inflammatory disorders of the intestine. the role of the pro-inflammatory p mapk signalling cascade in the pathogenesis of ibd is highly controversial. we therefore aimed to investigate the role of p mapk in chronic dextran sodium sulfate (dss) induced colitis, an experimental model of ibd. chronic intestinal inflammation was induced by oral cyclic administration of % dss in sjl mice. clinically, the dss treatment produced episodes of colitis manifested by diarrhoea, gross intestinal bleeding, marked loss of body weight, and shortening of the colon. at the molecular level, this was accompanied by an up-regulation of tnfa, il- â, il- , il- , kc, cox- , igg heavy chain, and phospho-stat in the dss treated mice.the clinical and molecular features described above recapitulate findings reported in human ibd. in order to assess the role of p mapk, the activation of the p mapk signalling cascade was analysed by western blot analysis. the expression and phosphorylation levels of both p mapk and of mk and hsp , two down-stream targets, were not increased in dss treated animals compared to controls. leo , a potent inhibitor of p activity in vivo, was dosed as pretreatment and after completion of dss treatment. pretreatment had a deteriorating effect on all measured cytokines, whereas treatment after disease induction had no effect on any measured parameters. collectively, these results strongly suggest that the p kinase pathway only plays a minor role, if any, in the dss model. (sp) were gmcsf differentiated, dcs purified through gr + cell depletion, and spleen tcells isolated by pan tcell negative selection. spdcs or bmdcs were stimulated +/- ng/ml lps. for mlr, balb/c tcells were added for days. cells were incubated with sb ( -( -fluorophenyl)- -( -ethylsulfinylphenyl)- -( -pyridyl) h-imidazole, sb) or ml ((rs)-{ -[ -( -fluorophenyl)- -methylsulfanyl- h-imidazol- -yl]pyridine- -yl}-( -phenylethyl)amine, ml) and washed prior to lps stimulation (bmdcs) or cell mixing (t cells). hthymidine incorporation was measured, cell viability by mtt assay, tnfa and il- production by elisa. mlr tcell proliferation inspdcs or bmdcs was inhibited by sb (ic spdc . mm, bmdc . mm) and ml (ic spdc . mm, bmdc . mm). preincubation with dcs had no effect, despite reduced lps stimulated il- and tnfa synthesis by sb (ic il- . mm, tnf . mm) and ml (ic il- . mm, tnf . mm). preliminary data shows that preincubation of t cells with sb and ml modifies the mlr response. p plays a role in the interaction of dcs and t cells in antigen recognition. however, pre-incubation of drugs with dcs was ineffective. the role of t cell p mapk in the mlr is under investigation. p inhibitors may possess disease modifying properties because of reduced tcell antigen reactivity to dc antigen presentation. ( ), s luik( ), s laufer( ), m seed ( ), v holan( ), s fiorucci ( ) ( ) synovo gmbh, tübingen, germany ( ) university of tübingen, germany in vivo anti-inflammatory activity of certain p kinase inhibitors is limited by bioavailability. however, it is possible that they may be useful in the therapy of ibd should it be possible to mediate there uptake in and around bowel lesions. we reasoned that activity could be especially increased if drug physical properties were altered to allow preferential partition into macrophages and neutrophils (wbc) associated with lesions. we prepared prodrugs of p inhibitors and screened them using whole human blood, murine spleenocytes and peritoneal macrophage. pharmacologically inert macrocycle (azilide) conjugates were assessed for enhanced efficacy in murine collagen induced arthritis either therapeutically (after onset of signs) or prophylactically ( d post boost) or in a dss or tnbs model of ibd in the mouse. in both types of models, the prodrugs achieved improved suppression of arthritis and inflammatory score in colon sections at tolerated doses with optimal activity at mmolkg- d- . we propose that the prodrugs increase efficacy via improved pharmacokinetics partly related to biased disposition of the prodrug toward immune cells. despite the potent anti-inflammatory and immunosuppressive properties of glucocorticoids its applying in the management of severe necrotizing pancreatitis is still controversial. the plasma levels of interleukins (il- , il- ) and adhesion molecules (e-selectin and icam- ) were measured in patients with necrotizing pancreatitis. the measurement was performed immediately after admission, at the , , and day. all patients were divided on two groups: first group compiled patients, in which dexamethasone ( mg/day during - days) was applied in the complex management of acute pancreatitis, and control group - patients that did not receive corticosteroids. all patients received the initial therapy. the increased levels of il- , il- , il- , icam- , and eselectin were noted in both groups of patients at the time of admission. the gradually increase of all proinflammatory mediators plasma levels up to seventh day was noted in patients of the control group. its levels clear correlated with the severity of mods and spreading of necrotic processes confirmed by ct. starting from the third day the gradually decrease of mediators levels were noted in the patients of the first group. the incidence of contamination of necrotic foci had no difference in both groups of patients. the ability of glucocorticoids to inhibit expression of proinflammatory mediators due to the glucocorticoids-mediated repression of nf-kappa b pathway provide the pathogenetic substantiation for the applying of glucocorticoids in the complex management of necrotizing pancreatitis. the objective of this study was to examine whether t cell specific overexpression of the th transcription factor gata- can inhibit th /th cell mediated experimental mbsa arthritis. mbsa-immunized wild type mice developed joint inflammation which gradually increased in time with a maximum at day . at day , t cell specific gata- tg mice did not show any difference in arthritis score compared to wild type mice. however, at day , wild type mice had developed severe joint inflammation having the maximum arthritis score. in contrast, gata- tg mice showed only mild joint inflammation, suggesting that t cell specific overexpression of gata- protects against development of severe joint inflammation. facs analysis revealed low levels of il- +/ifn-gammacells in wild type as well as in gata- tg mice at day . as expected, il- positive cells were higher in gata- tg mice compared with wild type mice. interestingly, at day , the percentage of il- +/ ifn-gamma-cells were markedly increased in wild type mice but not in gata- tg mice, suggesting prevention of th expansion under gata- overexpression in vivo. these data revealed that t cell specific overexpression of the th transcription factor gata- protects against progression of severe joint inflammation during mbsainduced arthritis. furthermore, il- +/ifn-gammacells play a critical role in the progression of joint inflammation in this model and gata- overexpression in t cells prevents expansion of the il- +/ifn-gamma-t cell subset. pingping jiang ( ), pt sangild( ), t thymann ( ), hh-y ngai ( ), w-h sit ( ), k-l chan ( ), jm-f wan ( ) ( necrotizing enterocolitis (nec) is a severe intestinal inflammatory disease for which the disease etiology and progression remain unclear. preterm delivery and enteral milk formula feeding are factors predisposing to nec. to understand the pathophysiology of nec, two-dimensional gel electrophoresis ( d page) proteomic approach was applied in studying changes in intestinal protein pattern in preterm piglets with spontaneous nec occurring in response to days of parenteral feeding followed by day of enteral formula feeding. the intestinal proteomes of pigs with clinical symptoms of nec (n = ) were compared with corresponding pigs remained healthy (n = ). syproruby staining was used and differently expressed proteins were identified by maldi-tof ms or maldi-tof/tof ms. the proteins with significantly different expression between nec and healthy pigs involve in energy metabolism (sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase and chain a, medium-chain acyl-coa dehydrogenase with -thiaoctanoyl-coa etc.), inflammation (peptide-binding protein (pbp ) and snail homolog ), signal transduction proteins (thyroid hormone binding protein precursor, park protein and chain b, structure of the rho family gtp-binding protein cdc in complex with the multifunctional regulator rhogdi etc.) and anti-oxidation (manganese-containing superoxide dismutase(sod)). these data underscore the significant impact of intestinal proteomics in unraveling nec pathophysiology and biomarker discovery. blood are used to monitor the progression of inflammation. the aim of our study were to investigate systemic markers of disease in a rat model of lps induced pulmonary inflammation to provide a link between preclinical in vivo research and early clinical research. animals were exposed to bacterial lipopolysacharide (e.coli :b ) by inhalation. the lungs were lavaged hours post provocation and the level of cell influx was determined. relevant mediators of acute pulmonary inflammation were analysed with standard elisa technology in bronchoalveolar lavage fluid and in blood. in addition, measurement of changes in body temperature were performed at different time-points post provocation in order to monitor the systemic inflammatory responses to the local pulmonary inflammation manifested as alteration in body-temperature. results showing the effects of lps challenge on local and systemic parameters will be presented and the possible link to lps responses in man discussed. pulmonary inflammation models are widely used in pharmacological research. however, provocations and treatments aimed directly at the lung are often invasive which limits the possibility to perform repeated administrations of test agents and compounds. also, results derived from bronchioalvelar (bal) fluid are subject to variability if the retrieval techniques are non standardized. here we describe a non-invasive standardized method for retrieval of bal fluid to be used in mice. we present the characterisation of these techniques using the inflammatory response to lps and propose that this non invasive method can be used to refine lps and other challenge models. the objectives were to evaluate the dynamic response after a single intra-tracheal administration of of mg ( ml/animal) of lps (p.aeruginosa) to c bl/ j mice. control animals received a single dose of sterile ml . % saline/animal. the mice were terminated , , , and h after instillation using a non invasive and operator independent lavage technique. results showing the effects of single lps challenge on bal parameters, excised lung gas volume and lung weight will be presented showing reliable dynamic responses. these techniques open the possibility to run repeated treatments and chronic provocations and are not subject to variability from bal fluid retrieval. the human psoriasis xenograft scid mouse model is one of the most accepted and well characterized models for screening of novel anti-psoriatic compounds. the model has primarily been applied for testing novel treatment principles via systemic or intradermal administration routes. in order to evaluate the model for topical treatment, psoriatic keratome biopsies were grafted to immune-deficient scid mice. transplanted mice were treated with daivonex /dovobet (calcipotriol) and bms (betamethasone). the results show a strong antipsoriatic efficacy after treatment with bms (epidermal thickness reduced by %). treatment with daivonex / dovobet also showed an anti psoriatic effect with a % reduction in epidermal thickness. serum did not contain test compounds, indicating that the observed effect were not due to systemic exposure. the observed effects are in concordance with clinical results of treatment of psoriasis. it is concluded that the model is useful for testing topical treatments. we have demonstrated that adult rats offspring of dams submitted to protein restriction during early lactation, presented impaired acute immune responses probably related to an imbalance in glucocorticoids and insulin secretion (barja-fidalgo; inflamm res ( ): ) . here, we evaluated the innate immunity mediated by neutrophils and host defense against infection in adult rats offspring of dams fed with either a protein free diet (un-group) or % protein diet (c-group) during the first days of lactation. un rats showed lower number of blood pmn, though no difference in bone-marrow neutrophils number was observed. blood neutrophils from un-group presented a significantly reduced phagocytic activity against opsonized zymosan, constitutively expressed inos and spontaneously produced o -, no and tnf-alpha. in vivo treatment with lps, at non-lethal doses, significantly increases tnf-alpha and superoxide production by neutrophils, compared with controls. lps increased no production by neutrophils from both groups, inducing inos expression in control cells, but no further increase in inos expression in un rats. nucleare nf-kb is constitutively augmented in un rats. un animals presented a higher survival rate in a model of clp-induced severe sepsis. these results indicate that a metabolic programming induced during early lactation affects the innate immune responses in adult rats, which are unable to properly mount an inflammatory response, may predispose to chronic diseases in adult life. transgenic mice over-expressing vascular endothelial growth factor (vegf) in the epidermal basal layer under the human keratin (k ) promoter have previously been reported to develop a psoriasis-like inflammatory condition in the skin. important hallmarks of psoriasis are epidermal hyperplasia in association with infiltration of t-cells in the dermis and epidermis and also increased dermal angiogenesis. the aim of this study was to describe the epidermal hyperplasia and the infiltration of the skin with t-cells in transgenic k /vegf mice. we induced a cutaneous inflammation in the ear skin by repeated topical treatments with -o-tetradecanoylphorbol- -acetate (tpa), in order to investigate the inflammatory response. the in vivo pharmacological effect of topical treatment with a number of reference compounds, including betamethasone- -valerate, was also investigated. the ear thickness was significantly increased in transgenic animals compared to wild type animals following tpa-induction. the epidermal thickness measured in histological sections of biopsies from the ear skin was also significantly increased in transgenic animals. furthermore, increased dermal vascularisation was observed in the histological sections of the ear skin. a marked infiltration with cd -positive cells was observed in both dermis and epidermis, and this was highly correlated with the increase in epidermal thickness. finally, topical treatment with betamethasone- -valerate significantly reduced the ear swelling and epidermal thickness. we conclude that over-expression of vegf in the epidermal basal layer plays an important role in skin inflammation and for the development of important psoriatic hallmarks. the model may furthermore be used as an in vivo screening tool for novel anti-psoriatic compounds. background and aims: the diabetes-prone bb (bbdp) rat spontaneously develops insulin-dependent diabetes resembling type diabetes (t d) in man. the bbdp rat is t-cell lymphopenic with a profound lack of regulatory t cells. the recent thymic emigrants in bbdp rapidly undergo apoptosis unless rescued from apoptosis by tcr stimulation. the increase in apoptosis is due to a frameshift mutation in gimap which causes a severe truncation of the protein. the mutation is the strongest genetic factor for rat t d. we aim to detect how gimap affects the lifespan of t cells. results: overexpression in c cells of both wt gimap and gimap with the bbdp mutation causes an increase in apoptosis -the latter with a very rapid onset. reduction of human gimap by rna-interference in jurkat cells did not affect the number of apoptotic cells. overexpression of human gimap causes apoptosis in jurkat cells and primary naïve t cells but not in activated t cells. finally, gimap -mrna is upregulated in in vitro activated human primary t-cells (detected by rt-pcr). conclusions: based on the phenotype of the bbdp, rat gimap was expected to be anti-apoptotic. however, we report here that overexpression of both mutated and wt gimap causes rapid death of the cells. this suggests that gimap is pro-apoptotic. the results with human wt gimap support this conclusion: recently, much focus has been on the cellular cd / cadpr signaling system during inflammatory processes. the cd /cadpr system has been shown to be regulated by interferon, estrogen and the proinflammatory cytokine il- . to our knowledge, the expression and function of the cd /cadpr signaling system in the human detrusor muscle have not been described. cd protein expression in cultured (explant technique) human detrusor smooth muscle cells (hdsmc) was demonstrated by western blot (wb) and confocal microscopy (cm). cytosolic free ca + concentration ([ca +] i) in hdsmc and isometric force in human detrusor strips were measured by spectrofluorometry and myograph technique, respectively. wb and cm showed that hdsmc expressed cell surface cd which could be upregulated by il- ( ng/ml). in hdsmc briefly activated with il- ( ng/ml) cadpr induced a rapid, transient dose-dependent increase in [ca +]i. cyclic adpr-mediated ca + increase was greatly reduced in ca + free medium suggesting ca + entry as well as ca + release. cyclic adpr -elicited ca + increase was mimicked by -deaza-cadpr, and blocked by -bromo-cadpr, a cadpr antagonist, but not by nifedipine or verapamil. in the presence of il- , cadpr caused concentration-dependent relaxations of detrusor muscle. we report for the first time that ) hdsmc express cell surface cd , ) the expression and function of cd are augmented by il- , ) externally added cadpr elicited a rapid, il- -dependent, and -bromo-cadpr-inhibitable ca + mobilization, ) cadpr induces relaxation of human detrusor muscle. the study indicates a role of cd /cadpr in human urinary bladder inflammation. miao lin is a formulation of sen miao san and lingzhi that consists of cortex phellodendri, atractylodisa rhizoma, radix achyranthis bidentatae, and ganoderma lucidum. these ingredients are reported to have anti-inflammatory and analgesic effects. in this study, we have investigated the anti-arthritic property of miao lin in an animal model of arthritis induced by unilateral injection of freunds complete adjuvant (fca) into rat knees. contents of the miao lin capsules were dissolved in saline and administered to the rats daily by intraperitoneal or oral route for days before induction of arthritis and days after. extension angle, size and blood flow of the rat knee joints were measured to give indexes of algesia, oedema, and hyperaemia, respectively. assessments of the extent of cell infiltration, tissue proliferation, and erosions of cartilage and bone provided additional indexes of the arthritis condition. single unilateral injection of fca into rat knees produced significant oedema, algesia, hyperaemia, immune cell infiltration, synovial tissue proliferation, and erosions of cartilage and bone in the ipsilateral knees compared with the contralateral saline-injected knees. intra-peritoneal injection of miao lin ( mg/kg/day) suppressed oedema, pain and hyperaemia in the inflamed knees, and oral administration ( mg/kg/day) suppressed oedema and hyperaemia. histological examination showed that both routes of administrations of miao lin reduced immune cell infiltration and erosions of cartilage and bone, and intraperitoneally administered miao lin also attenuated synovial tissue proliferation. these findings suggest treatment with intra-peritoneal or oral miao lin could provide significant anti-arthritic effects. an extract of the anti-arthritic thermalife cream contains trace elements. diffusion studies were undertaken to assess the permeability of human epidermis to the trace elements. non-penetrating trace elements were discarded from the test formula (t ), and compared with the original formula (t ) for in vitro anti-inflammatory efficacy (tnf-a secretion in lps-challenged human monocytes). methods: human epidermis was mounted in vertical franz type diffusion cells (stratum corneum facing up). t cream (n= ) or no cream (n= ) was applied to the donor compartment of diffusion cells, with pbs in the receptor compartment ( . ml ; stirred continuously at c). min after administration the receptor fluid was analysed for presence of metal ions by icp-ms. a replication study used a different skin donor. subsequently, human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ,) or not in the presence of % t , % t , or no treatment. hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). statistical analyses used a treat by lps anova (p < . ). results: zinc was the only trace element to penetrate the human epidermis significantly. both formulations strongly suppressed lps-induced tnf-a secretion. t with zinc only was more effective than t (treat:f , = . , p < . ; lps:f , = . , p < . ; treat by lps:f , = . , p < . ). conclusions: anti-tnf efficacy from thermalife extracts was retained with zinc chloride as the only trace element. ( ) ( ) osprey pharmaceuticals limited, canada ( ) probetex, inc., texas, usa the ccl /ccr chemokine/receptor axis, infiltrating monocytes/macrophages (m/m), th cells and mast cells play a pathological role in tissue damage and fibrosis in kidney diseases. the eradication of the supernumerary activated leukocytes should curb the production of inflammatory mediators and modulate chemokine communications, thus ameliorating disease. a recombinant fusion protein comprised of the human ccl chemokine fused to a truncated form of the enzymatically active a domain of shiga toxin has been developed. the ccl portion binds specifically to ccr -bearing leukocytes and enters the cells, where the sa portion inhibits protein synthesis. the compound was tested in a model of anti-thymocyte serum (ats)-induced mesangioproliferative glomerulonephritis. male rats were injected with ats on day and treated intravenously with vehicle, or mg/kg of the recombinant protein q d from day until day . urine and blood collections were made prior to ats injection and on days and . animals were sacrificed on day . no treatment related effects on body weight or signs of clinical toxicity were observed. urine protein levels were decreased in treated animals. histopathological analyses of kidney sections revealed maximum reductions of , , , and % for glomerular lesions, m/m count, fibronectin and µ-smooth muscle actin, respectively. the latter two proteins are markers for extracellular matrix synthesis and mesangial cell activation, respectively. these results indicate a significant renal-protective effect in this model of nephritis. further observations suggest that different chemokine-ligand toxins may be used in the treatment of diseases modulated by other chemokine/receptor axes. inflamm. res., supplement ( ) posters immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cytokines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. coronary artery disease (cad) is characterized by enrichment of inflammatory cells in the vessel wall. we hypothesized that an altered transmigration and activation of monocytes may contribute to plaque build up. in vivo transmigration was studied by use of a skin blister model. blisters are raised by suction and cells are analysed the following morning ( h blister) and after additional ten hours of incubation with pbs or autologous serum, corresponding to intermediate and intense blister. monocytes were analysed by flow cytometry for the expression of cd b, before and after in vitro fmlp stimulation. chemokines in serum and blister fluid was analysed in parallel. cd b expression on resting monocytes harvested from h blister was lower in patients as compared to controls (p= . ). lower expression of cd b in patients was also observed in the intermediate and intense blisters after stimulation with fmlp (p= . and p= . , respectively). the number of transmigrated cells was similar in both groups and increased with the intensity of inflammation. serum concentration of mip- µ was higher among patients (p= . ) and similar levels were seen in blister fluids. concentration of mcp- was similar in both serum and blister fluid. we demonstrate that monocytes from patients with cad have a reduced expression and ability to up-regulate the adhesion molecule cd b at sites of inflammation. these differences may modulate events related to the transmigration process and indicate a changed activation pattern. to which extent this feature might contribute to monocyte entrapment at the atherosclerotic site needs further studies to be delineated. in inflammation, nitric oxide (no) is produced by inducible nitric oxide synthase (inos) induced by bacterial products and cytokines, and no acts as a regulatory and proinflammatory mediator. one of the anti-inflammatory mechanisms of glucocorticoids is the inhibition of no production. the aim of the present study was to investigate the mechanisms how glucocorticoids inhibit inos expression and no production. dexamethasone and a dissociated glucocorticoid ru inhibited no production, and inos protein and mrna expression in murine j macrophages exposed to bacterial lipopolysaccharide (lps). in the presence of a glucocorticoid receptor (gr) antagonist mifepristone, the effects of dexamethasone and ru on no production were reduced. the role of histone deacetylation in the glucocorticoid effect was studied by using three inhibitors of histone deacetylases (hdacs); non-selective trichostatin a and apicidin, and hdac selective mc . hdac inhibitors reversed the effects of dexamethasone and ru on inos expression or no production. stably transfected a / cells containing human inos promoter were used in promoter-activity studies. cytokine-induced inos promoter activity was inhibited by dexamethasone and the inhibitory effect was reversed by trichostatin a. these results suggest that glucocorticoids inhibit inos expression and no production by a gr-mediated and gre-independent manner possibly through histone deacetylation and transcriptional silencing. we are investigating mechanisms involved in tnfainduced hyperalgesia in the mouse paw. previously, we have seen that tnfa causes a trpv -dependent bilateral hyperalgesia. here we investigate the role of cox in this process. female cd mice ( - g) were given intraplantar injections (i.pl.) of tnfa ( pmol/ microl) and tyrode (as vehicle, contralateral paw; microl). thermal hyperalgesic thresholds were measured using the hargreaves technique before and h after injection. indomethacin ( mg/kg) was co-injected with tnfa whilst contralateral paw was injected with tyrode and corresponding amounts of indomethacin vehicle ( % nahco ). another group of mice were injected with tnfa i.pl. plus % nahco with the contralateral paw injected with tyrode plus indomethacin ( mg/kg). results are expressed as mean ae s.e.m and statistical analysis performed using students t-test. tnfa ( pmol) leads to significantly reduced (p< . compared to baseline values) paw withdrawal latency in both paws h after injection i.e bilateral hyperalgesia. however, local injection of indomethacin ( mg/kg) with tnfa prevented this reduction in paw withdrawal latency in both paws suggesting that prostaglandins are important in the development of hyperalgesia. interestingly, indomethacin co-injected with tyrode in the contralateral paw did not prevent the reduction in paw withdrawal latency in both paws. the same results were seen using the selective cox- inhibitor, nimesulide. in conclusion, cox- derived prostaglandins are important in the development of hyperalgesia. local cox- inhibition at the site of tnfa-induced inflammation prevents the bilateral hyperalgesia suggesting that local prostaglandin production is sufficient to cause hyperalgesia in the contralateral paw. hydrogen sulfide (h s) is synthesized naturally in the body from cysteine by cystathionine g lyase (cse). h s has been variously reported to exhibit both pro-and antiinflammatory activity. in an attempt to obtain further information about the role of h s in inflammation we examined the effect of dexamethasone on lipopolysaccharide (lps)-mediated endotoxic shock. male sprague dawley rats ( - g) were administered dexamathasone ( mg/kg, i.p.) either h before or h after lps ( mg/kg, i.p.) injection. animals were killed h after lps administration and plasma and tissues harvested. as expected, lps injection significantly increased plasma tnfa and il- b as well as liver and lung myeloperoxidase (mpo) activity. lps also increased plasma nitrate/ nitrite (nox), h s concentration and liver and kidney h s synthesis from exogenous cysteine indicative of upregulation of cse in these tissues. either pre-or post treatment of animals with dexamethasone reduced signs of inflammation and also reduced the increase in plasma h s and tissue h s synthesizing activity. in separate in vitro experiments, exposure of rat peripheral leucocytes to lps ( ng/ml, h, oc) resulted in upregulation of both cse and inos (measured by western blot). dexamethasone ( nm) significantly (p< . ) reduced expression of both cse and inos. these data provide further evidence that h s is synthesised during endotoxic shock and suggest, for the first time, that at least part of the anti-inflammatory effect of dexamethsone may be related to inhibition of h s production. ( ), u jalonen ( ), h kankaanranta ( ), r tuominen( ), e moilanen ( ) ( ) the immunopharmacology research group, medical school, university of tampere and tampere university hospital, tampere, finland ( ) the division of pharmacology and toxicology, faculty of pharmacy, university of helsinki, helsinki, finland tristetraprolin (ttp), also known as nup , tis , g s and zfp , is a factor that binds to utr of mrna of some transiently expressed inflammatory genes and regulates mrna stability. ttp has been implicated in the posttranscriptional regulation of e.g. tumor necrosis factor a and inducible nitric oxide synthase. however, the regulation of the expression of ttp itself is largely unknown. in the present study, we investigated the role of classical protein kinase c (cpkc) isoenzymes in the regulation of ttp expression. in j macrophages ttp expression is induced by lipopolysaccharide (lps) and this can be further enhanced by addition of nm phorbol myristate acetate (pma). this additive effect of pma on ttp was abolished by a prolonged preincubation with a higher s inflamm. res., supplement ( ) posters concentration of pma for h, which also downregulated the expression of pkca, pkcbi and pkcbii isoenzymes. pkc inhibitors ro (inhibits pkcb, & and e), gÖ (inhibits pkca, b and &) and cgp (inhibits pkcbii) reduced lps + pma -induced ttp protein and ttp mrna expression. pkcbii inhibitor cgp did not affect ttp mrna half-life and therefore we measured the effects of cgp on the activation of transcription factors involved in ttp expression. cgp had no effect on the activation of nf-kb, egr or sp . in contrast, cgp reduced the activation of transcription factor ap- , which may explain its inhibitory action on ttp expression. the results suggest that pkcbii is involved in the regulation of ttp expression in activated macrophages, possibly through the activation of transcription factor ap- . the most widespread gracilaria verrucosa in the sea of korea is the attached form of red algae growing on a rockly substrate. in this study, we isolated fourteen compounds from g. verrucosa and investigated their inhibitory effect on the production of inflammatory markers (tnf-a il- , il- and no) in raw . cells. among them, -oxooctadec- -enoic acid and -oxooctadec- -enoic acid inhibited the production of tnf-a, il- , il- and no at the concentration of mg. also, these two compounds showed inhibitory activity on the mrna expression and protein level of inflammatory markers (tnf-a il- , il- and inos) in a dose-dependent manner. these results suggest that g. verrucosa may have anti-inflammatory activity through the inhibition of inflammatory cytokines and inos. lene jensen( ), p hjarnaa ( ), j fensholdt ( ), p-p elena( ), k abell ( ), tk petersen ( ) ( ) discovery, leo pharma, ballerup, denmark ( ) iris pharma, la gaude, france angiogenesis is known to play an important role in many inflammatory diseases including arthritis. additionally, inflammation is known to play a role in the angiogenesisdriven disease age-related macular degeneration (amd). we have synthesized a potent angiogenesis inhibitor, leo-a, targeting kinases related to angiogenesis, e.g. vegfr- . additionally, leo-a has potent effects on a broad panel of other kinases, whose normal functions are related to inflammation and immunity. the compound was tested systemically in inflammatory in vivo models in mice and rats. the in vivo models selected include the cia arthritis model (mice and rats), the local gvh rat model, the lps induced tnfa model (mice and rats), the anti-cd induced il- response mouse model and the rat argon laser-induced choroidal neovascularisation (chnv) model, a model for amd. the following results were obtained after systemic treatment with doses of up to mg/kg i.p. or mg/kg p.o. once daily: in the local gvh model, leo-a significantly inhibited the growth of the local lymph node by %. in the cia model, leo-a had a significant inhibitory effect on the progress of arthritis both in mice and in rats when dosed early (pretreatment). in the lps induced tnfa model in mice, high doses of leo-a were found to inhibit the tnfa release. in the chnv model, a significant effect was obtained following systemic treatment. in conclusion, leo-a has an interesting profile for the treatment of diseases in which inflammation and angiogenesis are involved. mice lacking pi kg and d isoforms display severe impairment of thymocyte development, but the outcome of this developmental defect has not been investigated. we show here that mice harbor pi kg gene depletion and pi kd kinase-inactive mutation, pik cgd koi, exhibited thymus atrophy, similar to previously reported pi kg and d double knockout (p g/d-/-) mice, and profound peripheral lymphoid depletion, markedly reduced lamda chain production and seemingly lymphopenia-provoked effector/memory t cell activity. in particular, serum igg / igg a ratio and ige level were elevated in pik cgd koi mice corresponding to a skewed th profile in vitro. histological analysis revealed eosionophil-and t celldominated inflammation in stomach and salivary gland as well as occasionally other organs of pik cgd koi mice, but organ-specific auto-antibody was not detected in circulation. on the contrary, when mature wt t cells were treated with pi k d or together with pi k g selective inhibitors, while th cytokines were suppressed th cytokines were not augmented in vitro. thus, t cell development, but not peripheral t cell proliferation or cytokine production, requires cooperativity of pi kg and d. genetic inactivation of these two isoforms leads to the development of severe lymphopenia, skewed type ig and t cell response, and increased susceptibility to eosinophilic multiple organ inflammation; whereas pharmacological inhibition at the adult stage would probably not promote th reaction but attenuate th medicated disorders. platelet activating factor (paf) is an important mediator in several pathophysiological processes. paf receptor activation can causes a series of cellular and tissue modifications and can lead to the production and/or release of diverse molecules, including cytokines, chemokines and receptors, amongst others, which are capable of amplifying the inflammation. paf can up-regulate kinin b receptor expression by various mechanisms. our aim was to investigate the role for kinases in paf-induced kinin b receptor up-regulation. wistar rats were treated with paf, or left untreated as controls, h before i.d. injection of . ml pbs containing des-arg -bradykinin (dapk, nmol right hind paw) and . ml pbs (for control, left paw). various kinase inhibitors were administered to the rats after paf treatment and oedema was measured by the use of a plethysmometer (ugo basile) - minutes after dapk-injection. oedema was expressed in ml as difference between right and left paws.additionally paw samples were taken for western blot analysis for total and phosphorylated forms of jnk and erk / . dabk-induced paw oedema after pafinjection is significantly inhibited by the selective jnk sp and erk / pd inhibitors. western blot analysis shows that phosphorylation of jnk and erk / is important in the up-regulation of b receptors. our results clearly show that the phosphorylation of both erk / and jnk mapkinases is an important step for the in vivo up-regulation of b receptors by paf. however, the exact mechanisms (transcriptional and post-transcriptional) by which paf can trigger kinase phosphorylation and then up-regulate the b receptor require further investigation. continued interest in development of small molecule inhibitors of p mitogen-activated protein (map) kinase is based on the central role this enzyme plays in inflammatory cell signaling. activation of p leads to increase production of pro-inflammatory cytokines such as tnf-a and il- b making it an prominent target for antiinflammatory drug discovery. a virtuell screening approach identified -( -chlorophenyl)- -(( -methoxyphenoxy)methyl)- [ , , ] triazolo [ , -b] [ , , ] thi adiazole as a potential hit. this was confirmed by synthesis and testing. to explore further sar, a first set of derivates was prepared by cyclization of the -substituted- -amino- -mercapto- h- , , -triazoles with carboxylic acids in presence of phosphorus oxychloride. the synthetic strategy used allows both variation at position and . synthesis and sar will be presented. cytokines like il- b and tnfa play central roles in inflammatory diseases like rheumatoid arthritis. production of cytokines in monocytes, macrophages and other cells is triggered by factors such as lps, uv-light, osmotic and cellular stress or physical and chemical attraction. in particular il- b and tnfa are key regulators as they amplify inflammatory stimuli in cells by induction and upregulation of further cytokines. involved in this signal pathway, p mapk as a pivotal enzyme is considered to be a validated drug target and therefore, p mapkinhibitors are of therapeutical interest. in this study, we developed and validated an economic in vivo whole blood assay for optimization and characterization of small molecule p mapk-inhibitors with promising in vitro activity. the assay procedure involves defined blood cell stimulation by lps and isolation of tnfa or il- b, which are subsequently quantified by tmb-elisa technique via photometric measurement. the validation of the assay conditions involved well characterized p mapk inhibitor sb and a highly active compound developed in our lab. a data set was generated by determining whole blood samples consisting of in each case three male and female individuals on three different days. statistical methods were used to analyze specificity, baseline-peak correlations, repeatability, robustness as well as gender specific intra-and interindividual differences. p mitogen-activated protein (map) kinase is required for the biosynthesis and release of pro-inflammatory cytokines il- and tnf a. inhibition of p map kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders, like rheumatoid arthritis and inflammatory bowel disease. trisubstituted pyridinylimidazoles are potent inhibitors of the p map kinase. scope of this work was to investigate -thio-ether moiety as a position to link the inhibitors to macrocyclic drug carriers. we synthesised -alkylsulfanyl, -( -fluorophenyl), -( -aminopyridin- -yl) substituted imidazoles as p map kinase inhibitors. as substitution at this pyridinyl moiety allows both increase the anti-inflammatory activity as well as selectivity. the synthesis and biological testing of effective the -aminopyridin- -yl imidazoles with low inhibitory concentrations are described. biological data demonstrate both the imidazole derviates and the linked imidazoles lead to highly efficient inhibitors.variation at the -thio-ether moietywhich interacts in the phosphate binding region of the enzyme -with polar groups shows no loss of activity. studies underscored the importance of hydrogen bonding with the backbone nh group of met , for inhibitory activity. less clear is the importance of the hydrogen bond between n of the imidazole ring and lys of p map kinase.to investigate the role of lys in interacting with the scaffold we prepared two sets of , diaryl-substituted isoxazoles. these data suggest a dynamic interaction of the core heterocycle with lys , contrary to the observation on the compound vk- and p map kinase, that a nitrogen atom bearing a lone pair in position of the imidazole ring could be necessary to avoid a repulsive interaction with the positively charged side chain of lys rather than to form an attractive interaction with p map kinase. to complete our study, we focused on the interdependency of biological effects exerted by substitution at the pyridine ring for a series of -substituted and unsubstituted , -diarylisoxazoles investigating the interaction with the hydrophobic pocket ii of p . these data indicate that the isoxazole has better scaffold properties compared with imidazoles, suggesting that heterocycles that are stable as regioisomers, such as isoxazole (in contrast to tautomeric imidazoles), are worthy of further investigations. despite of the intensive research effort, sepsis is still the leading cause of death in critically ill patients. it is a consequence of acute inflammatory response to lipopolysaccharide (lps), a major component of the outer membrane of gram-negative bacteria. natural products are known sources of bioactive components exerting antioxidative and anti-inflammatory effects. in this study, we investigated the effect of ferulaldehyde (fa), a natural compound of red wine, on lps-induced endotoxic shock in mice and on lps-stimulated murine macrophage-like raw . cells. treatment of c bl/ mice with fa significantly attenuated the lps-induced inflammatory response in the gastrointestinal tract, and decreased the level of the two major pro-inflammatory cytokines tnf-a and il- b in the serum. the serum level of the anti-inflammatory cytokine il- was higher in mice treated with fa and lps compared to lps treatment alone. lps-induced phosphorylation and thereby activation of akt, and jnk was also strongly inhibited by fa treatment whereas the phosphorylation level of erk / and p mapks remained unaltered. activation of nuclear factor-kappab (nf-kb) in liver of fa-treated mice were significantly suppressed. although fa had no effect on the production of inflammatory cytokines, or on inhibition of signal transduction pathways in raw . cells either, it decreased the lps-induced ros and nitrite production in a dose-dependent manner. our results suggest that fa has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems, which in turn decrease inflammatory cytokine response and suppress nf-kb activity via the down-regulation of akt and jnk. myeloperoxidase (mpo), stored in the azurophilic granules of the neutrophil granulocyte, is a heme enzyme with the unique property of oxidising chloride ion to the powerful reactant hypochlorous acid in the presence of hydrogen peroxide. therefore, it plays an important role at inflammatory loci in killing invading micro-organisms. on the other hand, hypochlorous acid reacts with a variety of biomolecules as amino acids or membrane lipids and causes therefore host tissue damage resulting in widespread diseases like atherosclerosis or rheumatoid arthritis, e.g. the formation of chloramines from taurine or ammonium ions is one possibility to reduce tissue toxicity while maintaining bactericidal properties. membrane charge alterations during apoptosis provide docking sites for the kationic enzyme myeloperoxidase and this close contact to the membrane lipids opens the possibility for lipid alteration pathways even though these reactions will normally not take place because of their slowness. we investigated alterations in phospholipids after reaction with hypochlorous acid or the myeloperoxidase-hydrogen peroxide-chloride system by matrixassisted laser desorption and ionisation time-of-flight mass spectrometry (maldi tof ms). specific reaction products play an important role in the modulation of the immune response. comparative pathobiology of the disease is also discussed within the context of current human and animal reoviral disease models. objectives: to study the safety and efficacy of infliximab plus leflunomide combination therapy in adult rheumatoid arthritis (ra). methods: twenty patients with active ra received leflunomide mg for days followed by mg daily for weeks. at week all patients started infliximab mg/kg, and received a further four infusions at weeks , , and . results: the commonest adverse event was pruritis associated with an eczematous rash. there was no relationship between the serum concentration of a , the active metabolite of leflunomide, and adverse events. the mean disease activity score (das ) fell from . at week to . (p< . ) at week and remained between . and . up to week . in those patients remaining on treatment, more than % achieved an acr response from week to week , and up to % achieved an acr response. conclusions: infliximab plus leflunomide combination therapy appears to be highly efficacious in the treatment of adult ra. however, widespread use may be limited by adverse events, which were common and in some cases severe. objective: the transcription factor nuclear factor-kb (nfkb) regulates the expression of proinflammatory cytokines such as tnfa and il- those play pivotal roles in pathogenesis in rheumatoid arthritis. parthenolide, a sesquiterpene lactone, was reported to inhibit the dna-binding of nfkb. the objective of this study is to investigate the potential of parathenolid to inhibit the pathogenesis of collagen-induced arthritis. methods: mice were injected i.p. with a cocktail of anticollagentype ii mabs on day , followed by i.p. injection of lps on day to induce anti-collagen mab-induced arthritis. the mice were orally administrated with parathenolide ( mg/kg/day) starting on the day of first immunization (day ) in prophylactic treatment group and after the onset of arthritis (day ) in the therapeutic treatment group. clinical disease score, radiographic and histological scores were evaluated. mrna expression of il- b and tnfa in the affected joints were measured by real-time pcr. results: clinical disease scores were significantly reduced both in prophylactic treatment group ( . ae . ) and therapeutic treatment group ( . ae . ) compared to untreated group ( . ae . , p = . and p = . respectively). histological scores of joint destruction were significantly reduced in prophylactic treatment group compared to untreated mice (p< . ). steady state mrna levels of il- b and tnfa in isolated joints were significantly decreased in prophylactic treatment group compared to untreated mice (p< . ). the results in this study suggest that nfkb is an important therapeutic molecular target for treatment of inflammatory arthritis. fibrinogen is a soluble plasma glycoprotein, multifunctional, that participates in haemostasis and has adhesive and inflammatory functions through specific interactions with other cells. the concentration of this glycoprotein increase in inflammatory conditions. a fundamental paradigm involved in the acute inflammatory response is neutrophil migration to the affected tissues to mount an initial innate response to the aggression. the objective of this study is to characterize how fibrinogen modulates the pattern of neutrophil activation. neutrophils from healthy donors were isolated from peripheral venous blood and loaded with the fluorescent probe dihydrorhodamine ( ìm) to detect oxygen free radical production. the cells ( , x cell/ml) were then incubated with a range of concentrations of fibrinogen ( - mg/dl) for minutes. our results show that posters inflamm. res., supplement ( ) s fibrinogen leads to an increase in neutrophil activation as measured by free radical production. this effect becomes evident at borderline-high concentrations ( - mg/ dl), and in some of the individuals it was possible to differentiate two subpopulations of low-responsive and high responsive neutrophils to activation by fibrinogen. we hypothesize that, in this regard, the concentrations of fibrinogen identified as a risk factor might promote the setting of an inflammatory microenvironment in the circulation and facilitate cardiovascular disease progression. cyclooxygenases (cox- and cox- ) are isoenzymes involved in the first steps of the biosynthesis of prostanoids. the constitutively expressed isoform cox- is mainly involved in homeostatic processes, while the inducible isoform (cox- ) is associated with inflammatory reactions. various in vitro assays have been developed in order to define the selectivity against cox- and cox- of nonsteroidal anti-inflammatory drugs (nsaids). however, these in vitro assays can give discordant results related to several parameters. the aim of this study was to optimize and standardize two distinct in vitro methodologies to evaluate new nsaid candidates. first, in an enzymatic cox assay, the arachidonic acid concentration (aa; cox substrate), and the species of cox enzymes tested (ovine vs. human), two factors able to conceal the anti-cox activity of nsaids, have been evaluated and optimized. next, we developed an in vitro cell-based assay using human whole blood depleted from plasma and reconstituted in saline solution. this cell-based assay allows concomitant measurement of anti-cox- and anti-cox- effects by prostaglandin e (pge ) measurement after a (calcimycin) and bacterial lipopolysaccharide (lps) stimulations, respectively. both assays have been calibrated and compared by testing reference nsaids, selective or not for cox- or cox- . fifty % inhibitory concentration (ic ) values against cox- and cox- and cox- :cox- ratios obtained were in accordance with the previously described nsaid specificity and coherent between both assays (r= . ). in conclusion, both in vitro assays are optimized to determine the efficiency and the selectivity of new nsaid candidates against human cox- and cox- . for increasing the success of preclinical drug candidate molecules, there is a need for translatable animal models. the human serum skin chamber technique and the rodent carrageenan induced air pouch model are two wellestablished methods for measuring interstitial inflammation in respective species. we aimed to study the translational aspects of these models. material and methods: in humans, epidermal skin chambers were stimulated with autologous serum for hours. in rats, a dorsal subcutaneous air pouch was stimulated with autologous serum on day . the inflammatory response was measured after , and hours. the cellular distribution of in vivo transmigrated cells, the expression of cytokine receptors, adhesion molecules and inflammatory mediators was investigated. results: at / hours the cellular distribution was similar in air pouch and skin chambers. the major population constituted of granulocytes, followed by monocytes/macrophages and lymphocytes. both in human and rats the concentrations of mpo and mcp- were increased. furthermore, transmigrated cells displayed a different chemokine receptor pattern. in rats transmigrated cells expressed cd b, were cd lo, ssclo and rp- + (granulocyte marker). in humans, transmigrated granulocytes expressed cd and cd b. these cells had a significantly higher cd b expression compared to corresponding cells in peripheral circulation. our results indicate that the serum induced human skin chamber technique and rodent air pouch model translate well to each other. these models may be useful for bridgingpreclinical and clinical drug discovery. furthermore, they may work as translatable proof of mechanism (pom) models for drug candidates targeting different inflammatory components. objectives: to analyse if neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke. material and methods: we investigated consecutive patients with mean age ae . years who were admitted within h after ischemic stroke. a control group of patients with mean age ae . years without ischemic stroke was also tested. measurement of serum neopterin levels were performed using enzyme linked immunosorbent assay. results: patients with acute ischemic stroke had significantly higher serum levels (mean value+sd) of neopterin than those without acute ischemic stroke: . ae . and ae . nmol/l. correlation analysis revealed p< . . discussion: immune mechanisms contribute to cerebral ischemic injury. the finding of higher serum levels of neopterin, which is regarded as a humoral component of the immune-mediated inflammatory response sustains the hypothesis that patients with ischemic stroke may show higher levels of inflammatory markers like neopterin. our results indicate increased macrophage activation after ischemic stroke. in patients with stroke it has been shown that neopterin was determinant of endothelium-dependent vascular dysfunction. however, these preliminary results need be confirmed by controlled studies. produced a marked (p< . ) reduction in the number and duration of ventricular tachycardia (vt) during both ischemic and reperfusion phases. the total number of ischemic ventricular ectopic beats (vebs) reduced from ffae in the control to ffae at the concentration of ffmg/ml (p< . ). in the ischemic phase, cynodon dactylon ( ffmg/ml) also decreased the incidence of vt from % (control) to %. in addition, incidences of reperfusion-induced vt, total vf and reversible vf duration were significantly lowered by the same concentration (p< . for all). the results show that cynodon dactylon has a protective effect against i/r-induced cardiac arrhythmias in isolated rat hearts. regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their potential role in the scavenging of oxygen free radicals, it seems that the cardioprotective effects of cynodon dactylon probably is due to its anti-inflammatory properties.key words: cynodon dactylon; arrhythmias; anti-inflammatory; isolated heart; rat objectives: intestinal ischemia-reperfusion (iri) is well known to be associated with distant organ dysfunction; but no evidences to date have focused either the brain or skeletal muscle. we thus decided to investigate the effects of iri on nos and cox isoforms, neutrophil infiltration (mpo), lipoperoxidation (tbars) and protein tyrosine nitration (nt) in different brain areas and diaphragm muscle of wistar rats. methods: iri comprised the occlusion of superior mesenteric artery during min followed by h of reperfusion. sham animals were submitted to the surgical procedure with no interference on the blood flow. results: iri resulted in increased expression of mrna for nnos (cortex) and cox- (hypothalamus) associated to a marked reduction of ca +-dependent nos activity in cortex, hypothalamus and hippocampus (but not in cerebellum). tbars contents were also reduced in cortex and hypothalamus. neither mpo activity nor nt was altered by iri in the brain. diaphragms from animals with iri exhibited increased mpo and ca +-dependent nos activities, as well as tbars content and nt. in contrast, enos protein expression and both gene and protein nnos expression were reduced. no effects were observed on cox isoforms or enos gene expression. conclusions: these findings suggest that, within the first h of reperfusion following intestinal ischemia, an oxidative response is observed in diaphragm, involving both lipid and protein modifications. in the cns, distinctive susceptibility to the iri seems to occur in the different areas, probably as a defensive strategy aimed to counteract the iri-mediated systemic injury. anne-sofie johansson ( ), h qui ( ), m wang ( ), i vedin ( ), jz haeggstrçm ( ), j palmblad ( ) ( ( ), r carnuccio( ), p romagnoli ( ), f rossi ( ) ( ) second university of naples, italy ( ) university of naples, italy ( ) university of florence, italy we previously found that several inflammatory markers, e.g., nuclear factor-kb (nf-kb), were increased and a neointima was formed in a model of carotid surgical injury ( ) . the purpose of the present study was to determine if chronic treatment with rosiglitazone protects rat carotid artery from surgical injury induced by an incision of the vascular wall. to this aim we measured cox- , nf-kb, platelet aggregation and neointima formation in rats administered rosiglitazone ( mg/kg/ die, by gavage) for days before carotid injury and days after injury. control rats received physiological solution. days after injury cox- expression, evaluated by western blot, was significantly lower in the treated carotid versus controls (p< . ). rosiglitazone also caused a significant decrease of nf-kb/dna binding activity, evaluated by electrophoretic mobility shift assay, in nuclear extracts of treated carotids at all time points considered. platelet aggregation was reduced by % in treated versus control carotids (p< . ). the influx of inflammatory cells in response to injury, monitored by electron microscopy and immunohistochemistry, was lower in treated than in control carotids starting days after rosiglitazone treatment. the results indicate that rosiglitazone inhibits molecular and cellular inflammatory events induced by vascular injury. the aim of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (eae). rats of eae-susceptible dark agouti and eae-resistant albino oxford strain were immunized with guinea pig spinal cord homogenate (dagpsc and aogpsc), while non-immunized rats served as controls (danim, aonim). on day after immunization rat peritoneal macrophages were tested for adherence capacity, zymosan phagocytosis and respiratory burst. macrophages from aonim rats exhibited lower adherence capacity and higher phagocytosis and h o production then macrophages from danim rats. immunization decreased adherence and phagocytosis and increased h o production in macrophages from ao rats, but did not influence these activities in macrophages from da rats. our results suggest that inflammatory activities of macrophages from ao rats could be considered as regulatory mechanisms connected with the resistance to eae induction ( ( ), b sehnert ( ), h lanig( ), s päßler( ), r holmdahl ( ), h burkhardt ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander university of erlangen-nuremberg, germany ( ) lund university, sweden objectives: the aim of the present study was to characterize the interaction sites between the prototypic arthritogenic murine igg mab ciic that is highly somatically mutated and its epitope on type ii collagen (cii, aa - ). methods: the establishment of a dynamic simulation modelling of a ciic single-chain fragment (scfv) in complex with the triple helical ciic epitope permitted structural insights into immune complex formation. the computer-based data were experimentally tested by mutations of predicted critical residues into alanine in the c scfvs and the respective ciic epitope that were produced as recombinant constructs. the binding affinities of the mutated scfvs were determined by elisa and surface plasmon resonance measurements. the mutation experiments confirmed the predicted interaction sites of cii in the cdr and cdr regions of both heavy andlight chain. surprinsingly also the model prediction, that the conversion of the c scfv sequence into the respective germline does not affect cii binding affinity (kd x - ) could be confirmed experimentally by the mutagenesis of (!) positions. our data indicate that potentially harmful cartilage specific humoral autoimmunity is germline encoded. the molecular modeling further demonstrate that the rigid collagen triple helix restricts the likelihood of molecular interactions with the corresponding cdrregions of the antibody considerably compared to globular antigens. these sterical constraints might provide an explanation why somatic mutations have no obvious impact on cii recognition by the arthritogenic autoantibody. moreover, the structural insights into cii-autoantibody interaction might be useful in future developments of collagenomimetic ligands for therapeutic and diagnosistic purposes. we observed a significant association between the mbp-elicited cd + t-cell proliferation and active brain lesions, on the one hand, and il- , il- and ifn-gamma, on the other. when grown in the presence of standard serum from a healthy donor, pbmc from healthy individuals responded to mbp with a higher il- production than pbmc from ms patients. thus, normal pbmc respond to mbp with production of tnf-alpha, ifn-gamma and il- , but ms is associated with enhanced tnf-alpha-, ifn-gammaand decreased il- responses, and disease activity is associated with mbp-induced proliferation of cd + t cells. ( ), k goula ( ), p georgakopoulos ( ) ( ) renal unit, st. anrdew hospital, patras, greece ( ) intensive care unit, st. anrdew hospital, patras, greece background: urethritis is an infection of the urethra. most cases are sexually transmitted. haemodialysedpeople seem more prone to all kinds of urinary tract infectionsthan others. patients with underlying diabetes are also a specific population at risk. urethritis may be caused by some sexually transmitted diseases (chlamydia, gonorrhea, and ureaplasma urealyticum infections) and by the same organisms that cause urinary tract infections (e. coli or klebsiella). viral causes of urethritis include herpes simplex virus and cytomegalovirus. neisseria gonorrhoeae and c trachomatis account for most cases of urethritis in men ( %). the aim of our study was to determine all cases of urethritis of haemodialysed patients at our unit during the last five years. we also determined diabetes as a coexisting factor in the infected patients. we retrospectively reviewed all cases of urethritis of maintenance haemodialysis patients at our center over the past years. the diagnosis was made according to patients symptoms and signs but also using urine specimens for culture. patients ( . %) from the study group were diabetic. results: cases of urethritis were determined. all infected patients were diabetic. isolated microorganisms were e. coli ( cases), enterobacter aerogenes ( case objectives: to explore the ability to use paquinimod as a steroid sparing drug in an animal model for sle. methods: mice were initially treated with a high dose of prednisolone ( mg/kg/day). thereafter the amount of steroid was reduced to . mg/kg/day and a low dose of paquinimod ( . mg/kg/day) was added. the development of glomerulonephritis was measured as hematuria during the experimental period. serum was collected for analysis of anti-dsdna antibodies. kidneys were collected and histopathological observations were performed. organ weight and lymphocyte sub-populations were assessed in the spleen. results: when treatment with high dose prednisolone was replaced by low dose prednisolone andpaquinimod a steroid sparing effect was seen in a number of variables. a significant reduction in the level of hematuria, in spleen enlargement and in the total number of cd +, cd + and on cd -cd -t cells was observed in mice treated with paquinimod and low dose of prednisolone compared to mice treated with high dose prednisolone alone. the development of glomerulonephritis was also significantly reduced in these mice. an almost complete inhibition of anti-dsdna in serum was seen in all treated groups. conclusions: when high dose prednisolone was replaced by low dose prednisolone and paquinimod a steroid sparing effect was seen when a number of variables e.g., hematuria, t-cell sub-populations and development of glomerulonephritis were examined. this setting could be of great importance in future treatment of human sle in order to reduce the steroid dose needed in the treatment of this disease. and la(ssb). the purpose of this study was to screen for novel antibodies against cell surface antigens in primary sjs. proteins (mp) were isolated from cell membranes (hela cells), and were tested with sera from sjs patients or healthy blood donors individually in western blot (wb) at : . mp were separated on -d gels and tested in wb ( : ) to locate the appropriate spots for mass spectrometry (ms) analysis. paraformaldehyde fixed hela cells were incubated with sera from patients or blood donors and examined by fluorescence microscopy. antigens were isolated at around , , , kda ( total positive/ tested patients). the dominant antigen was at kda. large quantities of endogenous proteins were obtained and the membrane fraction was enriched. one of the main obstacles to further study possible surface antigens as m muscarinic receptor was overcome. proteins were separated on d-gels and tested in wb to locate the relative spots for ms. the correct localization of the patients antibodies on the cell surface was confirmed by fluorescence microscopy. in conclusion, membrane or membrane-associated antigens were recognised by sera from sjs patients. one of them might correspond to m muscarinic receptor. this identification might help in developing a diagnostic assay for sjs. osamu handa, s kokura, k mizuahima, s akagiri, t takagi, y naito, n yoshida, t yoshikawa aim: various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration. however the precise mechanism of cytotoxicity of these additives are not known. in this study, we investigated the effects of ultraviolet-b (uvb) exposure on additives-treated human normal skin keratinocytes (hacat). most popularly used additives in cosmetics such as methylparaben (mp), octandiol (od) and phenoxyethanol (pe) were used. hacat keratinocyte was cultured in mp-containing medium for h, exposed to uvb and further cultured for another h. subsequent cellular viability was evaluated by fluorescent microscopy and flow cytometry using double staining method with hoechst and propidium iodide or annexin-v. same experiments were done using od and pe respectively instead of mp under same condition. in addition, gene chip analysis was performed in each group. results: uvb exposure enhances cytotoxicity of these additives even at low concentration. gene chip analysis showed that the expression of apoptosis-related genes, oxidative stress-related genes and transcription related genes were significantly upregulated in each group. these results indicate that some additives, which have been considered safe preservatives in cosmetics, may have harmful effects on human skin when exposed to sunlight. these kinases in the pathogenesis of psoriasis. recently, increased focal activation of the downstream target mitogen-and stress-activated protein kinase (msk ) was demonstrated in psoriatic epidermis. the purpose of this study was to investigate msk and the transcription factor camp-response-element-binding protein (creb) in psoriatic skin and in cultured normal human keratinocytes. keratome and punch biopsies were taken from patients with plaque-type psoriasis. normal human keratinocytes were cultured and stimulated by interleukin- â (il- ß) or anisomycin. some of anti-inflammatory plant flavonoids as a form of whole plant extracts have been used topically for skin inflammatory disorders. on human skin inflammation, matrix metalloproteinase- (mmp- ) plays a pivotal role on unbalanced turn-over or rapid breakdown of collagen molecules. in the present study, for establishing a therapeutic potential against skin inflammatory disorders, the effects of natural flavonoids on mmp- activity and mmp- expression were examined. from the results, the flavonols including quercetin and kaempferol were revealed to be strong inhibitors of human recombinant mmp- with the ic s of . - . ìm, while the flavones such as apigenin and wogonin showed weak inhibition. when the effects of flavonoids on mmp- induction were studied, it was found that quercetin, kaempferol, apigenin and wogonin ( . - . ìm) strongly inhibited mmp- induction from tpa-treated human dermal fibroblasts, but naringenin (flavanone) did not. by gel shift assay, these flavonoids were also found to inhibit the activation of the transcription factor, ap- , whereas naringenin did not. among mapks, quercetin inhibited the extracellular signal-regulated protein kinase (erk) and p mapk activation, and kaempferol inhibited the p mapk and c-jun n-terminal kinase (jnk) activation. on the contrary, the flavones and naringenin did not inhibit the activation of these three mapks. all these results indicate that the capacity of mmp- inhibition and mmp- down-regulation of flavonoids may block collagen breakdown in certain pathological conditions and certain flavonoids are useful to treat skin inflammation, especially by topical application. ( ) ( ) university of valencia, spain ( ) istituto di chimica biomolecolare cnr, napoli, italy avarol is a marine sesquiterpenoid hydroquinone with several pharmacological properties including antioxidant, anti-inflammatory, and antipsoriatic effects. recently, its derivative avarol- -thiosalicylate (ta) also demonstrated interesting perspectives as anti-inflammatory drug in vitro and in vivo.it is interesting to note that avarol and ta inhibited nf-Þb activation in hacat keratinocytes. now, the effect of avarol and ta was investigated in the tpa-induced hyperplasia murine skin model, which presents some similarities with psoriatic lesions. topical treatment with ta ( mg/ml) produced a % inhibition of oedema and a strong reduction of pge ( %), ltb ( %) and mpo activity ( %) in skin homogenates. the inhibitory effect of avarol at the same dose was % for oedema, % for pge , and % for ltb and mpo activity. histological study for both compounds showed a decrease in epidermal hyperplasia as well as leukocyte infiltration respect to tpa treatment. besides, the reduction of cutaneous tnf-a by avarol and ta was also detected by immunohistochemical analysis. these compounds were also capable of suppressing nf-Þb nuclear translocation in mouse skin. in summary, our results suggest that inhibition of proinflammatory metabolites by ta and avarol might be beneficial for the treatment of the inflammatory component of psoriasis. its mechanism of action is related to the inhibition of nf-Þb activation and can be mediated by the downregulation of intracellular signal-transduction ( ), ams silva( ), cmm santos( ), dcga pinto( ), jas cavaleiro( ), jlfc lima ( ) ( ) requimte, departamento de química-física, faculdade de farmµcia da universidade do porto, porto, portugal ( ) departamento de química, universidade de aveiro, aveiro, portugal -styrylchromones are a novel class of chromones, vinylogues of flavones ( -phenylchromones), which have recently been found in nature. several natural and synthetic chromones have demonstrated to possess biological effects of potential therapeutic applications. however, the anti-inflammatory potential of -styrylchromones has not been explored so far. thus, the aim of this work was to evaluate the putative anti-inflammatory properties of several synthetic -styrylchromones by studding their influence on different systems that are related to the inflammatory process. the putative inhibitory effects of several -styrylchromones on the proinflammatory enzymes cyclooxygenase (cox- ), cyclooxygenase (cox- ) and -lipoxygenase ( -lox) was evaluated in vitro and compared with structurally related flavonoids. the capacity of the studied -styrylchromones to scavenge reactive oxygen (ros) and nitrogen species (rns) was also assessed by different in vitro assays, which allowed to identify the influence of those compounds in each reactive species, separately. from the tested -styrylchromones, those having a catecholic bring where shown to be the most effective scavengers of ros and rns, being, in some cases, more active than flavonoids. no considerable correlation was found between the scavenging profile of these compounds and their interactions with pro-inflammatory enzymes. the results obtained from the present study indicate that some of the tested compounds are promising molecules with potential therapeutic value. the usefulness of -styrylchromones in the prevention or control of inflammation can only be clarified with additional studies concerning their influence on other relevant mechanisms of this pathology. the importance of tumor-associated inflammatory cells, able to affect different aspects of neoplastic tissue, is a current matter of debate. primarily monocytes are recruited from the circulation into solid tumors and metastases where they differentiate into macrophages with several phenotypes and, e.g., may significantly contribute to uptake of certain radiotracers. we therefore sought to characterize the uptake of various radiotracers used for positron emission tomography (pet) in a well characterized in vitro model of human monocytes/macrophages in comparison with that in various human tumor cells. uptake of radiotracers f-fluorodeoxyglucose (fdg), -o-methyl- - f-fluoro-l-dopa (omfd), and f-labeled native/oxidized low density lipoproteins (nldl, oxldl) in single-or cocultivated human myeloid (monocytic) leukemia cell line thp- was compared with that by squamous cell carcinoma (fadu), mamma (mcf- ) and colorectal adenocarcinoma (ht ) cell lines (without or in the presence of specific inhibitiors). several thp- phenotypes along the monocytic pathway (monocytes, differentiated macrophages, retrodifferentiated cells) were studied before, during and after incubation with phorbol myristate acetate. differentiated thp- cells show, when compared with tumor cells, a comparable fdg accumulation, a considerably lower omfd uptake, and a significantly higher oxldl uptake. on the other hand, during differentiation and retrodifferentiation thp- cells obviously establish a distinct sequence of biological processes also reflected by considerable alterations in radiotracer uptake. the observed differences in uptake of several radiotracers in vitro in-between thp- phenotypes and between thp- phenotypes and tumor cells, respectively, stimulate studies on the contribution of macrophage radiotracer uptake to the overall uptake in neoplastic or inflammatory lesions in vivo. genomic and full-length cdna sequences provide opportunities for understanding human gene expression. determination of the mrna start sites would be the first step in identifying the promoter region, which pivotally regulates transcription of the gene. although the mrna start sites of most genes show heterogeneity, this may reflect physiological, developmental, and pathological states of the particular cells or tissues. recently, we have developed a -end sage ( sage) that can be used to globally identify the transcriptional start sites and frequency of individual mrnas. a strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. another important factor in tumor development seems to be the epigenetic effects on tumor suppressor genes. because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the epigenetic drug such as histone deacetylase (hdac) inhibitor is currently in clinical trials. however, how epigenetic drugs mediate its effects is poorly understood. to assess the effects of epigenetic drugs, the gene expression by sage in colon cancer cell lines treated with epigenetically affecting agents, -aza- deoxycytidine, a potent inhibitor of genomic and promoter-specific dna methylation and trichostatin a, a hdac inhibitor was investigated. epigenetic modification induced not only the change of expression of several inflammation-associated genes and the cell cycle progression-associated genes in human colon cancer cells but also the gene expression with aberrant start sites. colon cancer is one of the most frequently diagnosed cancers in western societies. interleukin- (il- ) is a potent, pleiotropic, inflammatory cytokine that contributes to a multitude of physiological and pathophysiological processes. il- is produced by many different cell types. the main sources in vivo are stimulated monocytes, fibroblasts, and endothelial cells. a variety of studies have demonstrated that over expression of il- contributes to the pathogenesis of various inflammatory diseases as well as cancer. it has been reported that human colorectal cancer cells display a wide heterogeneity in their potential to express and produce il- . serum levels of il- are elevated in patients with colorectal cancer, however serum levels of il- were found to be independent of il- mrna expression in tumor tissue. in this study we analyzed il- mrna expression by real-time pcr in sporadic colon cancer tissue as well as corresponding normal mucous tissue. il- mrna expression in tumor tissue was lower than in the corresponding normal mucous tissue (p= , ). there was no correlation betweenil- mrna expression and tumor grade or stage. thus we can conclude that il- produced at the tumor site is not involved in sporadic colon cancer progression. ( ), t aiamsa-ard ( ), v chinswangwatanakul ( ), ki techatraisak ( ), s chotewuttakorn ( ), a thaworn ( ) ( material and methods: huvec were cultured as standard techniques and grown to confluence until use. serum was obtained from cholangiocarcinoma patients and normal healthy subjects. huvec were treated with % of serum and incubated for hours. cells were analyzed by using [ h] thymidine and immunoblotting assay for cell proliferation and cox- /nos- protein expression, respectively. results: serum of cca patients trend to have more effect on proliferation of endothelial cell than healthy control subjects. on the protein expression, cca serum significantly increased the expression of cox- but not nos- in hevec. however, the proliferate effect on endothelial cells by cca sera did not correlate with the expression of cox- . conclusions: this result suggested that some factors in serum of cancer patients could induce cox- protein expression in huvec. the increasing of cox- might be one of various factors involve in the proliferation process. aim: superoxide is responsible for the neutrophil-mediated tumoricidal activity. the aim of our work was to monitor the changes of superoxide production from neutrophil attributed to tumor development from the early phase to the advanced stage, and to investigate the effects of ok- @on neutrophil-derived superoxide production and tumor growth. methods: ah a rat hepatocellular carcinoma cells were implanted into the hind leg of male donryu rats. pmns were harvested from rat peritoneal cavity h after intraperitoneal injection of oyster glycogen. superoxide production were measured by the method of cladependent chemiluminescence, which has high sensitivity and specificity to superoxide. the counts of peripheral leukocytes were significantly increased during tumor progression, and there are significant difference between that of controls and tumor-bearing rats after days of tumor inoculation. both pma and oz-induced superoxide generation derived from neutrophils became significantly reduced in the advanced stage of cancer. the suppression of neutrophil-derived superoxide generation was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. the subcutaneous administration of ok- , a biological response modifier, prevented the suppression of neutrophil-derived superoxide generation during tumor progression, which might induce the tendency of tumor growth suppression. our results suggested that the decreased superoxide generation as well as the high leukocytes concentration in the peripheral blood could be considered as indicators of an advanced stage of cancer. furthermore, the effect of ok- on neutrophil-derived superoxide production in cancer-bearing rats may provide pharmacological evidence to the therapeutic effects of ok- . ( ), m jokic ( ), v zjacic-rotkvic( ), s kapitanovic ( ) ( ) university hospital sestre milosrdnice, bucharest, romania ( ) division of molecular medicine rudjer boskovic institute, bucharest, romania introduction: il- is a pleiotropic cytokine mapped to chromosome p - . its promoter snp - g/c is associated with high serum cytokine production, and according to current investigation can play a role in the development and progression of different gastrointestinal malignancies. we tested its genotypes in the gastrointestinal and pancreatic neuroendocrine tumors (gep-nets). patients and methods: dnas from patients diagnosed with gep-net and age and sex-matched volunteers were analyzed for - g/c snp of the il- gene. to analyze il - c/g polymorphism we used pcr -nlaiii rflp method. for statistical analysis Ä test and fishers exact test were used. the level of significance was . . results: there were no differences observed in the frequencies of the - high expression (gc and gg) genotypes between the patients and healthy volunteers (p= . ), as well as between patients with gastrointestinal or pancreatic endocrine tumors (p= . ). - g/c genotype was statistically more frequent among patients with non-functional pancreatic endocrine tumors (pets) than in those with functional pets (p= . ). conclusions: high expression genotypes of il- - snp are more frequent in non-functional pets and may be a marker for the mentioned malignancies. are important in inflammation, are found around and within a variety of human tumors. their number correlates with tumor vascularity and aggressiveness and is a negative indicator for patient survival. how mast cells influence tumor growth is not well understood. the neuroendocrine peptide, neurotensin (nt) is a potent secretagogue of mc that has tumor-promoting effects in animals and promotes the growth of a variety of human cancer cells, acting via its gpcr nt-type receptor (nts ). here we show that hmc- human mc express nt-precursor (pront) mrna and protein, and secrete immunoreactive nt when stimulated. rt-pcr on hmc- cell rna yielded a band with % sequence identity to pront and a band corresponding to the pront processing enzyme, pc a.immunocytochemistry on hmc- cells showed specific staining for pront. stimulation of hmc- cells with a + pma, pge , c / or mastoparan released immunoreactive nt.rt-pcr on hmc- cell rna yielded a band with % sequence identity to human nts . western blotting gave bands corresponding to unglycosylated ( kda) and glycosylated ( kda) nts .immunocytotochemistry on hmc- cells showed specific staining for nts . these finding have significance for the role of mast cells in tumor growth. ( ), j buddenkotte ( ), mp schçn( ), m steinhoff ( ) ( ) university hospital münster, germany ( ) university hospital würzburg, germany the proteinase-activated receptor par- has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. however, the role of par- in cutaneous cancerogenesis is still unknown. here we could show a protective role of par- in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. to this end, par- -deficient and wild-type mice were painted once with dmba ( , -dimethylbenz[a]anthracene) for sensibilization, followed by topical application of the phorbol ester pma (phorbol myristate acetate ( -o-tetradecanoylphorbol- -acetate)) twice per week at the same sites. tumors started to appear after eight weeks. after weeks, par- -deficient mice showed a significantly increased number of skin tumors ( per animal on the average) in contrast to the wild-type (eight tumors per mouse). analysis of possible signal transduction pathways activated upon par- stimulation in hacat keratinocytes showed an involvement of extracellular signal regulated kinase / (erk / ) and profound epidermal growth factor receptor (egfr) transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta (tgf-â ). thus, our results provide the first experimental evidence for a tumor-protective role of par- . ( ), ma arbós ( ), a fraga ( ), i de torres( ), j reventós ( ), j morote ( ) ( pathogenesis of benign prostatic hyperplasia (bph) and prostate cancer (pca) is still unresolved, although chronic inflammation may play a significant role in disease progression. prostate stromal fibroblasts may be contributing to the inflammatory process through the expression and secretion of pro-inflammatory mediators, in particular proteoglycan-bound chemokines and other chemoatractants, and the interaction with inflammatory cells such as monocytes. to better understand molecular mechanisms underlying functional differences among prostate fibroblast populations, our primary objective was to characterize proteoglycan and chemokine gene expression in human fibroblasts of different histological/ pathological origin cultured in normal and monocyteconditioned media. we analysed primary human fibroblast cultures from normal transition zone (tz), normal peripheral zone (pz), benign prostatic hyperplasia (bph), and pathologically confirmed prostate cancer (ca). cells of different origin displayed distinct mrna expression profiles for the core proteins of proteoglycans and both sdf /cxcr and mcp /ccr chemokine axis. when incubated with monocyte-conditioned medium all four cell types significantly changed sdf /cxcr and mcp /ccr expression in a fibroblast population dependent manner. monocyte-fibroblast cell adhesion and the chemotactic response of fibroblasts to human peripheral blood monocytes were investigated in a coculture system. monocytes adhered rapidly to fibroblasts and preferentially to bph and pz cells. in addition, chemotaxis was significantly induced in both fibroblast cultures after incubation with monocytes. our results suggest that prostatic fibroblasts have a key inflammatory role associated to a distinctive proteoglycan gene expression and chemokine induction, which is dependent on their histological and pathological source. supported by the spanish urology society (madrid, spain). we have recently shown that paf-receptor is involved in phagocytosis of apoptotic and necrotic but not viable cells, possibly through its interaction with paf-like molecules present on the surface of these cells. removal of altered cells by macrophages could modify the microenvironment at an inflammatory site, and thus influence tumor growth. in the present study we investigated the impact of apoptotic cells or treatment with paf-r antagonist on ehrlich ascitic tumor (eat, ip) and melanoma b f (sc). paf-r antagonist, web ( mg/kg, ip) was given daily for days. we found that eat growth was significantly reduced by pretreatment with web , and that inoculation of apoptotic cells (thymocytes) before tumor implantation stimulated tumor growth, an effect reversed by web pretreatment. eat growth was accompanied by increased production of prostaglandin e , vegf and no which was reduced significantly by web treatment. in b f melanoma, web , alone or in association with an apoptosis inducer chemotherapeutic agent, dacarbazin (dcb, ug/kg,ip) significantly reduced tumor mass volume and the number of intratumoral small vessels. in association with dcb, web- reducedactive caspase- expression in the tumor andmarkedly increased the survival of tumor-bearing mice. the data obtained here show that during tumor growth, activation of paf-r by molecules present in the surface of apoptotic/necrotic cells, or by paf produced in the milieu, favors tumor growth and suggests that pafantagonists could be useful in tumor treatment, particularly when in association with chemotherapy. financial suport by fapesp and cnpq. ( ), mt quiles ( ), a figueras( ), r mangues( ), f vinals( ), jr germa( ), g capella ( ) ( ) institut de recerca vall de hebron, barcelona, spain ( ) translational research laboratory, idibell -institut cataladoncologia, spain the malignant potential of tumor cells may be influenced by the molecular nature of k-ras mutations. we have previously shown that codon mutations associate with an increased resistance to apoptosis. we hypothesized that their different malignant potential in vivo could be also related to the generation of a distinct angiogenic and inflammatory profile including vascular structure, macrophage infiltration and expression of angiogenic modulators, proteolytic mediators and the cxcl (sdf- )/ cxcr chemokine axis. to do so we have combined in vitro and in vivo studies using stable cys and asp nih t transfectants. cys tumors showed a higher microvessel density associated with shorter latency period. prominent vessels with µ-smooth muscle actin positives cells surrounded by f / macrophages were only observed in asp tumors associated with a shorter growth period. asp tumors displayed increased vegf expression both at the rna and protein levels, mainly produced by tumor cells. tsp- protein levels were similarly diminished in both transfectants. higher mmp and mmp activities and expression were observed in asp tumors probably produced by macrophages or stromal cells. total and active mmp levels were higher in cys tumors. the expression of sdf- and cxcr remained unchanged while sdf- g isoform was selectively induced in cys tumors, suggesting sdf- a or b are induced in asp tumors. these results show distinct k-ras mutations induce specific angiogenic phenotypes. the differential stimulation of vegf expression, metalloprotease activities and sdf- expression observed is the result of the joint action of tumor cells and the local microenvironment. contact information: dr maria a arbos via, institut de recerca vall de hebron, unitat de recerca biomedica, barcelona, spain e-mail: maarbos@ir.vhebron.net incisional hernias (ihs) represent a common complication of laparotomies, involving remarkable healthcare costs. representative ih animal models are lacking and characterization of human tissue resources is scant. this limited understanding of fundamental mechanisms regulating the destruction of the abdominal wall currently limits the prevention and treatment of ihs. here, we compared tissue specimens (carefully obtained > cm of the defect) and primary fibroblasts cultures from fascia and skeletal muscle of subjects with/without ih hernia. the most prominent morphologic characteristics of ih tissue were: alterations of the microstructure of the connective tissue and loss of extracellular matrix (ecm), and a paucity of fibroblasts. in ih muscles, inflammatory infiltrates were observed. other significant changes were: decreased collagen type i/iii ratio; differential proteoglycans mrna expression; enhanced metalloproteinases/ endogenous inhibitors ratio (mmps/timps); and upregulation of apoptosis effectors (caspase- and substrates; tnf-alpha; il- ). in vitro, hernia fibroblasts (ihfs) exhibited significantly higher ( -fold) cellular proliferation and migration rates and decreased strength of adhesion as compared to control fibroblasts, even after several passages. moreover, ihfs ultrastructure analysis revealed accumulation of autophagic vacuoles, autophagolysomes-like structures and multilayered lamellar and fingerprint profiles, as well as mitochondrial swelling. based on these descriptive results in human tissues, a novel hypothesis emerges regarding ih formation. specifically we propose that inflammation-related mechanisms triggering proteolytic and apoptotic effectors regulate cell turnover and eventually contribute to atrophy and progressive tissue insuffiency. overall, this may be causally involved in the mechanisms of ecm destruction yielding ih (supported by fis pi_ and gencat_agaur_ xt_ ). ( ), m spinola( ), c pignatiello( ), w cabrera ( ), og ribeiro ( ), n starobinas( ), t dragani ( ) ( ) butantan institute, sao paulo, brazil ( ) istituto nazionale tumori, milan, italy airmax and airmin mice are phenotypically selected for maximal or minimal subcutaneous acute inflammatory response, respectively, and display high inter-line differences in protein exudates and neutrophil infiltration, as well as in bone marrow granulopoiesis, inflammatory cytokines, and neutrophil apoptosis. in a combined experiment of urethane-induced lung inflammatory response and lung tumorigenesis, airmin mice developed a persistent subacute lung inflammation and a fold higher lung tumor multiplicity than airmax mice, which showed a transient lung inflammatory response. we have analyzed gene expression profiles of these outbred lines in comparison to the lung cancer resistant c bl/ and lung cancer susceptible a/j mouse strains. gene expression profile analysis of urethane-treated and untreated animals was performed using the applied biosystems mouse genome survey microarray containing , mouse transcripts. mrna expression of candidate differentially expressed genes was validated by quantitative real-time pcr and the over-represented biological themes were analyzed with the ease software. urethane treatment modulated the gene expression profile in all four lines. among the confirmed genes, vanin (vnn ) and major histocompatibility antigen e alpha (h -ea) resulted common to both mouse models. the most represented gene categories in air model were acute phase response, immunoresponse, electron and lipid transport, complement activation and tissue repair. mhc/antigen process and presentation and immunoresponse were the major themes in the inbred model. moreover, a gene cluster in chromosome ( . cm) was observed. the study suggests that the expression of a subset of genes may show a strain-and line-specific modulation pattern during inflammatory response and lung tumorigenesis. inhibition of tumour induced angiogenesis constitutes very attractive anti-cancer therapeutic approach.it is well established that the vegf signal transduction pathway is one of the key drivers of deregulated angiogenesis and selective inhibition can lead to inhibition of tumour growth. however, multiple angiogenic growth factors and pathways are involved, leading to a phenomenon of redundancy and overcoming of an inhibition of vegf signalling only. we have developed a nanomolar inhibitor (compound a) of the receptor tyrosine kinase vegfr-r (kdr), which was subsequently shown to be a potent inhibitor of closely related kinases (vegfr- and - , pdgfr, kit, csf- r) but also unrelated soluble tyrosine kinases (src-familily of kinases and raf). compound a potently inhibits vegf stimulated endothelial cell proliferation but has no effect on non-ec proliferation, which is suggestive of a selective antiangiogenic potential. the unique kinase inhibitory profile of compound a combined with excellent oral bioavailability ( %) has translated into superior in vivo anti- inflamm. res., supplement ( ) posters tumour efficacy when compared to the relatively selective kdr inhibitor ptk . thus, treatment of nude mice implanted with either commercial atcc derived tumour cells (a and du- ) or low passage patient derived tumors (cxf ; colon cancer, rxf ; renal cancer) with compound a resulted in inhibition of tumour growth which was significantly better than for ptk treated mice. compound a is fairly well tolerated by rodents and extended toxicological studies have been initiated to determine the therapeutic index, which also may allow for exploration of other non-cancer indications. ( ), p bobrowski( ), m shukla ( ), t haqqi ( ) ( ) albany medical college, usa ( ) rainforest nutritionals, inc, usa ( ) case western reserve university school of medicine, usa background: the amazonian medicinal plant sangre de grado (croton palanostigma) has traditional applications for wound healing and inflammation. we sought to characterize an extract (progrado) in terms of safety, proanthocyanidin profile, antioxidant activity and anabolic/catabolic actions in human cartilage explants. methods: acute oral safety and toxicity was tested in rats according under oecd protocol # . proanthocyanidin oligomers were quantified by hplc and progrados antioxidant activity assessed by the orac, norac and horac assays. human cartilage explants, obtained from surgical specimens, were treated with il- b ( ng/ ml) to induce matrix degradation and glycosaminoglycans (gag) release. progrado ( - mg/ml) was tested for its ability to maintain optimal igf- transcription and translation in cartilage explants and cultured chondrocytes. results: progrado displayed no evidence of toxicity ( mg/kg po) leading to gsh safety rating of /unclassifiable. oligomeric proanthocyanidin content was high ( mg/kg) with the majority of oligomers > mers.progrado was a remarkably potent antioxidant and in an ex vivo model of inflammation-induced cartilage breakdown, progrado was exceptionally effective in reducing both basal and il- b induced glycosaminoglycan release from human cartilage explants. progrado prevented il- b induced suppression of igf- production from human cartilage explants as well as stimulating basal igf- production (p< . ). comparable changes in igf- gene expression were noted in cultured human chondrocytes. conclusions: progrado has a promising safety profile, significant chondroprotective and antioxidant actions, and promotes the production of the cartilage repair factor, igf- . this suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. the solvent extracts from korean fermented soybean (chungkukjang) were evaluated for their protective effects against the generation of free radicals and lipid peroxidation. the activities of chungkukjang were compared with several antioxidants and soybean isoflavones including genistein and daidzein. in addition, the protective effects against h o -induced cytotoxicity and oxidative dna damage in the nih/ t fibroblasts line were examined. the extracts from chungkukjang and soy isoflavones inhibited the generation of , -diphenyl- picryl hydrazine (dpph) radicals, and had an inhibitory effect on ldl oxidation. the extracts from chungkukjang and soy isoflavones strongly inhibited h o -induced dna damage in the presence or absence of endonuclease iii and fpg. furthermore, they showed cytoprotective effects against h o , without cytotoxicity except for the hexane extract at high concentrations (> mg/ml). the ethanol and n-butanol extracts appeared to have most potent antioxidant activities. these in vitro results show that the extracts of chungkukjang may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative dna damage without having cytotoxic effect. moreover, the extracts of chungkukjang inhibited mda formation in the liver, dna damage assessed by comet assay and the microucleated reticulocyte formation of peripheral blood in kbro -treated mice. these in vivo results were similar to those of in vitro experiments. therefore, chungkukjang containing soy isoflavones is a promising functional food that can prevent oxidative stress. (supported by bk project from korea research foundation). sirt is a histone deacetylase, involved in oxidative stress and aging. because the role of aging and exercise on sirtuins activity in rats is unknown, we investigated the effects of exercise on age-related changes in the sirt activity, comparing heart (h) and adipose (a) tissue of sedentary young (n ), sedentary old (n ) and trained old (n ) rats. the trained old rats performed a -weeks moderate training on treadmill. on h and a tissue of all rats sirt activity was evaluated by assay kit, peroxidative damage measuring malondialdehyde (mda) and protein-aldehyde adducts -hydroxynonenal ( -hne), mnsod, catalase and foxo a by western blot, and gadd a, cyclin d and foxo a mrna by rt-pcr. aging reduced sirt activity in h (p< . ) without effects in a, producing an increase of mda (h, p< . ; a, p< . ) and -hne (h, p< . ; a, p< . ), and a decrease of mn-sod (p< . ) and catalase (p< . ) expression in both h and a. aging did not affect foxo a protein expression in h, and foxo a mrna in a. exercise produced an increase in h foxo a protein expression (p< . ) and in a foxo a mrna, associated to higher mn-sod (h, p< . ; a, p< . ) and catalase (h, p< . ; a, p= . ) levels in both h and a of aged rats. in heart exercise-induced higher sirt activity bring on decrease in cyclin d and increase in gadd a mrna expression. in a we found a similar decrease in cyclin d , without changes in gadd a mrna expression. these findings suggest that exercise is able to increase sirt activity in aged rats. ( ), t horiguchi( ), k abe( ), h inoue( ), t noma ( ) ( ) institute of health biosciences, the university of tokushima graduate school, tokushima, japan ( ) minophagen pharmaceutical co. ltd, japan objectives: glycyrrhizin (gl) is a major component of glycyrrhizae radix (licorice) that is generally used for treatment of hepatitis. gl has a regulatory activity on arachidonic metabolism, immunological function, and anti-viral effects. however, the molecular mechanisms of the effects remain unclear. to analyze the molecular basis of gl signaling, we performed the microarray analysis using ccl -induced mouse hepatitis models. methods: eight-week-old icr male mice were treated intraperitoneally with f×l/ kg bw of ccl w/wo mg/ kg bw of gl. after hours and hours, livers and serum were collected and analyzed. for microarray analysis, the expression patterns of genes between hour-treated-livers (ccl or ccl and gl) and no treated-livers were compared. results: gl-treatment dramatically decreased the gpt activity in plasma at hours compared to that in ccl treated plasma. however, the levels of mrna expression of inflammatory genes such as phospholipase a , hsp , and procollagen were still very high in gl-treated liver. after hours, the mrna levels of them were significantly reduced in gl-treated mice compared to those of ccl -treated liver. then, we screened , genes by microarray and found that genes were up-regulated and genes were down regulated in ccl +gl compared to ccl treatment. interestingly, ros scavenger-related genes were significantly up-regulated in ccl + gl. detail analysis is currently ongoing. we found the unique relationship between gl activity and ros regulation. this finding suggests a novel way to treat inflammatory diseases including hepatitis. objectives: experimental autoimmune encephalomyelitis (eae) is a demyelinating autoimmune disease that results from an immunological reaction against different myelin components at the cns. it is widely employed as an animal model of human multiple sclerosis. interestingly, the number of studies relating these diseases with peripheral organs is limited. we thus investigated the consequences of eae on the degree of lipoperoxidation (tbars) and mpo activity in different rat peripheral organs (eg. lung, spleen, liver, stomach, duodenum, colon, ileum, kidney and bladder). university of waikato, hamilton, new zealand mitochondria play a fundamental role in the life and death of all eukaryotic cells. cells with dysfunctional mitochondria are known to have higher levels of a molecular stress protein (cpn ). this protein is increasingly being implicated to play a role in modulating cellular inflammation. we have developed an in vitro model cell system using thp- monocyte cells with compromised mitochondrial bioenergetic functions to investigate the relationships between mitochondrial dysfunction, cpn expression and modulation of proinflammatory cytokine responses. we have found that the ability of cpn to modulate tnf-a expression was strongly correlated with the loss of mitochondrial bioenergetic functions in our thp- cells. we also demonstrate that such modulation involves both erk / and p mapk pathways. the significance of these results in relation to the role of mitochondria as modulators of inflammation will be discussed. ( ), b arnold( ), g opdenakker ( ) ( ) jagiellonian university, department of evolutionary immunobiology, krakow, poland ( ) german cancer research center, department of molecular immunology, heidelberg, germany ( ) rega institute for medical research, university of leuven, laboratory of immunobiology, leuven, belgium we showed that in mice genetically deprived of metalloproteinase (mmp- -/-) at least one compensatory mechanism operates as there are elevated levels of pge of cox- origin expressed by peritoneal macrophages during zymosan peritonitis; and this leads to increased early vascular permeability observed in those animals. also infiltration of peritoneal cavity by inflammatory neutrophils is changed in mmp- -/-mice as at hrs of inflammation, when otherwise highest numbers of neutrophils are detected in peritoneum, the cell numbers are significantly lower in the mice in comparison to their controls. in contrary, at hrs of peritonitis, when normally resolution of peritonitis takes place, no decrease in neutrophil counts is observed. thus the aim of the present study was to evaluate if impairment of neutrophil apoptosis could account for this latter phenomenon in mmp- -/-mice. for this numbers of apoptotic (annexin v) and necrotic ( -aad) peritoneal leukocytes were evaluated and levels of active caspases were tested by application of either caspase detecting antibodies or fluorochrome-labelled inhibitors; all analyses were performed by flow cytometry. the results revealed that both, numbers of apoptotic cells and levels of active caspase were significantly lowered in mmp- -/-mice while levels of caspase , and were significantly elevated in comparison to control animals. we conclude that an impairment of apoptosis is observed in mmp- -/mice during zymosan peritonitis and it is due to the decreased levels of active caspase . the increased activity of other examined caspases is most probably independent of apoptosis. ( ), h james ( ) the selective inhibition of nitric oxide generation by inhibiting the activity of nitric oxide synthase(nos) isoforms represents a novel therapeutic target for the development of anti-inflammatory agents. the aim of this study was to evaluate the activity of nos inhibitors in experimentally induced inflammation, pain and hyperalgesia. the effect on acute inflammation was studied in carrageenan-induced paw edema in rats. the effects on carrageenan-induced hyperalgesia, tail flick response to radiant heat and acetic acid-induced writhing were also studied. nos inhibitor ng-nitro-l-arginine methylester (l-name), and mg/kg produced a dose-dependent inhibition of paw edema ( % and % at h; % and % at h). a marked reduction in paw edema was observed with ng-monomethyl-l-arginine acetate (l-nmma), mg/kg( % at h; % at h). selective inducible nos(inos) inhibitor aminoguanidine hemisulfate inhibited the paw edema at a dose of mg/ kg( % at h; % at h) but not with a dose of mg/kg . the effects were comparable to nonselective cox inhibitor indomethacin mg and mg/kg ( % and % at h; % and % at h respectively) and selective cox- inhibitor rofecoxib, mg/kg ( % and % respectively). nos and inos inhibitors significantly increased the pain threshold latency in the tail-flick test. these inhibited the acetic acid-induced writhes, the effect being comparable to indomethacin. however, carrageenan-induced paw hyperalgesia was not inhibited. the results suggest that nitric oxide plays a role in carrageenan-induced acute inflammation and both nosand inos inhibitors have a potential anti-inflammatoryand anti-nociceptive activity. ( ), p hart( ), j edwards ( ), c quirk ( ) ( ) molecular pharmacology limited (usa), australian division, perth, western australia ( ) telethon institute for child health research, perth, western australia thermalife cream, an anti-arthritic biological product, has been successfully used off-label for sun burn recovery. a novel product, derived from thermalife, was assessed on its therapeutic potential in oxsoralen-uvb burns. as a possible mechanism for the sunburn efficacy, suppression of tnf-a and il- â production by human monocytes was assessed in vitro. methods: sunburn: four sites were marked on the arm of the subject. three sites were exposed to oxsoralen ( %) plus uva/uvb light, one site was exposed to oxsoralen only. cream was applied at min, or at hrs after injury. a third injury site was not treated. photographs were taken before, hrs, and weeks after injury. cytokines: human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of % or % active ingredient. hrs after incubation, culture media was collected, centrifuged, and assayed (cytokine elisa). results: at hrs after oxsoralen-uv, the min treatment site showed slight erythema, the hr treatment site had pronounced erythema and slight blister formation, whereas the untreated site had pronounced erythema and strong blister formation. weeks after injury, the min site was normal, the hr site was a dark colour, whereas the untreated site had a significant scar. oxsoralen alone had no effect on the skin. the novel product suppressed lps-induced tnf-a and il â secretion by . % and . %, respectively. conclusions: a novel thermalife-derived product reduced total injury after oxsoralen enhanced uva/ uvb burns, which is possibly related to cytokine suppression. ( ), p hart( ), j snowden ( ), maud eijkenboom ( ) ( ) molecular pharmacology limited (usa), australian division,perth, western australia ( ) telethon institute for child health research, perth, western australia a mixture of bovine plasma protein fractions and zinc chloride (bov-zn) was assessed for its ability to regulate cytokine production by lps-stimulated monocytes. dosereponse curves for tnf-a suppression were generated. further, competition with fcs in the culture medium and the metabolism of monocytes under influence of bov-zn were assessed. in all experiments the culture medium environment was similar. human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of %, . %, %, . %, %, % or % bov-zn (two pooled experiments). hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). a competitive inhibition design for the standard tnf-a assay was set up for %, %, %, % fcs against %, . %, %, % bov-zn. the culture media were treated as above. metabolism of non-proliferating monocytes was measured via accumulation of bioreduced formazan (promega celltiter ) in treated and untreated cell cultures over - hrs at intervals. the ic for tnf-a suppression was reached at . % bov-zn in each of two experiments. fcs did not compete with bov-zn in suppressing tnf-a in lpsstimulated monocytes. at low fcs concentrations bov-zn stimulated tnf-a production in the absence of lps. this tnf-a increase was countered with increasing concentrations of fcs. metabolism of cells was not affected by % bov-zn. conclusions: bov-zn could reliably and effectively reduce tnf-a secretion in vitro, without competing with the fcs in the culture medium, and without disturbing the metabolism of monocytes. inflammatory diseases such as rheumatoid arthritis (ra) result from overproduction of cytokines including tnf-£\ and il- fÒ. these cytokines are known to be regulated by the stress-activated p fnfnmap kinase pathway. because of this, inhibition of p map kinase has been one of the most compelling targets for the treatment of inflammatory disease. over the last years, numerous groups have reported on the development of p map kinase inhibitors. x-ray co-crystallization with the enzyme suggests a propensity to accommodate structurally diverse molecules. regions of the binding site are known to be unique to p vs other kinases, enabling the development of p selective molecules. inflamm. res., supplement ( ) posters anti-inflammatory activities. a series of labdane-type diterpenoids ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) with various patterns of substitution were tested for potential anti-inflammatory activity.of these compounds, and were selected to evaluate their influence in targets relevant to the regulation of the inflammatory response. these derivatives displayed good in vivo anti-inflammatory activity, and maximum inhibitions of and % were noted in the -o-tetradecanoyl-phorbol- -acetate (tpa)-induced ear oedema in mice. in addition, inhibition of myeloperoxidase activity, an index of cellular infiltration, was also observed. the diterpenoids also reduced the production of nitric oxide, prostaglandin e , and tumour necrosis factor-alpha in bacterial endotoxin-activated raw . macrophage cells with ic in the range - mm. inhibition of these inflammatory mediators was related to reductions in the expression of inducible nitric oxide synthase, and cyclooxygenase- , as determined by western-blot analysis and rt-pcr. since nuclear factor-kappab (nf-kb) plays a central role in the transcriptional regulation of these proteins, we investigated the effects of these diterpenoids on this signalling pathway. our results indicate that both compounds interfere with the phosphorilation of ikbalpha and ikbß, resulting in inhibition of their degradation. in summary, the anti-inflammatory effects of these labdane diterpenoids are related to the inhibition of inflammatory mediators by blocking nf-kb activation and provide potentially therapeutic perspectives in inflammatory conditions. the aim of the study was a research of mechanisms of inflammatory action of a new drug fepolen at a dosage of mg/kg prepared from bee products (bee pollen and phenolic hydrophobic extract of propolis) for prostatitis treatment. to fulfill above mentioned task a model of zimozan induced oedema whose dynamics gives a possibility to estimate influence of a drug on both routs of arachidonic acid metabolism -via cyclooxiginase and lipooxigenase was used. a comparison with diclofenacum at a dosage of mg/kg and substance ffw- Ñ (inhibitor of cyclooxygenase and lipooxygenase and has a high antiinflammatory effect ( %) at a dosage of mg/kg) was made. the results of influence of drugs dynamics of zimozan inflammation show that anti-inflammatory action of fepolen is based on decrease of release of biogenic amines and activity of lipooxigenase and is higher then effect of diclofenacum. fepolen revealed the highest effect during first min and hour of inflammation that was higher then action of diclofenacum. these data proves that fepolen decreases lipooxygenase activity that is in charge for the inflammation during this period. during next hours therapeutic effects of fepolen and diclofenacum were at the same level. fepolen showed the same dynamics of anti-inflammatory action as ffw- Ñ that demonstrates a property to influence both routs of arachidonic acid metabolism. in summary with previous results in conditions of carageninic inflammation we can conclude that anti-inflammatory action of fepolen is based mostly on influence on lipooxygenase then cyclooxygenase. the aim of this study was to establish a method by which probiotic bacteria can be selected for their immuno modulatory properties, especifically the ability of certain strains to suppress an inflammatory response. the gastrointestinal inflammatory condition crohns disease involves a th -response with increased levels of proinflammatory cytokines like tnfa and il , and mouse models of crohns disease show that the balance of il / is crucial for disease progression. we have used mouse bone marrow derived dendritic cells (bmdc) to model a proinflammatory crohns disease like condition in vitro with cocktail-induced bmdc secretion of il , il and tnfaf jthe model was validated using anti-inflammatory molecules like dexamethasone and prostaglandin d , which were able to suppress the cocktail induced il secretion. further validation of the model is confirmed by the fact, that probiotic strains which are able to suppress tnbs induced colitis in mice in preventive studies, also show potent anti-inflammatory activity in our model. among clinically relevant as well as novel probiotic strains, we have selected strains with potent anti-inflammatory properties, and are currently investigating the possible mechanism of action of these strains. in summary, our established model is suitable for identification of anti-inflammatory activity of probiotic strains and potentially other immune suppressing components, for rational selection of candidates for further preclinical and clinical evaluation and development. p - inflammation and human incisional hernia pathophysiology maria antonia arbos via( ) eae was induced by immunization of female lewis rats with guinea-pig myelin basic protein (mbp) in complete freunds adjuvant (cfa) and the animals were studied at the stage iii of the disease (characterized by complete paralysis of the hind-limbs) compared to cfa rats, eae resulted in increased mpo activity (u/mg tissue) in kidney ( ae vs. ae ae ; p< . ), and higher tbars contents (nmol mda/mg tissue) in liver acknowledgements: capes, cnpq, fapesp. contact information: ms simone teixeira, university of s¼o paulo, department of pharmacology, campinas, brazil e-mail: mone@usp.br tolerability, investigator and subject global assessments and rescue medication consumption supported by bk project from korea research foundation) contact information: mr young hoon kim here we report on the development of the pge mimetic combination therapy (dp- ) that inhibits basal and lps/tlr induced tnf-?, il- ß, and mmp- , , , in human and murine synovial membranes and peripheral macrophages.in a murine model of chronic synovitis (dorsal skin air pouch), dp dramatically inhibited il- ß, tnf-?, mip , mcp- and il- expression, delayed the profile of leukocyte/neutrophil extravasation and reduced exudate volume.in a model of inflammatory arthritis (collagen induced arthritis, cia), dp markedly reversed the inflammatory pathology by reducing synovial hyperplasia, cartilage erosion and articular inflammation.in addition, tnf-?, il- ß, mmp- and to a lesser extent mmp- expression/synthesis levels were strongly suppressed as judged by rt-pcr and elisa measurements we have developed two rias, one for functional blood levels of the above mentioned anti-tnf-alpha constructs, and one for anti-abs (all isotypes), and we have used these methods to monitor patients treated with infliximab/remicade and etanercept/enbrel ; i shall present some of these data ( , anatomy-physiology, faculty of medicine, laval university, quebec, canada neutrophils, which are often the first leukocytes to migrate at inflamed sites, can generate ltb from the -lipoxygenase pathway, pge through the inducible cyclooxygenase (cox- ) pathway and cytokines/chemokines as tnf-alpha, il- beta, il- , mip- alpha, mip- beta, mip- alpha and mip- alpha. engagement of the adenosine a a receptor (a ar) blocks the in vitro synthesis of ltb while it potentiates the cox- pathway in fmlp-treated neutrophils. in addition, it selectively prevents the expression and release of tnfalpha, mip- alpha, mip- beta, mip- alpha and mip- alpha in toll-like receptor- -stimulated human neutrophils. little effect was observed on il- beta and il- . using the murine air pouch model of inflammation with a ar-knockout mice, we observed that the activation of a ar positively impacts the expression of cox- in vivo, with particular magnitude in inflammatory leukocytes. in mice lacking the a a receptor, neutrophils that migrated into the air pouch h following lps injection expressed higher mrna levels of tnf-alpha, mip- alpha and mip- beta than neutrophils from wild type mice. together, these results indicate that neutrophils are important mediators of adenosines protective effects. given the uncontrolled inflammatory phenotype observed in a ar-knockout mice and in view of the potent inhibitory actions of pge on inflammatory cells, an increased cox- expression and a prevented release of tnf-alpha, mip- alpha and mip- beta caused by a ar activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine. sepsis induced by endotoxins including lipopolysaccharide (lps) is a big problem in clinical medicine. for a better insight into the molecular pathways and to assess markers of endotoxin-induced sepsis, we applied thetwo dimensional gel electrophoresis ( d-page) and maldi-tof to follow the changes of significant proteins in a murine macrophage cell line -raw . after challenged with lps (escherichia coli :b ) for , and hours. we identified proteins from approximately detected protein spots with either increased or decreased in relative abundance as a result of lps treatment. the proteins identified with increased expression are the retinoblastoma binding protein , capg protein, poly(rc) binding protein , isocitrate dehydrogenase (nad+) alpha, lactate dehydrogenase , a chain, guanine nucleotide binding protein (g protein), beta polypeptide like , triosephosphate isomerase and proteasome alpha subunit); and ones with decreased expression are the acidic ribosomal phosphoprotein p , malate dehydrogenase, soluble, proliferating cell nuclear antigen, proteasome (prosome, macropain) subunit, alpha type and rho, gdp dissociation inhibitor (gdi) beta). many of these altered proteins have interesting functions in inflammation. with the information obtained with the proteomic approach, it is possible to improve current methods of monitoring endotoxemia and to identify new therapeutic targets. the ubiquitous mitogen-activated protein (map) kinases are important enzymes in signal-tranduction cascades which regulates diverse cellular events such as cell transformation, proliferation, differentation, and apoptosis. they are therefore potential drug targets for therapeutic intervention in the treatment of inflammation, cancer, and other immune diseases. based on a virtual screening approach we identified -amino- -benzyl- -( -bromophenyl)- -methyl- , -dihydropyrano[ , c] pyrazole- -c arbonitrile as a potential novel lead structure as p map kinase inhibitors. a set of compounds were prepared starting from different substituted pyrazol- ( h)-ones via a base-catalyzed condensation with aldehydes and ch acids, such as malononitrile, to provide them for biological tests in a p enzyme assay. first structure-activity relationship confirm the value of this novel lead. this study was conducted to determine the physiological c-reactive protein (crp) and alpha -acid glycoprotein (aag) levels for two groups of beagle dogs: healthy dogs of various ages and pregnant dogs. serum crp levels were measured by elisa and aag levels were measured in healthy beagles of various ages by tia, and then separately -in pregnant beagles -by srid. serum crp levels ranged from . to . ìg/ml in male, and from . to . ìg/ml in female dogs. no significant sex-related differences were observed in the values. further, there were no significant age-related differences either. serum crp levels increased during pregnancy and peaked at . - . ìg/ml or days after ovulation, demonstrating two characteristic features of crp levels change in pregnant dogs. serum aag levels ranged from to ìg/ml in male, and from to ìg/ml in female dogs, without any significant sex-or age-related variation. serum aag levels increased in all pregnant beagles and peaked in the middle of gestation at - , ìg/ml. despite a high value of , - , ìg/ml being observed for serum aag levels in pregnant beagles inoculated with staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of srid ( ìg/ml). no significant sex-/-age related differences were observed in serum both crp and aag levels and these levels increased during pregnancy. the results of aag levels in umbilical cords were below the detection levels suggest aag is not transported to the placenta. polymorphonuclear neutrophils (pmns) play a key role in the inflammatory response against infectious agents.however, they can elicit significant tissue damage and in this respect, anti-inflammatory drugs are of interest.in this study, we examined the effect of pbi- , a low molecular weight immunomodulatory molecule, on pmn activation by lps both in vitro and in vivo. we measured by elisa the production of tnf-a by human lps-activated pmn in the presence or absence of pbi- .the ability of pbi to modulate pmn activation and recruitment in vivo was assessed using a rat air pouch model of inflammation.exudates from different groups of animals (controls and pbi- treated animals, n= ) were used to assess leukocyte infiltration and to measure by elisa tnf-µ, mcp- and pge production.in vitro, pbi- is able to significantly decrease by . % ae . % (p< . ), tnf-a production by human lpsactivated pmn.in vivo, pbi- significantly decreased the production of tnf-a ( . % ae . %; p< . ), mcp- ( . % ae . %; p< . ) and pge ( . % ae . %; p< . ) induced by lps injection.however, pbi- did not significantly inhibit leukocyte infiltration.these results show that pbi- is able to modulate pmn activation and inflammatory response and suggest potential use as anti-inflammatory agent. ( ), lj lowenstine,( ), aj norris( ), t spangler( ), lm woods ( ) ( ) zoological society of san diego, usa ( ) department of pathology, microbiology, and immunology, university of california, davis, usa this study investigated the role of a novel reovirus in a outbreak of necrotizing typhlocolitis in american crows in california. included is a detailed characterization of the necrotizing and inflammatory characteristics of the disease, as well as a discussion of the implications of these findings upon proposed mechanisms of pathogenesis. complete histopathology including stains for lesion characterization and potential concurrent etiologic agents was performed on all outbreak crows. feces and ceca were submitted for culture, parasitology, and negative contrast electron microscopy. two control groups (n= each) were selected for parasitology and em (group ), and gross and histopathology (group ). all outbreak cases and group controls tested negative for west nile virus by pcr.all outbreak crows had marked, necrotizing heterophilic typhlocolitis, fibrinonecrotizing splenitis, and variable intestinal lamina proprial necrosis and hemorrhage.two cases had multifocal hepatic necrosis. negative contrast em revealed reovirus particles in % ( / ) of outbreak cases and in % ( / ) of controls. supplemental tests failed to suggest other concurrent or confounding etiologic agents.overall, the findings suggest association between the reovirus and the outbreak of typhlocolitis, and the absence of reovirus in controls suggests that it is not ubiquitous in the crow population.there was a noteable absence of similar typhlocolitis in archived crows submitted to the vmth from - , suggesting an emerging corvid disease in california, which bears further investigation. mitogen-activated protein kinase (mapks) pathways play an important role in the signalling system activated by proinflammatory cytokines. among the most important cascades the activation of erk / by mek / is reported to be responsible for inflammatory responses and degradation of osteoarthritic cartilage. as , a selective mek inhibitor, demonstrated anti-inflammatory properties in reducing tnf-alpha production induced by lps injection (ic mg/kg). therefore, primary aim of the present study was to assess the therapeutic strength of the as in a mouse model of collagen-induced rheumatoid arthritis (cia) assessing the effect of the compound on structural changes related to the cartilage. cia is characterized by severe polyarthritis affecting peripheral joints, synovial hyperplasia with persistent inflammation and cartilage erosion. as treatment was initiated when signs of arthritis were clinically visible (in terms of paw swelling and redness) and was continued for days (twice daily), by oral route at the doses of , and mg/kg. as at and mg/kg significantly reduced clinical arthritic read-outs such as clinical score and paw swelling. at histology, vehicle-treated animals showed severe inflammation and joint surface erosion. administration of as significantly decreased inflammatory infiltrates and treated cartilage surfaces that presented normal levels of proteoglycan content. in conclusion, the results obtained in this study clearly demonstrate that the selective blockade of mek could be considered as an innovative therapeutic approach to treat rheumatoid arthritis. experimental evidences have shown that the toxicity of ni salts may involve inflammatory processes, with a subsequent overproduction of reactive oxygen species (ros) and carcinogenicity. neutrophils are the most abundant leukocytes of blood, and participate actively in the inflammatory innate host defense response. however, relatively little is known about the potential of nickel salts in activating human neutrophils.thus, the aim of the present study was to evaluate the putative stimulation of oxidative burst in isolated human neutrophils by nickel nitrate. the measurement of neutrophil burst was undertaken in vitro, by chemiluminescence, by monitoring the oxidation of luminol by neutrophil-generated ros and reactive nitrogen species (rns). enzymatic inhibitors and specific reactive species scavengers were used to evaluate which species were involved in neutrophils activation by nickel nitrate. the obtained results showed that nickel nitrate stimulates human neutrophils burst in a concentration-dependent manner, within levels that may be attained in vivo. in the present experimental conditions, the reactive species involved in neutrophils activation by nickel nitrate were superoxide radical (o -.), hydrogen peroxide (h o ), hydroxyl radical (ho.) and perchloric acic (hocl). the observed activation of isolated human neutrophils burst by nickel nitrate and subsequent tissue damage due to a sustained formation of reactive species may contribute for the deleterious effects attributed to this transition metal, though this assumption needs to be confirmed in vivo. ( ), h spalteholz ( ), u reibetanz ( ), p salavei ( ), m fischlechner ( ), h-j glander( ), j arnhold ( ) ( ) university of leipzig, medical faculty, institute for medical physics and biophysics, germany ( ) university of leipzig, derpartment of dermatology, andrology training centre of the european academy of andrology, germany unintentional childlessness often caused by common reasons as inflammation affects - % of german couples. inflammations of the male genital tract lead to an infiltration of polymorphonuclear granulocytes (pmn), respectively induce a restricted spermatozoa quality associated with early triggered acrosome reaction (ar) and apoptosis as well as changes in the lipid structure and reduced mobility. stimulated pmn release the strongly cationic heme protein myeloperoxidase (mpo), which is able to bind to negatively charged membrane surfaces, e.g. apoptotic cell membranes with externalized phosphatidylserine (ps). a population of freshly prepared spermatozoa shows only a very small amount of cells with mpo binding ability as well as externalization of ps. the number of spermatozoa able to bind mpo raises considerably in samples containing predamaged cells or introducing the ar as could be observed with rhodamine b isothiocyanate (ritc)labelled mpo and antibody techniques by fluorescence microscopy as well as flowcytometry. the activation ofmpo with its substrate hydrogen peroxide (h o ) in the presence of chloride ions generates the powerful oxidizing and chlorinating species hypochlorous acid (hocl) and enhanced markedly the number of annexin v positive and non-vital cells. components of seminal plasma as well as serum albumin can protect spermatozoa for the deleterious effects of mpo. the coincidence of ps externalization and mpo binding to spermatozoa surfaces indicates an up to now unknown role of this enzyme in recognition and removal of apoptotic cells during inflammation. recent findings suggest a crucial role of proteinaseactivated receptor- (par ) in inflammation and innate immunity. par is the second member of a novel g protein-coupled receptor subfamily with seven putative trans-membrane domains. this subfamily is characterized by a unique mechanism of receptor activation. accessible serine proteases cleave the receptor to expose a new, previously cryptic, n-terminal sequence ("tethered ligand") which further interacts with the same receptor and activates it. tryptase, trypsin, and bacterial serine proteases are capable of directly activating par . par is expressed by human neutrophils, however its functions on these cells remained unclear. the data of our present study indicate that par agonists enhance interferon gamma (ifna)-induced up-regulation of cell surface fca;ri, one of the key receptors involved in neutrophil phagocytic activity. moreover, par agonists (serine proteases as well as synthetic activating peptide) and their receptor represent an additional system which controls neutrophil transendothelial migration and apoptosis in vitro. additionally, there is a significant increase of par expression on the neutrophil cell surface in the case of septic patients as compared to cells from healthy volunteers. together, our results indicate that par may be involved in the pathophysiology of acute bacteria-induced human diseases (sepsis or septic shock, for example) potentially by regulating neutrophil apoptosis, transendothelial migration and fca-receptor expression. aim: to ascertain the role of macrophages as direct inducers of regeneration after renal ischemia/reperfusion, and to establish whether inflammatory conditions contribute to the process. we determined whether adoptive transfer of macrophages at different stages of kidney inflammation after mouse renal i/r could restore reparation and assessed the influence of inflammation in the process.results: i/r provoked the increases in renal regeneration, as evaluated by inmunohistochemistry and pcr mrna of stathmin and pcna. the cytokine profile revealed the influence of the inflammatory environment on kidney repair. regeneration was macrophage-dependent, decreasing when depletion was provoked, and increasing with adoptive transfer of macrophages; however, administration of resting macrophages did not induce repair at the time points in which tissue was inflamed, and was only able to promote regeneration in the absence of inflammation ( hours). pro-inflammatory cytokines increased at the early stages of reperfusion, coinciding with low regeneration, and anti-inflammatory cytokines increased during the longer periods of reperfusion when regeneration was more evident.conclusions: macrophages directly induce renal regeneration after ischemia/reperfusion in an inflammationdependent manner. ( ), k bendtzen ( ), f sellebjerg ( ), ch nielsen ( ) ( antibodies against myelin basic protein (mbp) are present in sera from patients with multiple sclerosis (ms), but the role of these antibodies is controversial. we collected sera from ms patients and healthy individuals and found that both groups contained igm anti-mbp antibodies, while ms sera contained small amounts of igg anti-mbp. however, the two groups of sera did not differ significantly with respect to the content of either antibody subclass. addition of mbp to the various sera and subsequent addition of the mixtures to normal peripheral blood mononuclear cells (pbmc) resulted in a significant deposition of igm on cd + monocytes, indicating that formation of mbp/igm complexes had occurred. this deposition was strongly inhibited by addition of mm edta to the sera, indicating that it was complement dependent. the pbmc produced significant amounts of il- , tnf-alpha and ifn-gamma upon stimulation with mbp, and the extent of the cytokine production did not depend upon whether sera from ms patients or from healthy controls were present. however, disruption of the tertiary structure of mbp by boiling significantly reduced the production of all three cytokines, supporting a role for antibodies in the induction of cytokine responses to mbp. we propose that natural igm autoantibodies may form complexes with mbp, facilitating the uptake of mbp by antigenpresenting cells (apc). since sera from ms patients did not enhance this uptake and the subsequent cytokine production, the mechanism may be part of an appropriate peripheral regulation of self-reactivity. we currently investigate this possibility. loredana postiglione( ), g tarantino( ), a spanò( ), p ladogana ( ), fl perrone( ), s padula( ), a riccio ( ) ( ) federico ii university medical school of naples, department of molecular and cellular biology and pathology l.califano, naples, italy ( ) federico ii university medical school of naples, departmentof clinical and experimental medicine, naples, italybackground: hepatitis c virus (hcv) infection can induce immunological disorders with different clinical expression such asarthritis, sjogren sindrome and various form of vasculitis.aim: to study the prevalence of anti-cyclic citrullinated peptides antibodies (anti-ccp) in a group of patients affected by hcv-related arthritis and the eventual correlations with rheumatoid factor (rf) and/orantinuclear antibodies (ana), and articular involvement. study design: patients with arthritis were selected in a population of subjects affected by hcv infection. each patients was evaluated by clinical examination ( denoted poliarticular and mono-oligoarticualr involvement), by x-graphic aspects of joint involvement ( patients presented join erosions), by ana, rf and anti-ccp positiveness.results: , % of patients presented positivenessfor anti-ccp, without significant correlation between suchparameter and ana, rf and articular involvement. anti ccp resulted positive in out of the patients with joint erosions, and only in out of the patients without joint erosions. such frequency analyzed by chi square ended up in no significant differences. our patients presented an interesting prevalence of the positiveness for anti-ccp. these data suggest a consideration about the specificity, commonly attributed to this parameter in the diagnosis of rheumatoid arthritis. expression of nkg d on cd + t cells is generally rare in both mice and humans, but has been reported in a number of inflammatory diseases, including rheumatoid arthritis, crohns disease and an animal model of type diabetes. the monoclonal antibody cx recognizes murine nkg d and has been shown to block ligandbinding and mediate internalization of nkg d. furthermore, cx can inhibit and/or ameliorate disease in animal models of type diabetes and inflammatory bowel disease. thus, it is very likely that nkg d plays an important role in the development of inflammatory and autoimmune diseases. since little is known about the pharmacokinetics and pharmacodynamics of the cx antibody, we decided to study this in both regular balb/ c mice and immunodeficient cb .scid mice. different doses of cx antibody was injected intraperitoneally and pk and pd was measured by elisa (anti-cx elisa in serum) and flow cytometry (down-regulation of nkg d on cd b+ nk cells) for up to two weeks after administration. we found that cx very efficiently down-regulate nkg d on cd b+ nk cells and that the effects of the antibody can be seen for more than two weeks after one single injection. finally, we propose a model which may be helpful in predicting the effects of different doses of cx antibody in vivo. ( ), k mehta( ), n deo( ), j chaudhary( ), p bobrowski ( ) ( ) albany medical college, usa ( ) vedic lifesciences, usa ( ) rainforest nutritionals, inc, us background: the efficacy and safety of reparagen, in treating osteoarthritis was compared to glucosamine sulfate in a mumbai-based multi-center, randomized, double-blind study.methods: subjects (n= ) were screened and randomized to receive glucosamine sulfate (n= , mg/day) or reparagen (n= , mg/day), a polyherbal consisting of vincaria (uncaria guianensis) and rni (lepidium meyenii) administered orally, twice daily. primary efficacy variables were womac scores, visual analog score (vas) for pain, and response to treatment defined as a % improvement in womac pain, with assessments at , , , and weeks. secondary variables were results: subject randomization was effective and both treatments showed significant benefits in primary outcomes within one week (p< . ), with a similar, progressive improvement over the course of the week treatment protocol ( - % reduction in total womac or vas scores). the response rate was substantial for both glucosamine ( %) and reparagen ( %), which exceeded placebo responses ( %, p < . ) and supported by investigator and subject assessments. tolerability was excellent and safety parameters were unchanged. rescue medication use was significantly lower in the reparagen group (p < . ), and serum igf- levels were unaltered.conclusions: both reparagen and glucosamine sulfate produced substantial improvements osteoarthritis symptoms. response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. we speculate that the high response rate to glucosamine sulfate may reflect higher baseline pain levels or synergy with dietary curcumin. inflammation accompanies and aggravates progression of all modern human chronic pathological conditions. growing evidence indicates the beneficial role of proper nutrition in controlling inflammation. we investigated the effects of selected essential nutrients in experimental inflammation and the molecular mechanisms involved. tested nutrient mixture (nm) consisted of green tea catechins, citrus flavonoids hesperidin, naringenin and quercetin, ascorbate, lysine, proline, arginine and cysteine. systemic inflammation in mice challenged with bacterial lipopolysaccharide (lps) was monitored by blood plasma levels of fourteen key inflammatory cytokines. two week supplementation with mg nm/kg body weight prior to lps challenge provided significantly greater protection than did supplementation with ibuprofen. induction of interleukin- (il- ) and monocyte chemoattracting protein- , two cytokines especially responsive to lps challenge, was reduced in nmsupplemented animals by % and %, respectively. corresponding reduction in ibuprofen group was % and %. protective mechanisms involved were assessed in human cultured u macrophages stimulated with lps.the cytokines most responsive were tumor necrosis factor alpha ( % and % reduction by supplementation with nm and ibuprofen, respectively) and il- ( % and % in corresponding reduction). nm supplementation dramatically reduced prostaglandin e secretion by stimulated macrophages along with cyclooxigenase- (cox ) cellular protein expression. mrna levels forcox and inflammatory cytokines were also dramatically reduced. quercetin was the most effective nutrient when tested individually. however, nm appeared to surplus the combined effect of individual components. we conclude that the tested combination of essential nutrients demonstrates strong beneficial effects in experimental inflammation by targeting responsible gene expression. ( ), hp kim ( ), kh son ( ) ( ) college of pharmacy, kangwon national university, south korea ( ) department of food and nutrition, andong university, south korea chalcones belong to flavonoid family from plant origin and some of them possess anti-inflammatory activity. recently, several natural and synthetic chalcones were reported to inhibit inducible nitric oxide synthase (inos)-catalyzed no production in cell cultures. in the present study, to find the optimal chemical structures and to elucidate their action mechanisms, synthetic chalcones having the substituent(s) on a-and b-rings were prepared and their effects on inos-catalyzed no production were evaluated using lps-treated raw . cells. among the tested compounds, -methoxy- , -dichlorochalcone (ch ), -hydroxy- -methoxychalcone (ch ), -hydroxy- -bromo- -methoxychalcone (ch ) and -hydroxy- , -dimethoxychalcone (ch ) potently inhibited no production (ic s, . - . mm). the favorable chemical structures were found to be a methoxyl substitution in a-ring at adjacent position ( or ) to carbonyl moiety with/without -(or -)hydroxyl group and -halogen substitution in b-ring. when the cellular action mechanisms of ch , ch and ch were further examined, it was revealed that ch and ch clearly down-regulated inos expression while ch did not. moreover, ch and ch were proved to suppress the nuclear transcription factor-kb activation. from the results, it is suggested that certain chalcone derivatives potently inhibit inos-catalyzed no production by the different cellular mechanisms, inos down-regulation or inos inhibition, depending on their chemical structures. these chalcone derivatives may be possibly used as lead compounds for developing new anti-inflammatory agents. an oligomeric stilbene alpha-viniferin (avf) was isolated from root of carex humilis (cyperaceae) as an inhibitor of cyclooxygenase (cox)- activity by bioassayguided fractionation. the avf was later found to downregulate lipopolysaccharide (lps)-induced cox- expression as well as to inhibit nuclear factor (nf)-kb activation, in addition to its inhibitory effect on cox- activity. furthermore, the compound exhibited antiarthritic effect in vivo. avf is a trimer of resveratrol and contains benzofuran moieties in its central part. starting from benzofuran and its related chemicals, cyclohexylimino- -methyl- , -dihydro- h-benzo [ , ] oxathiol- -one (lyr- ) was discovered to inhibit lpsinduced nf-kb transcriptional activity in macrophages raw . . the lyr- reduced lps-induced dna binding activity and nuclear translocation of nf-kb as well as inhibited lps-induced degradation and phosphorylation of inhibitory kb (ikb) protein. these results suggest that lyr- could suppress lps signaling molecule, putatively ikb kinase (ikk) complex, upstream ikb degradation in nf-kb activating pathway. lyr- inhibited in vitro kinase activity, gst-ikb phosphorylation, of wild type ikkbeta or a constitutively active ikkbeta mutant (c/a, cys- to ala) but did not affect that of another constitutively active ikkbeta mutant (ss/ee, ser- and to glu). therefore, lyr- could inhibit lps-induced nf-kb activating pathway by targeting ser- and/or residues on the activation domain of ikkbeta. as pharmacological actions, lyr- prevented nf-kb-dependent expression of inducible nitric oxide synthase, cox- , or inflammatory cytokines at the transcription level in lps-stimulated macrophages raw . . furthermore, lyr- protected lpsinduced septic shock in vivo. faculty of medicine, institute of pharmacology, ljubljana, slovenia a part of anti-inflammatory action of antidepressants can arise from their effect on histamine elimination from the side of inflammation. in mammals histamine is mainly degraded by two enzymes: histamine-n-methyltransferase (hnmt) and diamine oxidase (dao). the aim of present investigation is to establish whether antidepressants amitriptyline and sertraline can affect histamine metabolism. their effects on enzyme activity and mrna expression were studied in guinea pig tissues. plasma and tissue homogenates were incubated with saline (control) and different antidepressant concentrations. specific enzymatic activities of dao and hnmt were determined by radiometric assay. in addition, guinea pigs were treated with saline or amitriptyline ( mg/kg, ip), afterwards dao and hnmt mrnas were detected by pcr in different tissues. results showed thatamitriptyline, nm, , and mm, increased guinea pig plasma dao activity by , , and %, respectively, while sertraline increased it at mm (by %). at higher concentrations ( and mm) sertraline decreased dao activity. in the guinea pig tissues hnmt activity changes were found only when incubated with amitriptyline; sertraline had no effect. at and nm amitriptyline the activity of hnmt increased by and %, respectively. in animals treated with amitriptyline an induction of dao and hnmt mrna expression was noticed in several tissues. our results suggest that in guinea pigs due to higher histamine metabolism antiinflammatory effects can be expected at lower concentrations of antidepressants. the effect might be the opposite with higher amitriptyline concentrations. steven hefeneider( , ), c macarthur ( ), d trune ( ), s mccoy ( ) ( ) oregon health and science university, portland, oregon, usa ( ) targeted gene delivery, inc., portland, oregon, usa engagement of toll-like receptors (tlrs) by bacterial components such as lps and dna initiates inflammation.the current study examines a novel anti-inflammatory peptide, termed p , for treatment of inflammation induced by either lps or bacteria.peptide p was derived from an immunoregulatory protein of vaccinia virus, and interferes with tlr signaling.in this study we examined the efficacy of p to limit inflammation in a mouse model of sepsis and a model of middle ear inflammation, termed acute otitis media (aom).we demonstrate in the sepsis model, that in vivo treatment of mice with p inhibited lps-induced production of serum inflammatory mediators.moreover, p treatment, administered after initiation of inflammation, significantly increased survival of mice injected with lps.in the aom model, peptide p significantly reduced in vivo middle ear inflammation and fluid accumulation initiated by h. influenza.assessment of route of administration and delayed treatment studies demonstrated the efficacy of peptide p .simultaneous injection of bacteria and peptide p resulted in a significant reduction in fluid accumulation, infiltrating cells, and tympanic membrane thickness.fluid accumulation within the eustachian tubes was also significantly reduced following p treatment.-subcutaneous and oral administrations of p , but not intravenous administration, were also efficacious in reducing inflammation. administration of p after initiation of an ongoing inflammatory response was effective at reducing inflammation and fluid development.taken together, these results demonstrate the therapeutic potential of peptide p to limit an inflammatory response and suggest a possible new treatment strategy for bacterial-induced inflammation. ( ), c zhou( ), y zhang( ), m sun( ), x wan ( ), h yu( ), x yang( ), rd ye ( ), j-k shen ( ) formyl peptide receptor-like (fprl ) is a structural homologue of fpr, which binds chemotactic peptides of as small as amino acids (e.g., fmet-leu-phe, fmlf) and activates potent bactericidal functions in neutrophils. in comparison, fprl ligands include peptides of - amino acids, such as trp-lys-tyr-met-val-[d]met (wkymvm) and other synthetic peptides. to determine the core peptide sequence required for fprl activation, we prepared various analogues based on wkymvm and evaluated their bioactivities in an fprl -transfected cell line. although substitution of d-met resulted in loss of activity, removal of val together with d-met produced a peptide that retained most of the bioactivities of the parent peptide. the resulting peptide, wkym, represents a core structure for an fprl ligand. further substitution of lys with nle slightly improved the potency of the tetrapeptide, which becomes a dual agonist for both fprl and fpr. based on these structure-activity studies, we propose a model in which the modified tetrapeptide trp-nle-tyr-met (wnleym) binds to fprl through aromatic interactions involving the side chains of trp and tyr , hydrophobic interaction of nle , and the thio-based hydrogen bonding of met , with the respective residues in fprl which have not been identified. the identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for fprl -related disorders.there is a growing awareness of the interaction of food constituents with the immune system. the present study aims to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin. mice orally supplemented with glycine, lactoferrin or a combination were injected intradermal (in the ear) with zymosan. ear swelling, as a measure for inflammation, as well as il- , tnf-a and il- levels in the ear and the number of tnf-a producing spleen cells were analyzed.-glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced number of tnf-a producing spleen cells).glycine effects ( , and mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses ( . and mg/mouse/ day) inhibited the inflammatory response significantly. surprisingly higher doses of lactoferrin ( and mg/ mouse/day) failed to influence the inflammatory reaction. a combination of both nutrients (lactoferrin . mg/ mouse/day in combination with glycine or mg/ mouse/day) inhibited the zymosan induced ear swelling synergistically. additionally an additive effect of both components was seen on the number of tnf-a producing spleen cells. the present data show anti-inflammatory activity of glycine and lactoferrin using the zymosan induced inflammation model.moreover a combination of both components demonstrated a synergistic effect on inflammation of the skin and an additive effect on the number of tnf-a producing spleen cells. ( ), p sambrook( ), k fukudome( ), m xue ( ) ( ) university of sydney, st leonards, nsw, australia ( ) saga medical school, saga, japan objectives: to investigate the i) expression of endothelial protein c receptor (epcr) in synovial membrane and peripheral blood monocytes from patients with rheumatoid arthritis (ra) and osteoarthritis (oa) and ii) role of epcr and its ligand, activated protein c (apc), on the function of monocytes from ra patients.methods: epcr, cd and pc/apc in synovial tissues were detected by immunostaining and in situ pcr. monocytes were isolated from peripheral blood of patients with ra and treated with apc, lipopolysaccharide (lps), and/or epcr blocking antibody, rcr . cells and supernatants were collected to analyze the expression/activation of epcr, nuclear factor nf-kb and tumour necrosis factor tnf-a.results: epcr was expressed by both oa and ra synovial tissues but was markedly increased in ra synovium. epcr was colocalized with pc/apc mostly on cd positive cells in synovium. in ra monocytes, apc upregulated epcr expression reduced monocyte chemoattractant protein- -induced chemotaxis of monocytes by approximately %. apc also completely suppressed lps-stimulated nf-kb activation and attenuated tnf-a protein by more than % in ra monocytes. the inhibitory effects of apc were reversed by rcr , indicating that epcr modulates the inhibitory effects of apc.conclusions: our results demonstrate for the first time that epcr is expressed by synovial tissues, particularly in ra, where it co-localizes with pc/apc on monocytes/ macrophages. in addition, apc inhibits the migration and activation of ra monocytes via epcr. these inhibitory effects on ra monocytes suggest that pc pathway may have a beneficial therapeutic effect in ra. key: cord- -xx imhzb authors: lambour, jennifer; naranjo-gomez, mar; piechaczyk, marc; pelegrin, mireia title: converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: xx imhzb monoclonal antibodies (mabs), which currently constitute the main class of biotherapeutics, are now recognized as major medical tools that are increasingly being considered to fight severe viral infections. indeed, the number of antiviral mabs developed in recent years has grown exponentially. although their direct effects on viral blunting have been studied in detail, their potential immunomodulatory actions have been overlooked until recently. the ability of antiviral mabs to modulate antiviral immune responses in infected organisms has recently been revealed. more specifically, upon recognition of their cognate antigens, mabs form immune complexes (ics) that can be recognized by the fc receptors expressed on different immune cells of infected individuals. this binding may be followed by the modulation of the host immune responses. harnessing this immunomodulatory property may facilitate improvements in the therapeutic potential of antiviral mabs. this review focuses on the role of ics formed with different viral determinants and mabs in the induction of antiviral immune responses in the context of both passive immunotherapies and vaccination strategies. potential deleterious effects of ics on the host immune response are also discussed. therapeutic potential of antiviral monoclonal antibodies monoclonal antibodies (mabs) have gained an important place in the therapeutic arsenal against severe human diseases. more than mabs have been approved or are under review for human use, and several hundred are currently being tested in the clinic, , most of them to treat patients suffering from a variety of cancers or inflammatory diseases. concerning antiviral mabs, only one, directed against respiratory syncytial virus (rsv), has been approved for the prophylactic treatment of pediatric infections. however, employing mabs as antiviral drugs is under consideration for the treatment of several chronic and acute severe viral infections, especially to address the public health emergencies such as the recent ebola virus and middle east respiratory syndrome coronavirus outbreaks. [ ] [ ] [ ] [ ] [ ] illustrating this trend, the number of antiviral mabs developed and tested in preclinical and clinical trials has grown exponentially in the past years and includes mabs directed against life-threatening agents, such as human immunodeficiency virus (hiv), hepatitis b virus (hbv), hepatitis c virus (hcv), influenza virus, dengue virus, ebola virus and severe acute respiratory syndrome virus coronavirus, among others. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] importantly, recent clinical data have also demonstrated the efficacy of anti-hiv mabs in controlling viremia, when administered to hiv-infected patients, lending strong support to the idea that mabs could broaden the therapeutic arsenal against severe viral infections. , their use as antiviral agents is all the more likely to be considered given that multiple biological activities may account for their therapeutic effects. although a few mabs have been developed to inhibit the recognition of viral receptors or co-receptors on the surface of target cells, most antiviral mabs have been selected for their ability to neutralize virions through the binding of their antigen-binding (fab) fragment to viral surface antigens essential for entry into host cells. however, the biological activity of antibodies is also mediated by the fragment crystallizable region (fc) moiety. thus, it is interesting to note that most antiviral mabs in use are immunoglobulin (ig)-gs displaying a variety of effector functions, including binding to both complement and fcγ receptors (fcγrs). different types of fcγrs are expressed in a regulated manner by many cells of the immune system, including b cells, natural killer cells, dendritic cells (dcs), monocytes/macrophages, granulocytes and mast cells, and their engagement by the fc antibody moiety is essential for regulating the antibody effector functions. , upon recognition of their target antigens, antiviral mabs can facilitate virus elimination via two types of complementmediated mechanisms: (i) inactivation of viral particles and/or phagocytosis of opsonized virus mediated by cells of the innate immune system ( figure ) and (ii) opsonization and subsequent lysis of infected cells, when viral antigens are also expressed on the cell surface (for example, envelope (env) glycoprotein of lentiviruses such as hiv) via complement-dependent cytotoxicity. in addition to complementmediated actions, recognition of fcγrs can entail antibody-dependent cellular phagocytosis and antibody-dependent cell-mediated cytotoxicity ( figure ). , [ ] [ ] [ ] [ ] finally, antiviral mabs also have a role in viral blunting by inhibiting cell-to-cell viral transmission. in addition to controlling the viral propagation by these mechanisms, the opsonization of viral particles and/or infected cells by therapeutic antiviral mabs of the igg type leads to the formation of immune complexes (ics) recognizable by the fcγrs expressed on antigen-presenting cells (apcs) such as dcs. this can potentially affect the endogenous antiviral adaptive immune response of passive immunotherapy-treated individuals. despite the fact that the immunoregulatory functions of antibodies (as well as ics) have been known for a long time, and have been reported in different experimental settings and physiopathological situations, , - the immunomodulatory role of mabs with clinical potential as antiviral drugs has only recently been considered. this review mainly focuses on the induction of antiviral immune responses by ics in both passive immunotherapies and vaccination strategies. the potential deleterious effects of antiviral antibodies on the host immune dysfunction and/or viral propagation are also discussed. only recently have studies addressed whether and how passive immunotherapies utilizing antiviral mabs are able to enhance the antiviral immunity in infected individuals. this is largely due to the limited availability of suitable immunocompetent animal models of viral infection that allow in-depth investigations of the endogenous immune response. the concept that passive immunotherapies utilizing antiviral mabs can induce long-term protective immunity has recently been established using an immunocompetent mouse model, consisting of short immunotherapies given to young animals infected with the frcase murine leukemia virus. the induction of such 'vaccine-like' effects by antiviral mabs, as well as some of the mechanisms involved, are reviewed in detail elsewhere. in brief, the inoculation of mouse pups with frcase is fatal because the antiviral immune response is too weak to control the viral propagation. in contrast, treatment with a neutralizing mab for several days shortly after infection blunts viral propagation and induces a lifelong protective antiviral immunity composed of both a highly neutralizing humoral response and a cytotoxic cd + t-cell response. [ ] [ ] [ ] [ ] [ ] [ ] this induction of protective immunity strictly depends on the fc fragment of the neutralizing mabs. , moreover, the formation of ics composed of the administered mabs and infected cells rather than virions is crucial for the enhanced antiviral immune response. such ics are recognized by the fcγrs expressed by dcs, which facilitate ics internalization and lead to stronger activation and more efficient antigen presentation by dcs, eventually leading to stronger cytotoxic t-lymphocyte (ctl) responses. an fc-mediated effect that occurs concurrently is the inhibition of regulatory t-cell (treg) expansion. this depends on the mab effector functions and occurs rapidly. moreover, it is necessary for the development of the protective humoral and cellular responses, as treg-mediated immunosuppression is observed in all cases of chronic viral infections, where it dampens antiviral immune responses, thereby permitting the establishment of chronicity. finally, breastfeeding and placental transfer of maternal anti-frcase igs induced by mab immunotherapy not only led to the viral propagation blunting in infected pups, but also to the induction of long-lasting protective humoral immunity in these animals. this is a particularly interesting observation when one considers that the frcase model is reminiscent of perinatal infection by hiv, including breastfeeding-mediated mother-to-child virus transmission. other evidence for the induction of 'vaccine-like' effects by antiviral mabs comes from studies in several preclinical models of human viral infections and from hiv-infected patients. in a mouse model of rsv infection, the administration of a neutralizing mab directed against the virus attachment protein g induced a shift in the adaptive immune response from th -to th -type, leading to sustained and enhanced humoral and cd + t-cell responses. however, this effect was not fc-dependent, but rather due to the ability of the therapeutic mab to counteract the intrinsic immunosuppressive activity of the rsv g protein. mab-driven enhancement of the humoral response has also been reported in two preclinical models of henipavirus infection in african green monkeys. , recovery from both hendra and nipah virusinduced disease correlated with the development of host antibody responses consequent to the administration of the highly neutralizing . mab. this hendra and nipah virus cross-reactive mab is currently being considered for human use. finally, anti-hiv antibodies can modulate immune responses in infected organisms. such effects were initially reported in several nonhuman primate models of hiv infection and then observed in infected humans. macaques were infected with different strains of simian immunodeficiency virus (siv) or simian hiv (shiv, a chimeric virus in which hiv env substitutes for that of siv and allows for the assessment of the antiviral effects of anti-hiv antibodies) following different protocols. these experiments showed that the administration of highly neutralizing antibodies (either mabs or polyclonal igs) enhanced both the humoral and cellular antiviral immune responses of treated animals. , [ ] [ ] [ ] interestingly, recent clinical data describe the elicitation of host humoral responses in viremic subjects upon single injection of the potent bnc anti-hiv mab. however, the mechanisms leading to the stimulation of antiviral immune responses in these preclinical models of hiv infection or in infected patients remain uncharacterized. moreover, it is unknown whether these antiviral responses have genuine protective vaccine-like effects. in any case, these important observations open new avenues for the improvement of mab-based antiviral hiv therapies. moreover, as the in vivo activity of anti-hiv- bnabs, including viral load control, was recently shown to crucially depend on fc effector functions, , an important issue is identifying that fc-fcγrs interactions are involved in the induction of vaccinelike effects by antiviral mabs. to understand the mechanisms underlying the enhancement of antiviral responses by ics, several in vitro studies have addressed whether antibody-mediated viral uptake by dcs could lead to stronger activation of these cells and the development of stronger virus-specific cd + and cd + t-cell responses in an fc-dependent manner. such an increase in the cellular immune response has been reported in different infectious settings using ics made with different types of antigens, including recombinant viral proteins and whole virions, as well as infected cells (table ) . concerning ics made with viral proteins, several reports have shown that ics made up of anti-hbv mabs and the hepatitis b surface antigen (hbsag) can affect dc function and enhance t-cell responses. hbsag/anti-hbv ics significantly increased the uptake of the immunocomplexed hbsag antigen, and augmented the in vitro proliferation of virus-specific t cells and their production of interferon (ifn)-γ. moreover, dcs from hbv-infected patients incubated with hbsag/anti-hbv ics showed higher expression of major histocompatibility complex (mhc)-ii molecules and higher production of interleukin (il)- . ic-loaded dcs also enhanced production of il- and ifn-γ by co-cultured t cells. interestingly, the therapeutic efficacy of hbsag/anti-hbv ics has been tested in clinical trials (see below) in hbv-infected patients with encouraging results. [ ] [ ] [ ] more recently, in experiments aimed at visualizing immunopotentialization by hbsag/anti-hbv ics (see below), live-cell imaging revealed that ics were internalized via the fcγrs of apcs and were subsequently transported through early and late endosomes into lysosomes, where they co-localized with mhc-i and mhc-ii molecules. consistent with the latter observation, the administration of dcs loaded with hbsag/anti-hbv ics to mice increased the number of ifn-γ-and tumor necrosis factor-α-producing cd + and cd + t cells. similarly, in an siv infection setting, the incubation of apcs with ics made with a recombinant full-length gag p protein and an anti-p igg increased siv capsid cross-presentation. capsid cross-presentation was dependent on fcγr-mediated uptake of the immunocomplexed siv capsid protein, and required its proteasomal and endosomal degradation to generate stronger gag-specific cd + t-cell responses. from a mechanistic standpoint, these studies indicate that antiviral antibodies might enhance the priming and expansion of virus-specific cd + and cd + t cells by both promoting the secretion of key cytokines and facilitating the uptake and crosspresentation of viral ags by fcγr-expressing dcs. immune-complexed whole virions have also been shown to affect the functional activation of dcs. the stimulation of dcs with ics composed of siv virions and highly neutralizing siv-hyperimmune sera (svig) led to the increased virus-specific cd + t-cell responses in an fc-dependent manner. in contrast, dcs stimulated with ics made of hiv- and a polyclonal igg pool from hiv-infected subjects showed only weak hiv-specific ctl-stimulating activity. this suggested that opsonization of hiv- by iggs might be associated with decreased ctl-stimulatory dc activity. however, not all igg isotypes display equivalent effector functions. therefore, the undefined nature of the antibodies (both in terms of predominant isotypes and neutralization potential) used to form the hiv-ics in these experiments might explain these observations. whether hiv-ics made with highly neutralizing anti-hiv mabs of a specific igg isotype might induce stronger cd + t-cell responses is an important issue deserving further investigation (figure ) . moreover, the nature of the viral determinant present in ics might also be crucial in the stimulation of antiviral responses. interestingly, as mentioned above, in the mouse frcase infection model, ics made up of a neutralizing mab and infected cells, but not those made with virions, efficiently induce strong gag-specific cd + t-cell responses with high cytotoxic activity. this observation shows that the viral and cellular ics can trigger different immunologic outcomes. in the case of frcase, this is explained by the fact the frcase-gagl ctl immunodominant epitope is, at best, poorly incorporated into virions. taken together, these data indicate that the uptake of cellular ics might allow the presentation of a broader viral antigenic repertoire, leading to stronger effects on immunity ( figure ). ics formed with endogenous antibodies generated in virally infected mice have been shown to influence antiviral cellular immune responses in several models (table ) . notably, the highly neutralizing humoral response generated against the frcase retrovirus in mab-treated-infected mice was demonstrated to limit the viral propagation and to enhance memory cellular responses long after the disappearance of the therapeutic mab (which occurs within two weeks post administration, reflecting the natural igg lifespan in vivo). ic-mediated activation of dcs upon binding to fcγr was key for this effect. similarly, in an influenza virus infection model, ics formed with endogenous antiviral antibodies promoted more sustained antigen presentation by dcs, resulting in stronger cd + t-cell proliferation. interestingly, such prolonged antigen presentation by dcs was dependent on virus-specific, isotype-switched antibodies that facilitated the capture and cross-presentation of viral antigens by figure parameters to consider for achieving optimal ic-mediated modulation of antiviral immune responses. the optimization of vaccine-likeeffect-inducing protocols will require the consideration of several parameters such as the nature of the antigen (that is, purified viral proteins, whole virions and infected cells) and the antibody (that is, monoclonal vs polyclonal, nature of the isotype, engineered fc domain with improved effector functions and so on) used to form the immunogenic ics, as differences in these parameters might impact immunological outcomes. in addition, whether the optimized ics are used alone or in combination with immunostimulatory molecules might also be of paramount importance. several other parameters, including the ic dosage, the route of administration, the choice of adjuvant and the immunological status of patients, will also have to be considered. fcγr-expressing dcs. in addition, serum antibodies can affect the virus-specific cd + /cd + t-cell balance in an fc-dependent manner during rsv infection. an enhanced ratio of rsv-neutralizing to -non-neutralizing antibodies profoundly enhanced the cd + t-cell response. in a murine lymphocytic choriomeningitis virus (lcmv) infection model, endogenous virus-specific antibodies could stimulate innate immunity and thereby positively affect both the induction and the maintenance of the virus-specific cd + t-cell response. notably, anti-lcmv antibodies limited viral replication in peripheral organs, but allowed replication of the virus in the marginal zone of the spleen, promoting cd + t-cell priming. interestingly, anti-lcmv antibodies were also reported to be essential for long-term maintenance of the memory ctl response in infected mice. , these observations, together with the in vitro studies described above, demonstrate that virus-specific antibodies can promote the acquisition, processing and presentation of antigens that are subsequently instrumental for priming t-cell responses and programming functional cd + memory in an fc-dependent manner. they strengthen the concept that antiviral antibodies can regulate the quality and function of antiviral t-cell responses through the formation of ics. moreover, they also provide a rationale for developing novel ic-based therapeutic vaccination strategies. in , terres and wolins demonstrated the ability of ics to induce higher antibody titers than antigens alone. since then, the immunogenic potential of ics, alone or in combination with different types of adjuvants, has been tested in several viral infection systems, including animal models of human infections, for example, those involving hbv, hiv, rsv or flaviviruses. the immunostimulatory effects of ics are principally attributed to the ability of fc antibody fragments to recruit the host immune system. however, evidence also implicates fab fragments in modulation of the antiviral immune response, although the outcomes are less documented and were proposed to occur via alterations in antigen conformation and/or in the exposure of specific epitopes. we describe the enhancement in antiviral immune responses observed in ic-based vaccination experiments below (table ) . ics have been tested as vaccines to augment protective immune responses in different animal models of hbv infection. in ducks, ics made with duck hbsag and rabbit anti-duck hbsag (dhbsag/ anti-dhb) were used as immunogens in the form of solid matrixantibody-antigen complexes (smaa). such smaas contained killed staphylococcus aureus as a solid matrix and mab-opsonized viruses. they were initially shown to induce both humoral and ctl responses against the paramyxovirus simian virus in immunized mice. immunization of hbv-infected ducks with smaa-based dhbsag/anti-dhb ics led to the viral clearance in % of infected ducks. notably, the administration of dhbsag/anti-dhb ics lacking staphylococcus aureus showed decreased immunization efficiency, suggesting that the bacteria-based solid matrix functions as an adjuvant. ics have also been tested as a therapeutic vaccine against hbv infection in mice , , and woodchucks. balb/c mice immunized with hbsag/anti-hbv ics produced the increased levels of virusspecific antibodies. moreover, administering hbsag/anti-hbv ics to balb/c mice via intranasal inhalation induced both mucosal and systemic th -polarized immune responses, when administered with adjuvants such as cholera toxin or oligodeoxynucleotides containing modulation of antiviral immune responses by immune complexes j lambour et al immunostimulatory cpg motifs (cpg). this was not observed using hbsag alone. in addition, the administration of hbsag/anti-hbv ics to hbsag-positive transgenic mice decreased the serum hbsag levels and induced stronger ctl responses than hbsag alone. notably, the co-administration of ics and a plasmid coding for hbsag increased the antiviral immune response induced by ics, indicating the adjuvant effect of dna in this setting. a similar effect was also reported in a woodchuck model of hbv infection: immunization of woodchuck hepatitis virus (whv)-infected animals with whv surface antigen/anti-whv antibody ics combined with a dna vaccine resulted in a higher reduction of both viral load and antigenemia relative to ics alone. interestingly, the whv-infected animals were pretreated with lamivudine (a potent hbv antiviral drug able to enhance t-cell responses in chronically hbv-infected patients) before ic/dna immunization, suggesting that combination strategies should be considered in treating chronic hbv infections (figure ). the enhancement of antiviral immune responses by ics in vitro and in preclinical models of hbv infection paved the way for the development of ic-based therapeutic vaccination strategies against chronic viral hepatitis b infection. a therapeutic vaccine composed of yeast-derived recombinant hbsag/anti-hbv immunogenic complexes (yics) has been tested in a series of clinical trials. this vaccine approach was initially shown to be safe and to induce higher titers of hbsag antibodies, as well as to increase serum ifn-γ and il- levels in a phase i trial. importantly, a subpopulation of chronic viral hepatitis b patients showed a decrease in serum hbv viral load and hbsag levels together with an increase in anti-hbsag antibody titers in subsequent phase ii trials. , from a mechanistic standpoint, recent data showing that the administration of yic-loaded dcs to mice increased both cd + and cd + t-cell responses suggest that the improved immune responses induced by yics might account for the antiviral effect observed in a fraction of patients. in an attempt to enhance the immunogenic potential of yic-based vaccines, a phase iii trial tested the therapeutic effect of a higher number of ic doses. unfortunately, overstimulation with yic decreased the vaccine efficiency due to host immune fatigue. this suggests that vaccination protocols must be optimized and must take into account both the nature and the dose of ics, as well as other parameters such as the route of administration, the type of adjuvant and the immunological status of patients to achieve efficient protective immunization ( figure ). in , a study immunized healthy volunteers with hiv peptides. the authors found that compared with free antigen, recall immunization with ics induced stronger t-cell responses through uncharacterized mechanisms. other reports also describe alteration of the anti-hiv response by ics. [ ] [ ] [ ] in particular, the immunization of immunocompetent mice with ics containing a recombinant hiv- gp env glycoprotein and a mab ( -d mab) directed to the cd -binding site induced a higher virus-neutralizing antibody response relative to free antigen. as described above, humoral responses were further increased upon the co-administration of ics and monophosphoryl lipid-a/dimethyldioctadecylammonium adjuvants. notably, the interaction of the anti-cd -binding site mab with hiv- gp induced conformational changes in the latter, leading to the enhanced antigenicity and immunogenicity of neutralizing epitopes localized in the hiv- v loop. these observations highlight the ability of anti-hiv- antibodies to induce antigenic alterations in specific hiv- gp epitopes upon ic formation. interestingly, further improvement in the immunogenicity of the v loop was obtained in ics generated with gp mutants lacking site-specific n-linked glycans. , , taken together, these observations suggest that the ability of ics to stimulate the induction of neutralizing antibodies is dictated by the nature of the antigen, as well as the specificity and affinity of the mabs utilized. these results also indicate the potential contribution of fab-mediated activities in the enhancement of antiviral humoral responses by ics. tsouchnikas et al. investigated the influence of immunization with ics on the specificity of antibody responses using the e protein of the tick-borne encephalitis virus as an immunogen. mice were immunized with a dimeric soluble form of e (se) alone or in complex with mabs specific for each of the three domains of e. the antibody response induced by these ics was compared with that observed after immunization with se alone. unexpectedly, immunization with ics did not change the extent of the overall antibody response in immunized mice. however, substantially different antibody responses were observed between the different ics. these differences most likely reflected an epitope-shielding phenomenon and antibody-mediated structural changes that led to the dissociation of the se dimer. thus, such phenomena can profoundly influence the fine specificity of antibody responses to the same immunogen and must be considered in ic-based vaccination strategies. as mentioned above, serum anti-rsv antibodies can affect virusspecific t-cell responses. on the basis of this, kruijsen et al. tested whether ics made with the commercial rsv-neutralizing mab palivizumab could influence adaptive immune response priming after intranasal administration. substantial anti-rsv t-cell priming and b-cell responses were observed in mice receiving rsv-ics, resulting in predominant th -type cd + t-cell response and igg c antibody responses. importantly, the ics also primed anti-rsv cd + t cells. these data have important implications for the prophylaxis and treatment of pediatric rsv infections. nevertheless, interactions between ics and neonatal versus adult innate and adaptive immune systems still need to be investigated because mouse studies have revealed potential antibody-induced neonatal autoimmunity in certain settings. , ics and immune dysfunction in the course of viral infections, the formation of ics composed of viral determinants and the resulting host humoral responses can potentially produce deleterious effects. persistent ics are formed in a variety of chronic viral infections and may lead to unregulated and protracted fcγr signaling. this may lead to immune dysfunction instead of stimulating antiviral immune responses. in this regard, the high levels of ics formed during lcmv infection interfere with fcγr-mediated activities. , these endogenously formed ics were shown to outcompete the effector functions of exogenously administered therapeutic mabs, in particular binding to fcγrs expressed by immune cells. persistent endogenous ics are also linked to dysfunctional b-cell responses in hiv infection, including the suppression of antiviral iga responses and impaired production of neutralizing antibodies (reviewed in moir et al. ). the composition of ics might also negatively affect the efficiency of the antiviral immune response. for instance, the composition of ics has been shown to be dynamic throughout the course of hiv infections due to changes in both antibody specificities and virion levels. notably, circulating ics are initially comprised of antibodies that opsonize both infectious and non-infectious virions. this results in a decrease in the availability of antibodies able to blunt viral propagation. this phenomenon probably contributes to the reduced efficiency of the antibodies generated during acute infection. changes in circulating ics have also been reported in hcv infections. the level of circulating ics is low in acutely infected patients, whereas chronically infected individuals show a high proportion of immunocomplexed hcv, raising the possibility that ics may have a role in the pathogenesis of hcv, namely liver damage. moreover, the formation of ics with non-neutralizing antibodies may also lead to the antibody-dependent enhancement of viral infection of fcγr-expressing cells. this happens in several viral infections, including those by the dengue virus. [ ] [ ] [ ] [ ] along this line, the binding of ics to fcγrs on monocytes/macrophages can paradoxically suppress innate immunity, induce il- production and bias responses from th toward th . this in turn leads to the increased infectious outputs by infected cells via intrinsic antibodydependent enhancement. , finally, ics have also been reported to have a role in increasing viral loads in the context of gene transfer-based vaccination strategies. in the step hiv- vaccine trial, which evaluated a replication-defective adenovirus type vector vaccine, the ics formed with pre-existing anti-adv antibodies improved the environment for hiv- replication in t cells. this may have been due to the ic-driven activation of a dc-t-cell axis that induces the activation of cd + t cells and leads to a permissive environment for hiv- infection. this environment probably explains the increased propagation of hiv- infection among adenovirus type -seropositive vaccine recipients. several approaches can be considered to enhance the immunomodulatory potential of antiviral mabs, both alone and in the form of ics, in particular through combining neutralizing mabs and ic-based vaccination strategies with other therapies. a first possibility would consist of inhibiting immunosuppressive mechanisms in infected individuals by either depleting the treg response, as suggested by nasser et al. or targeting immune checkpoints, the latter strategy having already led to improved immune responses against both viral infections and cancer. [ ] [ ] [ ] [ ] in addition, the combination of antiviral mabs with different immunostimulatory agents can also be envisaged. because the primary structure and glycosylation pattern of the fc fragment are both essential for antibody effector functions due to their impact on the engagement of type i and type ii fcr family members, , fc engineering might also represent another approach, not only to improve direct antiviral effects, but also to induce stronger vaccine-like effects. in this regard, identification of the main fcrs and fcr-mediated mechanisms involved in enhancing the antiviral immune response will be of utmost importance. taking into account that the various igg isotypes display different effector functions and interact differently with fcγrs, the careful selection of antiviral mab subclasses is also crucial for enhancing antiviral immune responses. finally, as fcγr polymorphisms have already been associated with differences in viral disease progression and the therapeutic efficiency of anticancer mabs, it will be important to evaluate the extent to which such polymorphisms can affect the vaccine-like effects induced by mab-based antiviral immunotherapies. the therapeutic potential of antiviral mabs is now widely accepted, and their use as antiviral drugs is increasingly under consideration. the diverse biological activities of these mabs lead to the direct control of viral propagation and the modulation of antiviral immunity. this provides a novel rationale for their use in diverse prophylactic and therapeutic approaches. the improvement in both humoral and cellular responses achieved through the administration of mabs, either free or in the form of immunogenic ics, offers new therapeutic options. the challenge now is to improve our understanding of how ics convert mab-based immunotherapies from 'passive' to 'active' and to exploit the underlying mechanisms. this conversion will be crucial in reaching the goal of using antiviral mabs to induce longlasting protective immunity against life-threatening viral infections. molecular properties of human igg subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases trial watch: monoclonal antibodies in cancer therapy isolation of potent neutralizing antibodies from a survivor of the ebola virus outbreak protective monotherapy against lethal ebola virus infection by a potently neutralizing antibody prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus b -n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n / reversion of advanced ebola virus disease in nonhuman primates with zmapp therapeutic efficacy of potent neutralizing hiv- -specific monoclonal antibodies in shiv-infected rhesus monkeys antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology monoclonal antibodies for prophylactic and therapeutic use against viral infections broadly neutralizing antiviral antibodies broadly neutralizing antibodies abrogate established hepatitis c virus infection a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus clinical development of monoclonal antibody-based drugs in hiv and hcv diseases mechanism of human antibody-mediated neutralization of marburg virus cross-reactive and potent neutralizing antibody responses in human survivors of natural ebolavirus infection a single injection of anti-hiv- antibodies protects against repeated shiv challenges early short-term treatment with neutralizing human monoclonal antibodies halts shiv infection in infant macaques overcoming drug-resistant herpes simplex virus (hsv) infection by a humanized antibody the growth and potential of human antiviral monoclonal antibody therapeutics a novel humanized antibody neutralizes h n influenza virus via two different mechanisms therapeutic efficacy of antibodies lacking fcgamma receptor binding against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies viraemia suppressed in hiv- -infected humans by broadly neutralizing antibody bnc virologic effects of broadly neutralizing antibody vrc administration during chronic hiv- infection mouse and human fcr effector functions type i and type ii fc receptors regulate innate and adaptive immunity exploring the potential of monoclonal antibody therapeutics for hiv- eradication neutralizing antibodies and control of hiv: moves and countermoves which antibody functions are important for an hiv vaccine? fcgammar dependent mechanisms of cytotoxic, agonistic, and neutralizing antibody activities broadly neutralizing antibodies that inhibit hiv- cell to cell transmission properties of mouse and human igg receptors and their contribution to disease models how antibodies act as natural adjuvants antibodies as natural adjuvants antibody-mediated regulation of the immune response fcgamma receptors as regulators of immune responses intravenous immunoglobulin therapy: how does igg modulate the immune system? antibody-mediated immunomodulation: a strategy to improve host responses against microbial antigens antiviral monoclonal antibodies: can they be more than simple neutralizing agents? induction of long-term protective antiviral endogenous immune response by short neutralizing monoclonal antibody treatment endogenous cytotoxic t-cell response contributes to the long-term antiretroviral protection induced by a short period of antibody-based immunotherapy of neonatally infected mice efficient mother-to-child transfer of antiretroviral immunity in the context of preclinical monoclonal antibody-based immunotherapy a crucial role for infected-cell/antibody immune complexes in the enhancement of endogenous antiviral immunity by short passive immunotherapy long-lasting protective antiviral immunity induced by passive immunotherapies requires both neutralizing and effector functions of the administered monoclonal antibody control of regulatory t cells is necessary for vaccine-like effects of antiviral immunotherapy by monoclonal antibodies prophylaxis with a respiratory syncytial virus (rsv) anti-g protein monoclonal antibody shifts the adaptive immune response to rsv ra -line f infection from th to th in balb/c mice a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody neutralizing polyclonal igg present during acute infection prevents rapid disease onset in simian-human immunodeficiency virus shivsf p -infected infant rhesus macaques passive neutralizing antibody controls shiv viremia and enhances b cell responses in infant macaques an anti-hiv- v loop antibody fully protects cross-clade and elicits t-cell immunity in macaques mucosally challenged with an r clade c shiv hiv- therapy with monoclonal antibody bnc elicits hoost immune responses against hiv- broadly neutralizing anti-hiv- antibodies require fc effector functions for in vivo activity broadly neutralizing antibodies and viral inducers decrease rebound from hiv- latent reservoirs in humanized mice hbsag-serum protein complexes stimulate immune t lymphocytes more efficiently than do pure hbsag selective functional deficit in dendritic cell-t cell interaction is a crucial mechanism in chronic hepatitis b virus infection evidence for antibody-mediated enhancement of simian immunodeficiency virus (siv) gag antigen processing and cross presentation in siv-infected rhesus macaques polyfunctional cd + t-cell induction in neutralizing antibody-triggered control of simian immunodeficiency virus infection antibodies attenuate the capacity of dendritic cells to stimulate hiv-specific cytotoxic t lymphocytes vaccination with recombinant hbsag-hbig complex in healthy adults therapeutic effect of hepatitis b surface antigenantibody complex is associated with cytolytic and non-cytolytic immune responses in hepatitis b patients a randomized controlled phase iib trial of antigenantibody immunogenic complex therapeutic vaccine in chronic hepatitis b patients immuno-potentiating pathway of hbsag-hbig immunogenic complex visualized prolonged antigen presentation by immune complex-binding dendritic cells programs the proliferative capacity of memory cd t cells serum antibodies critically affect virus-specific cd +/cd + t cell balance during respiratory syncytial virus infections virus-specific antibodies allow viral replication in the marginal zone, thereby promoting cd (+) t-cell priming and viral control maintenance of memory ctl responses by t helper cells and cd -cd ligand: antibodies provide the key impaired antibody response causes persistence of prototypic t cell-contained virus enhanced immunological sensitization of mice by the simultaneous injection of antigen and specific antiserum. i. effect of varying the amount of antigen used relative to the antiserum antigen-antibody complex as therapeutic vaccine for viral hepatitis b enhancement of the immune response to hepatitis b virus vaccine by antigen specific igm therapeutic efficacy of hepatitis b surface antigenantibodies-recombinant dna composite in hbsag transgenic mice combination of an antiviral drug and immunomodulation against hepadnaviral infection in the woodchuck model immunization against hepatitis b virus by mucosal administration of antigen-antibody complexes the use of immune complex vaccines to enhance antibody responses against neutralizing epitopes on hiv- envelope gp improving immunogenicity of hiv- envelope gp by glycan removal and immune complex formation elicitation of broadly reactive antibodies against glycanmodulated neutralizing v epitopes of hiv- by immune complex vaccines intranasal administration of antibodybound respiratory syncytial virus particles efficiently primes virus-specific immune responses in mice immunization with solid matrix-antibody-antigen complexes containing surface or internal virus structural proteins protects mice from infection with the paramyxovirus, simian virus solid matrix-antibody-antigen complexes induce antigenspecific cd + cells that clear a persistent paramyxovirus infection results of a phase iii clinical trial with an hbsag-hbig immunogenic complex therapeutic vaccine for chronic hepatitis b patients: experiences and findings antigenic peptides recognized by t lymphocytes from aids viral envelope-immune humans targeting a neutralizing epitope of hiv envelope gp by immune complex vaccine immunization with immune complexes modulates the fine specificity of antibody responses to a flavivirus antigen cutting edge: ly c/i(-) neonatal nk cells predispose newborns to autoimmune ovarian disease induced by maternal autoantibody the unique neonatal nk cells: a critical component required for neonatal autoimmune disease induction by maternal autoantibody antibody effector functions mediated by fcgamma-receptors are compromised during persistent viral infection suppression of fcgamma-receptor-mediated antibody effector function during persistent viral infection b cells in hiv infection and disease dynamic antibody specificities and virion concentrations in circulating immune complexes in acute to chronic hiv- infection immune complexed (ic) hepatitis c virus (hcv) in chronically and acutely hcv-infected patients paradoxical role of antibodies in dengue virus infections: considerations for prophylactic vaccine development dengue antibody-dependent enhancement: knowns and unknowns intrinsic antibodydependent enhancement of microbial infection in macrophages: disease regulation by immune complexes how innate immune mechanisms contribute to antibodyenhanced viral infections antibody-dependent enhancement infection facilitates dengue virus-regulated signaling of il- production in monocytes activation of a dendritic cell-t cell axis by ad immune complexes creates an improved environment for replication of hiv in t cells restoration of hbv-specific cd + t cell function by pd- blockade in inactive carrier patients is linked to t cell differentiation a randomized, double-blind, placebo-controlled assessment of bms- , a fully human monoclonal antibody to programmed death- (pd- ), in patients with chronic hepatitis c virus infection immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential augmentation of hepatitis b virusspecific cellular immunity with programmed death receptor- /programmed death receptor-l blockade in hepatitis b virus and hiv/hepatitis b virus coinfected patients treated with adefovir key: cord- -dnsdg n authors: nan title: poster sessions date: - - journal: eur j immunol doi: . /eji. sha: doc_id: cord_uid: dnsdg n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx , the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th -oriented adaptive response. actually, ptx -deficient mice are highly susceptible to aspergillosis and develop th skewed responses; moreover, ptx -deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx , which does not show direct activity on fungal cells. finally, ptx alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx -mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx -opsonised conidia, cd b activation, internalization, recruitment to the phagocytic cup and cd b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx , fcgriia/cd mediates inside-out activation of cd b and consequently phagocytosis of c b-opsonised conidia. in vivo phagocytosis experiments performed with c q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx ) and the cellular arm (fcgrs, cr ). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx -/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd , immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx . we found high concentration of ptx in human colostrum ( . ± . ng/ml at day post-delivery) compare to the one found in human serum ( x ng/ml). the presence of ptx in human colostrum seems to be due to the secretion of ptx by human mammary gland since we report the production of ptx by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx supplying by breast feeding: (i) soluble ptx in colostrum (ii) hmc that can secrete ptx upon stimulation in the specific tissue, (iii) an increase of ptx production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx may participate to the beneficial role of breast feeding on the newborn health. a. m. baru , j. stephani , h. wagner , t. sparwasser twincore, institute for infection immunology, hannover, germany, technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of receptors in humans (tlr - ) and in mice (tlr - , - ). as transmembrane receptors, tlrs are expressed on the cell surface (tlr , , , , , ( ) ( ) ( ) ( ) and at endosomal membranes (tlr , , and ) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about years later to this, toll-like receptor (tlr ) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr (hutlr ) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr (mutlr ) is also expressed on conventional dendritic cells (cdcs). consequently, tlr ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr (henceforth called as hut mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr knock-out background. we expect that hut -mutlr -/mice mimic the human specific expression pattern of tlr , i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr . by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin- is a heterodimeric cytokine consisting of the two subunits p and p . the main inducers of il- p are microbial components activating toll-like receptors with the magnitude of il- p induction depending on the specific tlr engaged. differential induction of il- p upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il- p due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il- p promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il- p promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone and acetylation in specific regions of the proximal promoter region. acetylation of h showed a specific distribution pattern and occured mainly in regulatory elements within the il- p promoter, whereas acetylation of h took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue on h turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue , w. ellmeier medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi , m. buxadé , j. minguillón , r. berga , j. aramburu , c. lópez-rodríguez universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat , the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat . our work indicates that nfat is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr , tlr , tlr and tlr and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p and p nfxb subunits. interestingly, we observed that tlr stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr , tlr and tlr stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl- expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr , tlr , tlr and tlr . finally, in vivo tlr stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr , tlr or tlr triggering can directly favor tumor development whereas tlr signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms- ) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd + and cd + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd + and cd + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd + and cd + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: . innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . . adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni , r. oatuni , m. collini , m. caccia , p. castagnoli , g. chirico , f. granucci university of milano-bicocca, biotechnology and bioscience, milan, italy, university of milano-bicocca, physics, milan, italy, singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin- (il- ) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il- production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca +/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca + influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr engagement, depending instead exclusively on cd . we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase- (cox- ) that, by generating prostaglandins (pgs), such as pge , regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd . given the essential involvement of cd in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd represents a major step towards the development of potential treatments with modes of action involving interference with cd functions. we have examined the interaction of cd , a -kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd . human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd antibody coupled to biotin visualised by qd- -streptavidin and anti-cd antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd and cd , suggesting a new functional role of cd as a member of a multimeric lps receptor complex. l. lundvall , r.r. schumann charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase- induction leads to an increased release of mature il- b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d ) was established for assessing il- b induction during this disease. the murine raw . cell line, the human astroglial u- mg and the murine microglial cell-line bv- were stimulated with the tlr ligand lps, the tlr ligand pam cys, the nod ligand mdp, or the nod ligand c -ie-dap, and, as control, atp alone or in combination. we assessed il- b by elisa and caspase- and pro-il- b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il- b induction pathway. s. pneumoniae (d ) infected mice showed a significant increase in il- b release after hours. in vitro, an increase in il- b levels after costimulation with lps or pam cys, and mdp or c -ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il- b by tlr-and nlr-ligands was observed in raw cells, bv- cells, u- cells and primary astrocytes. active caspase- (p ) was induced by mdp or c -ie-dap, corresponding with high il- b responses when lps or pam cys was added. sirna experiments show that a knock-down of nod leads to a diminished il- b release after lps-and mdp-stimulation. the precursor forms of il- b and caspase- seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il- b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il- b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il- b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd and cd activation model system -tlr presence on cd + cells is found in mouse t cells, human t cells and jurkat cell lines. following cd /cd activation for hours we have identified a small but distinct populationof tlr + cells. further characterization indicates these cells to be cd +cd + cells. further characterization of the expression and functional acitvity of the tlr + t cells indicates co-expression of tlr with md- indicating a functional tlr receptor. in addition lps activiation did not lead to upregulation of tlr expression in t-cells. the data indicate that tcr activation leads to tlr expression. the expression appears to be associated with cd +cd + cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann , d. kaul , c. krüger , f. zipp , r. nitsch , s. lehnardt charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna ) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr (and human tlr ) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr is expressed in these cells. incubation of microglia with all three of the above mentioned tlr ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il -b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr knock out (ko) microglia by real- because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin- was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin- is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin- -transfected plb cells, coronin- overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd b labeling. concerning apoptosis, increased coronin- expression in dmf-differentiated plb significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin- significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase- and caspase- activities, but not caspase- or bid truncation suggesting that coronin- interfered with mitochondria-related events. to validate the prosurvival role of coronin- in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin- expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin- immunolabeling. we concluded that coronin- could constitute a potential target in resolving inflammation. p.-n. hsu national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw . macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf- sirna and traf- decoy peptide, indicating this pathway is traf- dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b -h and b -dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il- was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il- and ifn-g which could not be overcome by restimulation with acd . this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd t cells into t helper (th ) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il- receptor-related protein st could be implicated in lps tolerance. the natural ligand of st was recently identified as interleukin- (il- ), a new member of the il- family. in this study, we investigated whether il- triggering of st was able to induce lps desensitization of mouse macrophages. we found that il- actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il- enhancing effect of lps response is mediated by the st receptor since it is not found in st ko mice. the biochemical consequences of il- pretreatment of mouse macrophages were investigated. our results show that il- increases the expression of the lps receptor components myeloid differentiation protein- (md ), cd and tlr- and the myeloid differentiation factor (myd ) adaptor molecule. in addition, il- pretreatment of macrophages enhances the cytokine response to tlr- but not to tlr- ligands. thus, il- treatment preferentially affects the myd -dependent pathway activated by the tlr. c. klotz , b. lenz , r. lucius , s. hartmann humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., ) . the cystatin effect was blocked by the application of anti-il- receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin- (il- ). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il- in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il- production after avcystatin stimulation. application of specific inhibitors revealed that the il- induction was p and erk dependent, and inhibitor titration indicated a higher sensitivity towards p . western blotting experiments confirmed the phosphorylation of p and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il- is also regulated by the phosphatidylinositol- -kinase (pi k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il- and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose- -phosphate (s- p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose- p and xylulose- p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p /jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s- p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts , y. morias , p. de baetselier , a. beschin vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m ) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd , ifng, il- , ccr and nf-kb to the development and/or recruitment of pathogenic m in the liver was investigated using knock-out mice or by delivering il- in infected mice. results: we established that cd b+ly c+cd c+ tnf and inos producing dcs (tip-dcs) represent the major m liver subpopulation. tip-dcs differentiated in an ifng/myd -dependent manner from cd b+ly c+ inflammatory monocytes in the liver of infected mice. ccr promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il- enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il- in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p and il- play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m , in particular tip-dcs. the inflammatory activity of liver m is controlled by il- and/or p nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov , j. driesen , z. abdullah , a. niño castro , t. chakraborty , m. krönke , o. utermöhlen , c. wickenhauser , j.l. schultze limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, institute of molecular medicine and experimental immunology, bonn, germany, institute of medical microbiology, university of giessen, giessen, germany, institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine , -dioxygenase, cyclooxygenase- and cd and production of prostaglandin e (pge ) and interleukin . all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd , expressed by regulatory myeloid cells, acts as an il- scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd + ido + cox- + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin (il- ) and il- in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il- and il- or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il- and il- . in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il- and il- . therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel , m. rodriguez gomez , m. niedermeier , y. talke , n. göbel , k. schmidbauer , m. mack unversity hospital regensburg, internal medicine ii, regensburg, germany, university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il- and il- . activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp- cells infected with m. bovis bcg, the avirulent m. tuberculosis h ra strain and the virulent m. tuberculosis h rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva , l.d. miteva , s.a. stanilova trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il- is a heterodimeric cytokine composed of a p subunit associated with the il- / p subunit. like il- , il- is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il- p -related cytokines controls the appearance of normal th and pathological th mediated immune responses. in this study, we examined the dynamics of inducible il- p and il- p mrna expression and protein production in purified human monocytes and how jnk and p mapks inhibitors influenced il- p and il- production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il- p gene expression was higher than those observed for il- p gene at all time-points. the level of il- p mrna increased after th h and reaching a maximum level at th h ( . fold for c bgp and . fold for lps). c bgp and lps triggered il- p gene transcription were almost equal at the rd h ( . and . fold) and at th h ( . and . fold, respectively) after stimulation. the higher level of il- p gene expression was detected at th h in lps compared to c bgp stimulated monocytes ( . vs. . fold). however, il- p and il- protein production was increased in the highest level after c bgp stimulation. the inhibition of p led to the statistical significant augmentation of c bgp stimulated il- p production. the inhibition of the same map kinase enhanced lps stimulated il- p production without significant difference. the inhibition of jnk and p mapks significantly decreased c bgp stimulated il- production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c bgp and lps to induce the expression of il- p and il- p mrnas in purified human monocytes. we showed that inhibition of p mapk down regulated il- and upregulated il- p production in stimulated monocytes. we concluded that in human monocytes p map kinase activation has an opposite effect on the il- p and il- p expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd , cd and cd , and secrete il- , thus acquiring the ability to induce proliferation and th polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd c, bdca- , and cd . by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd + subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd + dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd + dc, including their survival and their ability to produce il- p . besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il- , il- and mcp- involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg , s. wolke , a. brakhage , m. gunzer otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and -photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor (irf ), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr . in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens , s. vander beken , p. bogaert , j. grooten ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th -driven immunological counterpart of the th -driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th -and th -driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd . + ram remained constant in cell number for the first days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker , a. stojanovic , a. cerwenka german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr- + cd b + f / + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas , f. marchesi , m. fabbri , s. schiarea , c. chiabrando , a. mantovani , , p. allavena istituto clinico humanitas, rozzano, italy, istituto mario negri, milano, italy, università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m and m . m macrophages act as a first line of defence against pathogens whereas m cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about % of the monocytes differentiated after days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd , cd and low levels of mhc class ii. tc-macro produced il- , il- , tnf but not il- , even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl , cxcl , ccl and cxcl . the transcriptional profile of tc-macro revealed that several genes in line with an m polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g . kda). il- and il- were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl- h rat mast cell line. we demonstrate that gr nuclear translocation begins within minutes and completes after minutes in dx treated rbl- h cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within minutes as non-genomic. studying gc-caused apoptosis, rbl- h cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl- h cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. minutes of dx treatment inhibited ca + -signaling in antigen stimulated rbl- h cells in the concentration range of nm - mm. moreover, minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl- h cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box (hmgb ) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin ( ) . extracellular hmgb can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb exerts antibacterial functions in human adenoid and testis ( ) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity ( ). we asked whether hmgb from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for or minutes with nm phorbol ester (pma), ng/ml interleukin (il- ), or ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h a-histone h b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb localizes on nets. we hypothesize that net-bound hmgb might exert a direct antimicrobial function, or that nets might concentrate hmgb locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation ( °c, h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor- induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr- and anti-vegfr- antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il- . altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd a and cholesterol-dependent lipid rafts impact on cd a surface expression and cd a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd -restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd molecules in multiple sclerosis. we first analyzed cd expression on monocytes from ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd a was not expressed on ms patient monocytes, while the other members of the cd family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd molecules in this context. c. ohnmacht , d. voehringer ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il- reporter ( get) mice as well as in vivo depletion of basophils are used to uncover their role during type immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th cells. these results demonstrate that basophils play a crucial role as effector cells in type immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl- in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il- beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl- -transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water ( mg and mg daily) for days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il- and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il- and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant ). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type- macrophages, mph ), and during resolution of inflammation (type- macrophages, mph ). methods: mph / were generated by culturing cd + human monocytes for days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph / of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph / were stimulated with lps and secreted il- , il- , il- , il- p and tnf were quantified by elisa. results: mph are relatively efficient phagocytes for apoptotic neutrophils whereas mph are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph and mph which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il- , il- and tnf production is suppressed while il- secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of years old and older (long-livers). intracellular ros and hsp levels were registered by flow cytofluorimetry upon labeling with ', '-dichlorofluorescin diacetate (invitrogen) and anti-hsp antibody (brm- , sigma), respectively. intracellular level of hsp was also estimated in neutrophils after heat shock (hs) performed at °c for min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at - min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for min at °c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp (hsp basal ) and ros level, both intracellular and extracellular. at the same time increased hsp level immediately after hs (hsp ( min)) correlated negatively with intracellular ros (initial and after hs). the hsp increase value (hsp ( min) -hsp basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp increase was registered in min after hs (hsp ( min) -hsp basal ). thus it was found that within this age group the alteration in hsp induced by hs in neutrophils but not basal hsp itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant # . d. goyeneche-patiño , z. orinska , f. mirghomizadeh , s. bulfone-paus forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type ifn. two novel genes, receptor transporter protein (rtp ) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during and h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine . stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr and , as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd and trif and the pathways that they represent are not relevant in the expression of rtp and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano , e. erba , r. frapolli , m. d'incalci , a. anselmo , s. pesce , p. allavena , a. mantovani , humanitas clinical institute, rozzano, italy, mario negri institute, milan, italy, institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et- ) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl , cxcl and the inflammatory protein pentraxin (ptx ) either at transcriptional and protein level, especially after tnfa/il b stimulation. down-regulation of ccl , cxcl , ptx and also of il and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd + vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin- (il- ) is a key cytokine of the t helper cell response. il- has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il- production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il- producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day - after birth, a small population of il- producing cells was observed in the absence of any stimuli. the il- + population was not detectable at or weeks of age. other cytokine producing cells (ifn-g, il- ) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il- + cell population. phenotyping of the neonatal il- + cells showed that they were ige + /mhcii -/cd cells. the occurrence of cd + il- producing cells after pma stimulation increased slowly with age and did not reach adult levels by weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il- at day after birth. neonatal pbmc collected before colostrum uptake did not produce il- in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il- responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il- response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel , d. voehringer ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat -dependent manner when stimulated with the th cytokines il- or il- . alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat -dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat -deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat -deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il- exposed macrophages from wild-type and stat -deficient mice. candidate genes will be expressed using retroviral transfections of stat -deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of pleural effusions, including malignant and nonmalignant tumors. the following human malignant cell lines were tested: a , ht , hct , sw , mcf , mda-mb , jurkat and hl . results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m , and the tams isolated from the malignant effusions similar to m . among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms a a and ms a a: highest trascriptional level after h of stimulation with - m dexamethasone. ms a murine genes are differently expressed respect to the human counterpart and only the homologs of ms a a (ms a b, c and d) have a similar regulation. finally, egfp-tagged ms a a, ms a a, and ms a expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein- (mcp- )/ccl and the gpcr ligand fmlp or the tlr agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il- b or ifn-g, significantly and dose-dependently synergized with ccl in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin- /cxcl , but not of the ccl receptor ccr in monocytic cells. in turn, the induced cxcl synergized with ccl for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr /cxcr , because cxcl receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, ) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for min at °c with gentle shaking, followed by spinning down of pmns for min, at °c and g. the supernatant was sedimented at g for min, °c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd low cd + blood monocytes (mo) in healthy individuals and the increase in cd +cd + blood mo in patients with inflammation has been reported. the functional activity of human cd + blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd and activation markers such as hla-dr and trem- . percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a . fold increase (p x . ) in the total number of circulating cd low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd + blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd + blood mo expressed increased levels of cd on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem- +) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd on cd high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m -and m -polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m and m macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m type and were cd negative but cd and mhcii positive and produced high levels of il- , tnfalpha and il- following lps stimulation. culture in the presence of mcsf resulted in induction of the m phenotype. these cells were cd positive with intermediate expression of cd and mhcii expression and produced high levels of il- , il- and il- following lps stimulation. interestingly, already baseline il- production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m -and m -polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma ( ng/ml) treatment for h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl cells expressed antigen receptors cd , tlr , tlr and cd , and displayed enhanced phagocytic activity and production of ros. expression of cd , cd and cd was also maintained however the cells were hla-dr and cd a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl cells to differentiate into macrophages in response to pma. hl may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta , n. ferrer , a. gómez , j. gonzalo , a. arbués , a. anel , c. martín , j. pardo , apoptosis, immunity and cancer university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so , a m. tuberculosis phop mutant strain that was shown (perez et al ) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al ) . in the present study, we compare the time course and phenotype of cell death induced by so , bcg and wild type m. tuberculosis on the murine macrophage cell line j and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase- activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl x deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type receptor for vip (vipr ) gene is highly conserved through species and, in humans, is highly polymorphic. vipr has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr after , , and h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from donors that had been typed for the relevant vipr snps. the down-regulation of vipr correlates with the presence of a t at rs mapping in the '-end of the gene (p= . ). the vipr protein level was decreased about % in monocytes of subjects typed as t/t at rs whereas subjects typed as c/c at rs maintained a high level of expression after h of lps treatment. the data show that different haplotypes of the vipr gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska , t. vavrochova , d. filipp , immunobiology institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e . - . ) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd in the early mouse embryo (me). facs analysis of cell suspension prepared from . day me showed that about . - % of cells were positive for cd b. these cells exclusively were also positive for cd , tlr , and cd antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days , - , . reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e , ), embryo-derived mhcii + macrophages start to appear in the embryo around day . multicolor facs analysis of cd b, cd , cd , f / , tlr , tlr , c-kit and mhcii surface markers revealed differential expression of tlr and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd b + tlr + cells isolated from the e , embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il /il -dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in out of these patients mutations in several ifng pathway genes, other candidate genes are being investigated for the other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp . we did focus on the study of genes which are considered as important pathogen recognition receptors of the innate immune system: tlr is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il- cytokine. using a case/household-contact cohort we did investigate polymorphisms of these genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to - % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, . hsp are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp were used. exogenous hsp was used in concentration - ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio : and : directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp on their surface. results demonstrating effect of exogenous hsp on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp on amplitude of respiratory burst. the cells expressing surface hsp impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd /cd and cd /cd double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd / subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd / subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević , i. pilipović , s. stanojević , k. mitić , k. radojević , v. pešić , g. leposavić , institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h o ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to -day-long propranolol treatment and measured both no and h o production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b -and a -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h o production. an increase in h o production in the presence of the a -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h o production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b -and a -adrenoceptors on peritoneal macrophages (a stimulatory effect on b -adrenoceptors and a suppressive effect on a -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd ++ cd -('classical') and cd + cd + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd + cd + monocytes display higher tlr and - expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd ++ cd monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr stimulation. methods: cord blood (n= ) and peripheral-blood (n= ) mononuclear cells were stimulated in vitro for hrs with peptidoglycan and subsequently analysed for cd and cd and intracellular il- p and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il- p , both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il- p was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il- p (% positive cells and geomfi) was significantly higher for cd + cd + cells than for cd ++ cd cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd ++ cd cells were positive for tnf to a significantly higher extent than the cd + cd + cells. in particular the tnf response to tlr stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd ++ cd monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a -year-old boy who admitted with complaints of seizures during the previous months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g ( mgr/m /day, subcutaneously every other day) was started. after months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf gene concerns the well-known gt deletion in the second exon of ncf gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = ) subjected to moderate brain injury were randomized, and received either . mg/kg ubiquitin or vehicle (placebo) intravenously within min after cci. levels of tnf-a, il- b, il- , il- and il- receptor antagonist were analyzed in brain tissue using real time rt-pcr at and hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at h and days. data were analyzed with the mann-whitney u test and a two-tailed p x . was considered significant. all cytokines were highly up-regulated hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il- were significantly lower in the ubiquitin treated animals, whereas the levels of il- and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day . the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight ( ) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p= . ), tpp (p= . ), osi (p = . ), mda (p = . ) were significantly raised but taa (p = . ) was significantly reduced in ptb patients compared with controls. the levels of mda (p = . ), neopterin (p= . ) and tpp (p= . ) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p= . ), mda (p= . ), neopterin (p= . ), osi (p= . ) were significantly reduced while taa (p= . ) was significantly raised in c+m after weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas , e.m. cunha , m.j. oliveira icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles ( nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage ( %) of macrophages contained the metal particles than neutrophils ( %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap- is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap- regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., ; sivalenka and jessberger, ) . swap- -/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., ; sivalenka and jessberger, ; sivalenka et al., ) . crucial regulators of these processes are members of the rho family of small gtpases such as rac and rhoa. swap- interacts with rac in vitro and preferentially binds the active gtp-bound rac . swap- supports the increase of active rac in vitro by a yet to be defined mechanism (shinohara et al., ) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap- and rac . it was found that fulllength swap- preferentially binds to constitutively active rac (rac q l) but not to its dominant negative form (rac t n). binding assays with swap- truncated mutants showed interaction of swap- 's n-terminus with gtpgs rac or rac depleted of guanine nucleotide, whereas swap- central or c-terminal regions do not bind to any form of rac . preliminary competitive-binding assays with overlapping mer peptides, spanning the entire swap- sequence, mapped the rac binding site near the n-terminus of swap- . full-length swap- site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap- with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap- . v. c. barbosa , c. d. polli , m.c. roque-barreira , m.c. jamur , c. oliver , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl- h cells were sensitized with ige anti-tnp and stimulated with dnp -hsa or artinm. artinm binding to rbl- h cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp- and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp- release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand (psgl- ), b and b -integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il- b, il- and tnf-a. herpes simplex virus (hsv- ) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il- and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv . methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) ( ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv- for h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot . software. results: in the first itraq experiment over human proteins were identified in the hsv infected cell supernatants. from these proteins had at least fold increase after poly(i:c) + hsv infection compared to the uninfected cells. hsv infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of . fold increase in the protein amount in the poly(i:c) + hsv infected cell supernatant and a . fold increase in the hsv infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand , il- , tnf-a induced protein , complement factor b, galectin- and mxa. at present, further experiments are on-going for more detailed analysis of the hsv infected macrophage secretome. h. p. prakash , german cancer research centre, translational immunology, heidelberg, germany, max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein- (ciap- ) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap- and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal- ( mg/ml) binding to monocytes, in the presence or absence of mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal , laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal or rpmi and monocytes ( x ) were added into each insert. when necessary, hrgal was pre-incubated with mm lactose or sacarose. mcp- ( ng/ml) was used as positive control. we observed that hrgal- binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal- is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal- , we observed that the association between these glycoproteins and hrgal- resulted in a % increase in the number of migrating cells. both n-and c-terminal domains of hrgal- are involved in the association between laminin or fibronectin and hrgal- , since the presence of lactose resulted in % and % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal- induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for weeks in the presence of bmp ,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd +cd + cells. the sorted cd +cd + cells were further cultured for - days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd , m-csf receptor cd and the scavenger-receptor cd , we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd and cd (fcgammariii) : the cd lowcd -and cd + cd +. trancscriptional, phenotypic and functional assays suggest the alternative (m ) polarization of cd +cd + embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi , p. kropf imperial college london, immunology department, london, united kingdom the balance between t helper (th) and th cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th or th responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg vd t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f -atpase) is expressed on many cell types and is a possible specific ligand for the vg vd tcr. the present study aims at understanding the role of f -atpase in antigen regognition. using video microscopy calcium imaging in single vg vd t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f -atpase. purified f -atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg vd cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f -atpase. thus the f -atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg vd t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f -atpase. by using purified f -atpase and peptides derived from vg vd tcr sequences, interaction sites between f -atpase and tcr were identified on both ligands. based on these findings a generalized model for vg vd t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg d activation in vivo. by transiently overexpressing the nkg d ligand rae- -beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma vdelta gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae induction. the primary systemic th response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg d receptor suggest that different ones may play unique roles. a novel nkg d-ligand, h c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae- induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th (ifn-g, tnf) and th (il- , il- ) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd -nk . -inkt cells producing high levels of the pro-inflammatory cytokine il- has been identified (inkt cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt cells in type diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il- as compared to c bl/ mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt cells present in nod mice were mainly cd -nk . -, express the ror-g transcription factor and il- receptor, both molecules being usually associated with th commitment. we are currently analyzing, using co-transfer experiments, whether these inkt cells play a beneficial, a deleterious, or any role in the development of type diabetes in nod mice. j. s. dodd , r. muir , s.s. affendi , p.j. openshaw imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd d-deficient mice with poor nkt cell responses have inefficient induction of cd t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th immunity (increasing il- and il- ), promoting pulmonary eosinophilia and ablating cd t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd t cell activity (as measured by cd expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d ) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d ) weight loss by a cd t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd + ab t cells, are regarded as immature or t h biased. vg + vd + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)- -hydroxy- -methyl-but- -enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg + vd + t cells towards these activators. because il- is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg + vd + t cells. herein, we observed that zoledronate induced neonatal vg + vd + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h -like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h -like cytokines such as il- and il- were not induced. addition of il- to zoledronate selectively costimulated ifn-g production from neonatal vg + vd + t cells. furthermore, zoledronate/il- treatment resulted in neonatal vg + vd + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il- (il- r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il- resulted in a further increase of t-bet expression in neonatal vg + vd + t cells. these changes in the expression of il- r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il- treatment. of note, in contrast to adult peripheral blood vg + vd + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg + vd + t cells. altogether, these observations show that neonatal vg + vd + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il- is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e ligases such as hhv encoded k , k or mhv encoded mk . these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e ligases manipulate them. we discovered that both the cellular march and viral e ligases ubiquitinate cd molecules. however, whereas viral molecules inhibit cd -antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd molecules. in contrast mhc class ii was only targeted by cellular and not by viral e ligases. furthermore cd molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd neg clones in vivo, we generated hypodermal ht tumors in immunodeficient mice. concomitant injections of vd neg clones, in contrast to vd + cells, prevented the development of ht tumors. vd neg clones expressed chemokine c-c motif receptor (ccr ) and migrated in vitro in response to chemokines secreted by ht cells, among which were the ccr ligands macrophage inflammatory protein (mip)- d and monocyte chemoattractant protein (mcp)- . more importantly, a systemic intraperitoneal (i. p.) treatment with vd neg clones delayed the growth of ht subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd neg clones were not able to inhibit the growth of a hypodermal tumors. our findings suggest that cmv-specific vd neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht cells expressing the luciferase and realized orthotopic injection of ht -luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint , currently the only known determinant of a canonical iel compartment, that is selectively required for vg vd + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint function; for example, we demonstrate that the constitutive expression of wild-type skint fully restores detc development in a skint mutant mouse, but does not rescue normal detc function. thus, skint provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd + cd hi cd lo cd c -) and splenic conventional dendritic cells (cdcs) (cd c hi cd a +/-cd b +/-b -) from wt and cd d -/mice as apcs for nkt cells from va -ja transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il- by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il- producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h response, whereas cdc-primed nkt cells rather favor a t h response. objectives: il- is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il- give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd d -/-) mice. we set out to investigate the activated b cells in il- injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il- ( mg) for days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day was evaluated by flow cytometry and immunohistology. results: mice injected with il- developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il- was increased in inkt cell deficient (cd d -/-) mice, in contrast to published data. an increased response to il- was also observed in ja -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il- induced antibody responses. further characterization of the recruitment of b cells in il- injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il- induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd d, are prone to autoantibody production and often involved in early immune responses. the il- induced antibody response in mzb deficient (cd -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il- induced antibody responses. we conclude that the role for inkt cells in il- induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c bl/ mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha -/-and cd d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il- . moreover, ifn-g production required the presentation of ehlppg by cd d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il- . similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd in cmv-infected individuals. cd is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that . ± . % of gamma-delta t cells from cmv-infected ktr expressed cd , when compared with only . ± . % in non cmv-infected ktr (p x . ). similarly, . ± . % of gamma-delta t cells from cmv-seropositive blood donors expressed cd compared to . ± . % in cmv-seronegative donors (p x . ). cd + gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd -dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il- and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd a on cd + gamma-delta t cell lines. cd is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd + gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a skin carcinoma cell line pre-incubated either with rituximab (anti-cd ) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd -dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il- , ifn-g, tnf-a, osm, ccl , cxcl , cxcl , and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd , cd , hla-dr, and dc-sign, and lost cd , ccr , ccr , and cxcr . addition of further microbial stimuli (lps, peptidoglycan) induced ccr and enabled these inflammatory dcs to trigger antigenspecific cd + effector ab t cells expressing ifn-g and/or il- . importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg /vd t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg /vd t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg /vd t cells stimulated with hmb-pp in the presence of il- express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il- regulate expression of the b cell attracting chemokine cxcl /bca- , its receptor cxcr , and co-stimulatory molecules involved in b cell help. purified peripheral vg /vd t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to days with and without hmb-pp, in the absence or presence of il- or il- , or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl protein were detected in co-culture supernatants only when both il- and hmb-pp were provided, implying an il- -dependent and tcr-dependent expression. vg /vd t cells were confirmed as producers of cxcl by flow cytometry and immunofluorescence. under the same conditions, activated vg /vd t cells expressed cd , cd , cd l, cd , icos and ox . in contrast, neither cxcr nor ccr changed markedly by il- stimulation of peripheral vg /vd t cells. conclusion: our findings confirm on the protein level that stimulation of vg /vd t cells with hmb-pp and il- induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto , m. emoto gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)- . although downmodulation of surface t cell receptor (tcr)/nkr-p c (nk . ) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il- stimulation. to determine whether failure to detect inkt cells after il- stimulation is caused by dissociation/internalization of tcr and/or nkr-p c or by block of de-novo synthesis of these molecules, and to examine the role of il- in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p c by inkt cells after stimulation with a-galcer or il- , and influence of il- neutralization on down-modulation of stcr/snkr-p c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il- neutralization. whereas s/cnkr-p c + inkt cells became undetectable after in-vivo administration of il- , s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p c remained unaffected by a-galcer or il- treatment, despite the down-modulation of ctcr and/or cnkr-p c protein expression. in contrast, ctcr + cnkr-p c + stcr -snkr-p c -inkt cells and cnkr-p c + snkr-p c -inkt cells were detectable after in-vitro stimulation with a-galcer and il- , respectively. our results indicate that tcr and nkr-p c expression by inkt cells is differentially regulated by signaling through tcr and il- r. they also suggest that il- participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg -gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region d (cdr d ) and cdr d repertoire, with a striking enrichment in a specific germline-encoded cdr d sequence. differentiated gd t cells and the enriched cdr d sequence were detected as early as after weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd -jd and vgi-jg . / . rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd -jd and vg -jg . selection was seen in pb tcrgd cells. detailed analysis of the cdr motifs revealed selection determinants in both vg -jg . (canonical length and cdr motif) and vd -jd (minimal cdr length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚ ) and tcrgd cb cells ( - ) to tcrgd pb cells (˚ or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type diabetes (t d). we have previously shown that transgenic expression of a cd d-restricted, va . -vb tcr in nod mice lead to an increase in cd d-restricted type ii nkt cells ( abnkt cells), and prevention of the development of t d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc . cd + t cells, in the presence or absence of selected cells from abnkt cell transgenic mice. results: in ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, abnkt cells expressed a high level of cxcr and a low level of ccr and cd l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd + bdc . t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from ab transgenic mice with bdc . cd + cells resulted in the prevention of diabetes development. the protection from disease required a minor cd + subset of ab+ nkt cells, but was independent of cd + t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs , induces in vitro inkt cell expansion and ifng and il- secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il- with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th -type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th immune response is characterized by the secretion of il- a and il- f. the il- locus encodes the highly conserved il- a and il- f cytokines that are syntenic in kb distance to each other. besides cd + th and nkt cells, approximately % of the il a producers are gd t-cells. like cd + th cells, il- producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il- production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il- or ifn-g. combined with the well known classification of il- producing gd t-cells along the markers cd and cd , our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th -type immune responses in vivo. in this context, the potential redundancy of il- a and il- f may complicate the analysis. so far, most studies were carried out with il- a single-deficient or il- f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il- a and il- f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg vgd t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b and il- differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp ) and, similarly to ab tregs, suppress the proliferation of anti-cd /anti-cd stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il- was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd b in the liver following a-galcer treatment, numbers of cells lacking cd b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma /vdelta t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma /vdelta t cells expansion upon stimulation with zoledronic acid (za) and interleukin- (il- ). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by ) the bioinformatic analysis of gene expression profiling data ) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in patients ( %) (responders, r), whereas patients ( %) were non-responders (nr). vgamma /vdelta t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt , whereas temra of r patients had an higher expression of the costimulatory molecule nkg d. the proliferative response of vgamma /vdelta t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, % of r patients were m, whereas % of um patients were nr (p x . ). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c bl/ mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il- p (clone r - f ; atcc) or anti-cd- d (clone b ) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il- p , a component of both il- and il- ; pretreatment with anti-cd d mab had no effect. il- rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il- was not a critical factor. this suggestion is supported by the increased expression of il- p and il- p (but not il- p ) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il- production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb ) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd expression. gd + cells secrete interferon-g, while gd cells are capable of producing il- . this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd -defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine . cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚ % of gd + cells but not at all in gd cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, - % of gd + cells were positive for pgp activity, although their gd counterparts remained largely negative (p x . ). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd . as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd + and gd cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma vdelta t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma vdelta t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma vdelta t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma vdelta t cells from healthy donors were stimulated with different compounds (zol, ipp) for hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma vdelta t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma vdelta t cells. moreover, mcp- depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma vdelta -mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma vdelta tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma vdelta t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg vd t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg vd t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg vd t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg vd t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg vd t cells and their effects on the viability of glioma cells, we expanded in vitro vg vd t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg vd t cell lines to kill three different glioma cell lines (t , u , u ) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg vd t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg vd t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg vd t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg vd t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e -induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e -expressing transplanted skin is dependent on interactions between populations of cd d-expressing cd c+/f + myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il- and a-galcer. expression of cd a, cd d and the costimulatory molecules cd and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il- ) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd d expression on the cell surface in comparison with control cells. in contrast cd a expression was decreased. cd and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd a, cd b and cd c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il- producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, . - % of circulating lymphocytes express a vg vd t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg -enriched genes compared to conventional mhc-restricted cd + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf & crtam in gd and ab t cells respectively. because igsf binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd + ab t cells express crtam, and that engagement of igsf on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf /crtam. we therefore sought to answer: . what is the function of igsf /crtam on gd t cells? . how is the igsf -crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf enrichment on resting gd t cells, with expression also detected on˚ % of ab t cells. the properties of those cells are being examined. however, igsf generally correlates with markers of activation/antigen experience such as cd ro. thus, igsf cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf and crtam within hours. however, engagement of igsf by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf interactions are sufficient for cd t cells to kill gd t cells and/or vice versa. instead, crtam-igsf interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd z is a kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately - % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd tcr variable segment associated with the vg segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg vd t cells without antigen presentation. in vitro stimulated vg vd t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg vd t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg vd t cell anergy observed in hiv+ patients. to this aim, cd z expression and ifn-g production by vg vd t cells from hiv+ and hiv-subjects were analyzed. we show that vg vd t cells from hiv-infected patients expressed lower level of cd z compared with healthy donors. a direct correlation between cd z expression and ifn-g production capability by vg vd t cell was found. however, pkc activation by pma is able to restore cd z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg vd t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner , m. swamy , s.l. clarke , a. hayday king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd aa or cd -cd cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to days. the cells are initially activated by plate-coated acd antibody and a cytokine cocktail and maintained further in medium containing low levels of il- . after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il- ) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg molecules in heterodimer with cd , we screened the presence of these receptors on t cell subsets in bd. the expression of nkg a/c/d molecules on gd and cd + t cells were analyzed in active and inactive patients with bd and healthy controls. expression of nkg molecules was evaluated on cd +, gd t and cd + nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls ( . vs. . %, p= . ). in addition to the increase of gd t cells, increased expression of activating nkg c molecules was also observed on gd t cells ( % vs. %, p= . ). nkg a expression on gd t cells was found to be higher than nkg c expression in patients and controls; but nkg a expression on the t cells was not statistically different in both groups ( . vs. %). nkg d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd + cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th and th cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg /vd + lymphocytes. we have screened a panel of lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg /vd + cells, and selected two susceptible ("target") cell lines (over % death in the assay) and two vg /vd + resistant ("non-target") cell lines (under % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs (non-target), and validated the results by rt-qpcr quantification. results: we identified commonly up-regulated and commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp , ifitm and prame, for example, are up-regulated, whereas cd and clec d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg /vd + target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd d, in the rat. mice and rats have very similar cd d and inkt tcr genes, with the exception of the va gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd d revealed a very similar pattern of cd d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il- production was - fold lower in the best responder rat strain (f ) compared to mouse (c /bl ). since nkrp a (rat homologue of mouse nk . ) and tcr are not appropriate markers for rat inkt, cd d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g d t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g d t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g d t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g d t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp- . a significant (p x . ) reduction in intracellular bacterial numbers was observed (n= ) in the presence of pbmcs cultured with picostim+il- in comparison with pbmcs cultured with il- or media alone. picostim+il- caused significant expansion and activation of gd t cells following culture of pbmcs for - days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers -fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il- , reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma /vdelta (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: ) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; ) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); ) to establish whether the same issues could be solved using a simplified protocol of dc generation. ) dc were generated from cd + cells of healthy donors/mm patients; immaturedc on day were induced to fully mature by incubation for hours with tnfa + il- b + pge in the presence or absence of mm za. after days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; ) idc generated from cd + cells of hla-a* + healthy donors/patients were pulsed with sv-peptide and stimulated for hours with tnfa + il- b + pge in the presence or absence of mm za; after rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd + t cells was determined by svpentamers staining; ) the same experiments were performed both with dc generated following a standard protocol and a h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [ - h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd + cd + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin- on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd + have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n= ) and inactive (bdna) (n= ), versus healthy controls (hc) (n= ) and patients with recurrent oral ulcerations (ru) (n= ). to determine gd cytokine profile and surface markers treg-related in bd (n= ) and hc (n= ). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta , vdelta , cd alpha, cd beta, nkg d, nkg a and cd . -intracellular expression of ctla- , and foxp . -intracellular expression of il- , il- , ifngamma, il- and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta + cells were significantly increased in ru. vdelta + and gdcd + lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg d was slightly increased in gd from bda. -nkg a expression by gdcd + was not different in bd versus hc. -most of gdcd + presented cd alpha-alpha homodimers in bd and hc and were negative for cd , foxp and ctla- . gdcd + and gdcd -subsets were (in bd and hc): -high ifngamma-producers without differences. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd + than in gdcd --very low producers of il in most of cases. the hallmark in bd was the increase of gdcd /vdelta +. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd +, except a lower percentage of il- + cells than in the gdcd -subset. gdcd + from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd in humans. a subgroup, the invariant nkt cells (inkt), expresses the va vb tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd + cd + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from healthy umbilical cord blood samples and stimulated with ifn-g ( ng/ml), anti-cd ( ng/ml) and il- ( ui/ml). these cells were cultured for days and the expanded cd + cd + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd + c + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x , was considered significant. results: we could significantly expand cord blood cd + cd + nkt cells from , ± , % to achieve an enrichment of , ± , % (p= , ). table shows the percentage (mean±sd,n= ) of phenotypic markers in cd + cd + cells at baseline (day ) and after days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb and vb families (p x , ). conclusion: our results show that cord blood-derived nkt cells are mainly cd + and cd + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb and vb families in these cells. l. marischen , d. wesch , p. rosenstiel , a. till , d. kabelitz institute of immunology, kiel, germany, institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod . peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp and pvama proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd + and cd + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd a + (lysosomal associated membrane proteins: lamp- ) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th and th cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th -like and th -like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n= ) and healthy controls (n= ) were determined by flow cytometry using anti-cd and monoclonal antibody specific for the cdr loop of the invariant tcr a chain of inkt cells (clone: b ). furthermore, after pma/ionomycin stimulation for hours, intracellular ifng and il- cytokines were detected in cd +cd -, cd -cd -(dn), cd -cd + and cd +cd + subsets of inkt cells by five colour flow cytometry in patients with ad (n= ) and healthy controls (n= ). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x . ) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x . ). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r= . and p x . ) and healthy controls (r= . and p x . ). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il- level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x . ). the frequency, the number of inkt cells and the cytokine producing capacity of the cd /cd inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il- that promotes the th differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting . %, . %, . %, . %, . % and . % of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells ( . %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd + nkt and cd -cd -nkt cells. nk . + nkt cells may release large amounts of il- , il- , ifn-g and il- after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il- had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il- . there was a significantly increase in the percent of cd + nk . + and tcrvb + nk . + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd molecule existed in . % of the cells from large-scale selection. the percent of cd + nk . + and tcrvb + nk . + nkt cell subsets in the giant lymphocytes were enhanced to . and . folds, respectively. under a light microscope at x magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than . and they are non-adherent cells. the differentiation pathway of the seb-activating cd + and tcrvb + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd + nkt cell and tcrvb + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo- am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h begfp reporter mice. stimulation with anti-gd clone gl or anti-cd clone c elicited activation of gd t cells suggesting that tcr gd and cd molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl in vitro produced ccl , ifng and tnfa. therefore, we were interested whether the ccl production of gd iiel influenced the homing of ccr cells such as lamina propria (lp) cd + foxp + cells (tregs). to test this, wt mice were i. p. treated with gl mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il- production in g reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il- gene. in the absence of any manipulation gfp was expressed from the il- locus in populations of immature thymic nkt cells (predominantly cd +cd lotcrhi cells on a balb/c background, and cd +cd lonk . -on a c bl/ background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il- production was induced predominantly in cd + nkt cell subsets of the liver and spleen, and after i. n. administration, in cd + nkt cells of the airways. spontaneous and a-galcer-induced expression from the il- locus occurred in the absence of stat signalling, and did not require initial exposure to il- protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin- (il- ) plays an important role in neutrophil recruitment. herein, we investigated the role of il- receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c bl/ (wt) and il- receptor gene-deficient (il- r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated hours after surgery. the ability of il- mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il- r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il- induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il- r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il- receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il- showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il- also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il- receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin- receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il- r associated kinases to activate nfxb and p mitogen-activated protein kinase. the il- -induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il- in different mast cell subsets. methods: different mast cells subsets (hmc- , human cbmcs and murine bmmcs) were stimulated with il- . the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk / , pakt, pnfxb, p and pjnk). additionally, we studied the signal transduction of il- in il- r transfected hek t cells. results: we found, that a tir family member, il- r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il- -induced cytokine production depends on c-kit transactivation. il- r and il- r accessory protein (il- racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il- r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il- -induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il- in mast cells in absence of iger activation. ( ) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase- activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease- (ho- ) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho- expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin and . recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr shows a bimodal kinetic, with the first peak at ''/ ' and the second at ' after stimulation. rna interference-mediated depletion of beta-arrestin specifically inhibits the occurence of the second wave of rap activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin is involved in rap activation and that the oscillations in the formation of rap -gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c g (rap gef) and spa (rap gap): preliminary results suggest that spa- has probably a role in the early activation peak. since this oscillatory chemokine-induced rap activation is present on other myeloid cell lines (hl , d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin , localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of different sh domains that revealed over putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)- and il- stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il- and il- receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il- , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: ( ) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna- expression is induced by activation of the toll-like/interleukin- receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna- a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna- a transfected cells showed significantly reduced levels of the active proapoptotic caspases and (casp / ). in line with this, mirna- a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna- a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna- a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna- a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il- , il- r a-chain and il- r a -chain genes in hiv disease progression. methods: we studied antiretroviral treated patients (progressors) and long term non progressors (ltnp). we analyzed snps in the il- gene, snps in the il- r gene and snps in the il- r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il- r aa and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il- r and il- r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd and cd t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art , and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art transcripts by rt-pcr analysis in human blood leukocytes. soluble art , released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek cells with art and tnf resulted in modification of tnf at at least distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r , a site that has previously been implicated in binding to tnfr . binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik , t.v.poroshina institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and control men were assessed for slpi, tnf-alpha, il- and free tgf-b in ejaculate by elisas using stat-fax plus. a to day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at - degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients ( . ± . pg/ml, p x . ) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid ( . ± . pg/ml; p x . ). the il- concentration was elevated in all patients ( . ± . pg/ml; p x . ) in seminal fluid. free tgf-b was present in normal seminal plasma in high concentrations ( . ± . pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b concentrations were . ± . pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il- and free tgf-b are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi , e. a. elgaaeid faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th or th cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod /card protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod /card gene in cd. we performed a cases /controls study upon cd patients and healthy controls. this study suggests that in northen tunisian population, insc mutation in nod /card gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il- polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il- cytokine genetic profile in patients with ibd. we examined the contribution of il- gene promoter polymorphisms (- and - ) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card /nod gene mutations in cd presentation and location. in tunisian population, the insc insertion in nod /card gene is a marker of susceptibility to cd, while the a allele at position - in the il- promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il- gene polymorphisms and card /nod gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il- -dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il- -driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il- molecule by ganglioside. but gangliosides apparently can also form complexes with il- r; such complexes influence on the signal transduction through il- r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il- r subunits. in our work we use il- -dependent cytotoxic t-cell murine line ctll- . two different approaches for study of possible interaction types between exogenous gangliosides and il- r subunits were applied: antibody staining of il- r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with i-dcp-gm followed by immunoprecipitation of il- r subunits. the fluorescence intensity of the antibody-labeled il- r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by . % after cells incubation with ganglioside gm , and by . % after incubation with gm . labeling of the cells with antibodies to the il- r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm ( . %) or with gm ( . %). to determine the mode of this impressive masking influence of ganglioside gm , photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm with subunits of il- r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il- r, but not for il- r a-subunit. these results demonstrate that exogenous ganglioside gm can interact with a-and bsubunits of il- r in different modes. interaction of il- r b-subunit with ganglioside gm requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa , j. bukur , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak . results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak expression due to a gene deletion on chromosome , whereas the expression and functionality of other ifn-g signal transduction components like stat and jak were not affected. jak blockade by two jak -specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak leading to a lack of jak expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of controls of the spanish population. methods: two il polymorphisms located on the il- promoter region, snps - a/c (rs ) and - g/c (rs ) were genotyped using taqman snp genotyping assays. the functional il- gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il- - p= , . il - p= , ) but may contribute to disease onset and aggressiveness: the genotype il- - cc genotype, which is associated with higher il- production, was significantly associated with higher tumor size (p= , ), grade (p= , ), t (p= , ), m (p= , ) and stage (p= , ). the influence of the il- - gg genotype was less relevant, however was correlated with higher tumor size (p= , ), grade (p= , ), t (p= , ) and stage (p= , ). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il- polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il- gene (il- - and - ) may be associated with an worse prognosis of rcc. high levels of il- production can play an important role in grow, invasion and metastasis of renal cancer. interleukin- (il- ) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il- is its ability to induce the production of ifn-gamma in presence of il- . moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l . then it seems that il- has a crucial role in immunity against brucella infection. since the expression of il- can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il- gene polymorph isms and brucellosis. methods: a total of patients with brucellosis and healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il- polymorphisms at positions - , - , , + , + and codon / using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il- polymorphisms at position - g/+ t/+ c (correlated with high production of il- ), codon / c and - g/- c (correlated with higher production of il- ) were significantly higher in the healthy controls than the patients (p= . , p= . and p= . , respectively). discussion: as data revealed genotypes that have correlation with higher production of il- are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il- at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor (ifngr ) mouse mutant. we have generated a mouse line carrying a conditional ifngr allele using the loxp -flp recognition target (frt) approach. a targeting vector with loxp sites flanking exons - and frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez , r. carretero , p. saenz-lopez , j. cantón , j. carretero , f. ruiz-cabello , l.m. torres , cts- hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca) allele of ifgn is significant more frequent in healthy control than in cin patient (p= , ) in contrast homozygosis for (ca) allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p= , and p= , respectively). conclusions: our study suggest that ifng (ca) allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin- (il- ), a th -related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il- single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: patients with brucellosis and healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il- (c t, g a, c a and a t) polymorph isms using pcr-rflp. results: at position , the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p= . and p= . , respectively). at position , the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p= . , p= . ). discussion: as shown the frequency of cc genotype and c allele at position were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position is lower in patients than the healthy controls and the frequency of c allele at position is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf- and skbr- , as well as their ability to respond to several cytokines and to the g- gpr agonist. the mcf- and skbr- human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il- ra, il- rg, il- /il- /gm-csfr, il- /il- r, il- ra, il- ra, il- r (gp ), il- ra, il- r, tnfr i, tnfr ii, ifngra, cxcr , cxcr , cxcr , cxcr , cxcr . cells were incubated with il- , ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr agonist. cytosolic ca + concentrations [ca + ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr- cells were found positive for a larger number of receptors than the mcf- cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf- cells with il- reduced the calcium response to g- , while pretreatment with ifn-g and tnf-a potentiated the calcium response to g- . tnf-b had no effect on mcf- g- stimulation. no direct effect on basal [ca + ] i stimulation could be noticed when administering the cytokines alone. in skbr- cells, pretreatment with il- or tnf-b had no effect on basal [ca + ] i and did not significantly alter the calcium response to g- , while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g- . pretreatment with tnf-a produced calcium oscillations and reduced the response to g- . conclusions: mcf- and skbr- cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd a -pdcs and cdcs were infected with mcmv, whereas after h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag -deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg , respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt -l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f / + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd t cells in response to an adova infection due to an impaired cd t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il- . to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp and vacv-gp expressing the gp epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p t cell receptor recognizing the gp epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p ifnar -/mice. upon adoptive co-transfer of p ifnar -/and p ifnar wt/wt t cells and subsequent challenge with mva-gp , a massive expansion of p ifnar wt/wt t cells was observed, whereas p ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp induced a rather similar expansion of ifnar competent and ifnar deficient p t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h n -specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h n induced an exceptionally nf-kb dependent antiviral response. irf is essential part of this interferon-response of human endothelia. furthermore, we identified hmga as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h n . finally, nfatc was found to be a transcriptional regulator for specifically h n -induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h n which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns , delta-ns influenza virus (a recombinant influenza virus lacking the ns gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting days post-infection, reached its maximum at day , and triggered t cell priming in vivo. a direct comparison of delta-ns virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns protein. thus, we propose that the virally encoded ns protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that - - proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of - - target proteins. thus, these results suggest that - - proteins have a regulatory role in host defence against viral infections. i. wessels , d. fleischer , l. rink , p. uciechowski institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)- b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il- b expression is not elucidated, yet. it is known that the il- b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il- b when stimulated. b-cells which are il- b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il- b promoter and the impact of methylation on il- b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl- cells were differentiated into monocytic cells after dihydroxyvitamine d treatment. the monocytic phenotype was confirmed by flow cytometry. the il- b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with -aza- -deoxycytodine (aza) and changes in il- b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl- cells, differentiated cells displayed upregulation of cd antigen and acquired the ability to express il- b. by comparing the accessibilities of il- b promoter we detected that the il- b promoter was not accessible in undifferentiated hl- cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl- cells, demonstrating that the chromatin remodeling of the il- b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il- b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il- b expression were found. our data indicate that the il- b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il- b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg , g. wetzel , h. arnold , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il- b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp , , , and mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. ( )). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. aug ; ( )). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. dec; ( ) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il- b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory t breast cancer cells over expressing il- b ( t /il- b) tumor cells, but rarely in untransfected t cells. secretion of il- b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il- ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array . having p x - in two independent cohorts each consisting of blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for h and we measured the levels of il- , il- , il- , tnf-alfa and il -ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed snps with p x , * - . to identify/replicate the association of cytokine production for these we reanalysed these on a cohort. a combined analysis revealed snps with p x , * - .these results are not genome wide significant. the snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp on adipocytes. for the first time we could show a hsp -stimulated release of the proinflammatory cytokines il- , cxcl and mcp- in a time-and concentration-dependent manner from murine t -l adipocytes. analyses of hsp -signalling in these adipocytes revealed that members of the mapk-family (erk / , p ) and the transcription factor nfkb are involved in hsp -mediated induction of the mediators il- , cxcl and mcp- . binding-studies with fluorescence-labelled hsp demonstrated that the interaction of hsp with adipocytes exhibits basic features of a receptor-mediated binding. hsp -binding to adipocytes was saturable and reached its maximum at . mm. binding was inhibitable only by the unlabelled ligand ( %), but not by unrelated proteins, thereby proving the specificity of hsp -binding. further analyses to characterize hsp -receptor structures on adipocytes revealed the presence of toll-like receptor (tlr) on adipocytes. tlr has been found to be expressed on macrophages and to interact with hsp , therefore suggesting tlr as a potential receptor candidate for hsp on adipocytes. in order to identify the responsible binding-epitope of hsp we investigated the effect of specific antibodies directed against different epitopes of the hsp -molecule. incubation with antibodies directed against the n-terminus of hsp (aa - ; - mg/ml) were capable of inhibiting the hsp -binding to adipocytes ( - %) indicating that the n-terminal region of hsp is involved in receptor binding. our experiments demonstrate that hsp stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp -mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf . we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with % of fetal calf serum at °c and % co . bone marrow nonadherent cells were removed after h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd , cd , cd , cd and stro- and were negative for cd . intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost % of msc. interleukin- , ifn-gamma and tnf (th cytokines) increased msc contractility, whereas il- (a th cytokine) decreased msc contractility. by immunofluorescence, we observed that il- , ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il- decreased that incorporation. our results suggest that th and th cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca + -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of × /ml in complete rpmi- medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as nm and nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents ( mm) at the -h culture interval reached the values of approximately ng/ml and ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of - h in rat pecs. the -h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p and erk / . it was not suppressed by the calcium chelating agents bapta-am and tmb- . the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr / / . pin is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin substrates and targeting sites. recent data show that pin interacts with apo-bec g (a g). the pin /a g interaction results in a reduced a g expression and a diminished a g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin gene (- g/c and - t/c) modulate pin expression; in particular, the - gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, ; - , ) . the - c/g and - t/c polymorphisms in the promoter of pin gene as well as pin protein levels were analyzed in exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; hiv-infected patients (hiv) and healthy controls (hc). the genotype and allele distributions of the - snp was skewed in esn (genotype: p= . ; allele: p= . ). in particular esn showed a significantly lower frequency of the - gg genotype compared to hiv and hc (p= . and p= . , respectively) and consequently a lower g allele frequency (p= . and p= . , respectively). no significant differences were found for the - snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, -(r)- [ -(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, -[ -(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of × /ml in complete rpmi- medium. secretion of cytokines was determined after the -h culture by elisa. production of no was assayed at the interval of h using griess reagent. approximately compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. -[ -(phosphonomethoxy)ethlyl]- , -diaminopurine derivatives, c) -[ -hydroxy- -(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) -heteroalkyl substituted -amino- -guanidinopurines, and f) -amino- -(purin- -yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip- a and cytokines tnf-a and il- . although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as to mm. the remarkably enhanced secretion of chemokines was reached within - h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant m . host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and million units of ifn-a treatment three times a week for months was initiated. before and after treatment: percentages of the il- and ifn-g in cd + t cells were assessed to determine intracellular t helper cell (th ) type cytokine expression. similarly, percentages of intracellular il- and ifn-g were detected to verify cytotoxic t cell (tc ) type cytokine expression in cd + t cells. percentage of th and tc type cytokine expression, (il- and il- ) were determined in cd + and cd + t cells, respectively. six ( %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th cells with respect to healthy controls before treatment. tc percentages, both tc and tc , were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il- expression was higher and the percentages of th type cells were significantly low. il- expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency ( , %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects ( , % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt , f. juengerkes , b. schumak , g. gielen , j. kalff , p. knolle , b. holzmann , a. limmer university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, university hospital bonn, department of surgery, bonn, germany, department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il- , the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il- and il- in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, h. pylori-infected du patients ( patients were positive for anti-caga antibody and patients were negative for anti-caga antibody), h. pylori-infected as carriers ( subjects were positive for anti-caga antibody and subjects were negative for anti-caga antibody) and healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il- and il- was measured by elisa method. the mean serum levels of il- in total du patients ( . pg/ml ± . ) was significantly higher than those observed in total as subjects ( . pg/ml ± . , p x . ) and healthy control group ( . pg/ml ± . , p x . ). in du group, it was found that the mean serum levels of il- in subjects with positive test for anti-caga ( . pg/ml ± . ) was significantly higher than those observed in subjects with negative test for anti-caga ( . pg/ml ± . ; p x . ). the mean serum levels of il- in du ( . pg/ml ± . ) and as groups ( . pg/ml ± . ) was significantly higher than those found in uninfected control group ( . pg/ml ± . , p x . and p x . , respectively). however, no significant difference was observed for mean serum levels of il- between du and as groups. moreover, in both du and as groups the mean serum levels of il- was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il- and il- in h. pylori-infected subjects as compared with control group. in du group the expression of il- influenced by the bacterial caga factor. a. aral , , a. atak gazi university faculty of medicine, department of immunology, ankara, turkey, gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il- levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il- elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il- levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the th hour and reached to maximum levels at the st week and decreased again at the rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the st week and started to decrease at the rd week. il- reached to its peak at the rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic , m. jurisic university of kragujevac, school of medicine, kragujevac, serbia, university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in radicular inflamed cysts and odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd , cd and cd . tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x . ) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. ) drawings blood from patients and healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. ) detection of the gene expression of prolactin and tlr- in cd + peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p . ) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr- and peripheral prolactin expression in cd + monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table- ). when tb household contacts and healthy controls were compared, cfp and esat seemed to be more useful than tst in tb contacts for displaying ltb (table- ) . although cfp spot numbers were much more than esat spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: , )( table- ). both esat and cfp spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g analytes immediately prior to liver transplantation and at sequential time points up to days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip- , il- and il- . notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/ / (h n ) (hk) differs from its putative avian precursor by amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk- aa-i r, n d, k n, s n, g a, human ( - ); rhk-r -l q, s g and rhk- aa-i r, n d, k n, s n, g a, l q, s g, avian ( - )). among these variants, the double mutant rhk-r and the seven mutant (rhk- aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l q and s g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip- and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r and rhk- aa as compared to rhk and rhk- aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il- , shedded adhesion molecules (cd , vcam- , icam- ), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk- aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il- , and neutrophilic cd levels, procalcitonin and il- were measured by elisa technique while, neutrophilic cd by single colour flowcytometric technique. of these "infected" infants, had positive blood culture (subgroup ia: culture-positive sepsis), and infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il- , cd , and crp. il- had the highest sensitivity and specificity, % and %, respectively, using cutoff n . pg/ml. for pct, the highest sensitivity and specificity, % and %, respectively, were at a cutoff value of n . pg/ml. neutrophilic cd had maximal sensitivity and specificity of % and %, respectively, at cutoff value of . %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il- and neutrophilic cd , which together provided sensitivity and specificity of % and %, respectively, and npv %. the combination of il- and crp had high sensitivity and moderate specificity, % and %, respectively. conclusions: il- and neutrophilic cd levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp , naip or ipaf and the adaptor asc are involved in caspase- activation in response to bacterial infection, triggering the processing and secretion of il- b and il- . recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi k, vps and can be inhibited with the pi k inhibitors wortmannin and methyladenine ( ma). in contrast, activation of akt, via class i pi k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il- b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with ma, wortmannin or an akt inhibitor. supernatants were analysed for il- b by elisa. results: ma enhanced il- b secretion by bmdc treated with the tlr and tlr ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il- b secretion was greatly reduced in bmdc from nalp -/mice compared to wild type c /bl controls. treatment with the akt inhibitor had no effect on lps-induced il- b secretion by bmdc. tlr-dependent secretion of il- a was also enhanced by treatment with ma. conclusions: these data demonstrate that il- b secretion by bmdc in response to treatment with pi k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp inflammasome. this response is limited to tlr and tlr agonists. inhibition of akt had no effect on lps-induced il- b production, suggesting that the effect of wortmannin and ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr signaling by the inflammatory lipid mediator sphingosine -phosphate (s p) through receptors and in human monocytes-macrophages, which could explain some of the s p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s p, and later analyzed by flow cytometry. a pharmacological analysis of the s p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam- expression was increased following lps and s p concomitant stimulation in both venous and arterial cells, suggesting that tlr and s p receptors cooperate in the expression of icam- . conversely, no cooperation was observed when tlr ligands were used. in order to elucidate which s p receptor subtype was involved in the increase of icam- expression, we used a pharmacological approach with s p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam- after lps and s p challenge was significantly reduced by blocking s p receptor and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s p receptors and , which suggest that s p receptor might be involved in the effect. conclusions: altogether these data demonstrate that tlr and s p receptors can interact to increase adhesion molecules such as icam- in human endothelial cells, and the s p receptor subtype involved in the effect differs between arterial and venous cells. with ssc without pah) and a pool of sera of healthy controls (hc) were tested. results: in dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with - , - and - protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized ± , ± , ± and protein spots respectively. twenty one protein spots were recognized by more than % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein and peroxyredoxin and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: patients suffering from different diseases were enrolled in our study. patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam- and vcam- were assesed by an enzyme immunoassay (elisa). results: out of the patients ( , %) had elevated levels of the intercellular adhesion molecule. out of the patients suffering from bone diseases ( , %) had raised values (mean value ng/ml) whereas patients out of the suffering from soft tissue diseases and diabetes ( , %) had raised values (mean value , ng/ml). reference value for icam- was - ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients ( %) having increasd levels of icam- . high icam- levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes ( , %) than in patients with bone diseases ( . %). mean values were found , ng/ml and ng/ml accordingly. those findings verify the positive correlation between icam- and inflammatory diseases and tissue damage but not for vcam- . colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: cases of ulcerative colitis (urc), adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, tubular or tubulo-villous adenomas with low grade dysplasia, and infiltrating adenocarcinomas. immunohisto- objectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). mg of pravastatina oral hours they were administered before the procedure (group study, n= ) or placebo (group placebo, n= ), and control (group control, n= ). samples of outlying veined blood were extracted to the hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in degrees: degree . without expression. degree . weak; degree . moderate; degree . intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree - . the placebo group: mixed pattern, degree - . group study ( mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree - . the percentage of cells that expressed cd was greater in the group study ( mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam and adam . we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam . these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam activity in the tissue. in the presence of the adam inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam -mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf- / vegf+ is associated with rcc risk ( p= , ), metastases ( p= , ), nuclear grade ( p= , ), tumor stage ( p= , ), and tumor size (p= , ). on the other hand, the polymorphism vegf - a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol , , -trisphosphate. therefore, pten is one of the main antagonists of the pi -kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il- as well as il- levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il- as well as il- and il- mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th- t cells, we measured th- cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il- and a strong reduction of il- mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il- exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il- ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il- . based on our previous observation that support a direct role of il- -activated stat in the enhancement of il- ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il- ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il- . quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il- ra promoter. crosslinked nuclear lysates were immunoprecipitated and min after il- addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il- ra promoter. chip assays showed that the pol ii recruitment to the il- ra promoter induced by lps is significantly increased by il- , further strengthening the concept that the rapid enhancement of lpsinduced il- ra gene expression by il- initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat immunoprecipitated dna showed statistically significant levels of stat binding to the il- ra promoter only in cells stimulated with lps in the presence of il- . surprisingly, anti-p and anti-p chip assays revealed enrichment of both p and p recruitment to the il- ra promoter when il- was added to lps-stimulated cells, suggesting that il- enhances the recruitment of nf-kb to the il- ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il- -treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il- ra promoter site is dependent on il- -activated stat , since it is greatly reduced when stat activation by il- is impaired. the molecular mechanism through which il- -activated stat promotes the recruitment of nf-kb to the il- ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il- acts as a specific negative transcriptional regulator of mouse mast cell protease- (mmcp- ). we examined the mechanisms underlying the repression of mmcp- gene expression. our data show that the "repressor" effects of il- on mmcp- promoter activity are still operating on the mmcp- bp long minimal promoter. moreover, il- deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy . furthermore, chromatin immunoprecipitation revealed that il- promoted specific reciprocal recruitment of c/ebpb but not yy to the mmcp- promoter. finally, il- deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp- . thus, we proposed that the expression of mmcp- and possibly other immunoregulatory genes may be regulated by il- through epigenetic modification and by balancing the content and binding of c/ ebpb and yy in mast cells. i. nagy , k. filkor , a. vörös , l. kemény , a. szász bzaka, baygen, szeged, hungary, university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir- , mir- a and mir- , which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir- expression; in contrast, pgn re-stimulation had no further effect on mir- a and mir- expression. next, we investigated the correlation between the expression of mir- and its two known direct targets: regulatory protein p and suppressor of cytokine signalling- (socs- ). although the gene-expression profile of neither p nor socs- changed, we found that the expression of mir- reversibly correlates with both p and socs- protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir- prior to pgn-treatment completely abolished both p and socs- down-regulation, revealing the involvement of mir- in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir- expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb +/-had a lower incidence and burden of benign papillomas when compared to tgfb +/+ animals. however, more scc developed in the tgfb +/-mice. after acute and chronic promotion, tgfb +/-skin showed a reduced proliferative response with no increase in epidermal tgfb or nuclear p-smad compared to tgfb +/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb gene dosage. further, pharmacological inhibition of alk suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb +/-skin, tgfb +/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb +/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb +/+ but not tgfb +/-keratinocytes, indicating that tgfb switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp- ) and fibroblast (mrc- ) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of ra patients and controls the levels of survivin correlated to urokinase (upa) (r= . ), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that / ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc- and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol , , -trisphosphate -kinase type b (itpkb) phosphorylates inositol ( , , ) trisphosphate (ins( , , )p ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca + responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h and h receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il- b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il- b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase- to generate the mature secreted active form. caspase- is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of genes involved in splicing with an average -fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified genes that significantly affect the production of il- b by thp- cells after a h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il- b secretion. tissue transglutaminase (tg ) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg inhibitor, in a transgenic mouse model cf and in the taz transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg in generating inflammation in two very different pathologies. this work underlines the critical role of tg in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge stimulated a-dc. preliminary results indicate no accumulation of hif- alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif- alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p but not erk / or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il- and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt a -but not wnt a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp in te. after infection with t. gondii, mice lacking neuronal gp (synapsin-cre gp fl/fl ) died significantly earlier in the chronic phase of infection than control gp fl/fl mice. death of synapsin-cre cre gp fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp -deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il- b, il- and il- have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il- , il- and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb further may generate more effective therapies. as tnfa mrna ' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb suppresses tnfa protein production by upregulating the rnabinding protein fxr , which can bind to tnfa mrna and inhibit translation. methods: using raw . cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb , we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb and luciferase expression was quantified. cells treated with lps and tgfb were also examined for fxr expression using pcr and western blot. following fxr knockdown using sirna, the influence of tgfb and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb . using the luciferase-tnf- 'utr vector we show that tgfb targets the 'utr of tnfa. furthermore, tgfb and il- both upregulate fxr mrna and protein; and treatment with tgfb and lps can synergistically upregulate mrna expression, more than tgfb alone. following sirna inhibition of fxr , tgfb can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp- and mmp- secretion from saecs, nhbes and fibroblasts to a peak of . +/- . ng/ml, at hours. interleukin- augmented comtb-stimulated up-regulation of mmp- and mmp- secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin- down-regulated mmp- secretion from saecs by %. interleukin- up-regulated mmp- and mmp- secretion from fibroblasts but not from saecs. timp secretion from saecs was enhanced by interleukin- but there was no effect of interleukin- . mmp up-regulation by interleukin- and comtb was inhibited by the pi kinase inhibitor ly and on western analysis akt (protein kinase b) was phosphorylated at minutes. chemical inhibition of the p d isoform of pi kinase with ic abrogated the il- and comtb driven secretion of mmp- from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome ) accentuated mmp- secretion. these inhibitory effects were confirmed with sirna. mmp- up-regulation was secondary to increased gene expression with promoter activity peaking h after stimulation. in summary, interleukin- and interleukin- drive transcription dependent mmp- and mmp- secretion from airway epithelial cells and fibroblasts. interleukin- also increases timp but down-regulates mmp- gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi kinase pathway is central in interleukin- driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto , l.s. moreira , e. gonzalez-rey , m. delgado institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper response. cholesterol metabolism is regulated by factors such as pparg (proliferator activated receptor g), srb (a class b scavenger receptor), cd or abca , that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg , srb , cd , and abca . we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il- in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il- and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il- , tnf-a, il- b, il- and il- , and the anti-inflammatory cytokine il- , in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il- was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il- and il- , and in some cases, of il- . the maximal plasma levels of il- and il- were found at hours and of il- between - hours. modest plasma levels of il- were also observed, with maximal production at various time points ( - hours). by contrast, production of tnf-a, il- b and il- did not occur to a significant extent, while production of il- occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il- , il- , il- and il- also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il- , il- , il- and il- , i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il- , activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il- is a novel il- cytokine family member that is expressed as an intracellular precursor (pro-il- ) and is thought to be cleaved by caspase- to yield a mature bioactive form of the molecule (mat-il- ). to date however, evidence of cell-associated proteolytic processing and caspase- dependent secretion of mat-il- has not been reported. here we show that pro-il- but not mat-il- is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il- to the il- r and also il- r-dependent bioactivity of pro-il- on mast cells. we propose a previously unrecognized role for pro-il- as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il- p mrna expression in myeloid cells, and markedly increase secretion of il- , but not il- , by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il- promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop . chromatin immunoprecipitation (chip) assays using anti-chop and isotype control mab were performed using nuclear lysates from u cells and il- promoter dna measured by qpcr. chop binding on the il- promoter was detected following stimulation of u cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il- promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop in il- gene transcription, u cells expressing shrna's specific for chop or non-specific gene target were tested for their ability to express il- following tlr and er stress stimulation. u expressing three independent shrna targets for chop exhibited significant reductions in il- p mrna (up to % reduction of the response to lps+tp) compared to u expressing a control shrna. chop shrna expression did not affect the expression of other lpsresponsive genes, including il- , il- , ccl and sod . to identify if er stress induction of il- mediated by chop expression plays a role in a more physiological setting, we examined the role of chop in the induction of il- p gene expression following chlamydia trachomatis (ct) infection. infection of u cells with live but not g-irradiated ct induced expression of er stress response genes, including chop . u infected with live ct exhibited increased il- p mrna expression compared to u infected with nonviable bacteria. chop silencing significantly reduced the ability of live ct to induce il- p mrna, confirming the important role of chop in this response. these data suggest that er stress induction of chop could contribute significantly to the pathogenesis of diseases in which il- plays an major role, through induction of il- and il- producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd b maintained a sustained activation of p kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd b-deficient mice showed only transient p activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd and cd were elevated on gadd b-deficient thymocytes. thus, we provide evidence that gadd b and a resulting persistent activation of p constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo institut pasteur, paris, france, monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il- is recognized as an essential factor for thymopoiesis, the nature of the thymic il- niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il- promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il transcripts (il- hi cells). il- hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl , ccl , cxcl ) and cytokines (il ) that are critical for normal thymopoiesis. in the adult thymus, il- hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il- hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il- levels. conversely, the frequency of il- hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il- -expression by tecs. > together, our temporal-spatial analysis of il- -expressing cells in the thymus suggests that thymic il- levels are dynamically regulated under distinct physiological conditions. this novel il- reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il- expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl -tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl -tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl -tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg plasma cells and serum igg levels were˚ - fold increased. importantly, serum from young slc-tg;em-bcl -tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in / and / cases, respectively. these values were increased when compared with control groups: / in fcgriib -/and / in bcl -tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd + cd + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd hi cells, representing multipotent progenitors, or cd + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb and ephb expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b and/or ephrin b , the ligands of ephb and ephb receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb or ephrinb genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb /b double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd + cells in the cortex, increased proportion of k +k + cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb and ephrinb in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink , d. vanhecke university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op -dl cell culture system ( ) . using this in vitro assay we obtain large numbers of human cycd + and cd + cd + double positive thymocytes starting from umbilical cord blood (ucb) derived cd + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il- and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd + cells upon notch signaling is cd followed by cd . t cell specification is accompanied by the induction of cd a and loss of cd on cd + cd + cells. these cd -cd a + cd + cells become dependent on continuous il and notch signaling for sustained survival and further differentiation into cd + cd ab + dp thymocytes. we found that flt l is not essential for the differentiation of cd + cd + human thymocytes but that addition of exogenous flt l in the co-cultures increases the number of cd + precursors and consequently result in higher yields of developing cd + cd ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd + cd + cd ab + dp subset in this in vitro assay suggesting that op stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn to dn stage, where bdnf and its receptor p are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga , c. lópez-rodríguez pompeu fabra university, barcelona, spain nfat is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat -null mice are unable to mount cd +-and cd + -immune responses. data from our laboratory indicate that nfat -null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat during the development of t lymphocytes, we developed mouse models that delete nfat at early (lck-cre + /nfat flox/flox ) or late (cd -cre + /nfat flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p , is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues - of the proline-rich domain of p . methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p null (p -/-) and wild type (p +/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p -/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p is not important for preventing thymic t-cell tumors. s. myrczek , r. pardi , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, vita-salute san raffaele university school of medicine, milano, italy jab is the catalytic subunit of the highly conserved cop signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab regulates the activity of ap transciption factors. to date jab is thought to be essential for every cell type as jab knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab . to investigate the function of jab in b cells we established a mouse strain deficient for jab selectively in b cells. mice with floxed alleles of jab kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb -locus (m. reth, freiburg). ablation of jab expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b and b cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab -deficient, bcl -transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab when apoptosis is prevented. t. nitta , s. murata , k. tanaka , y. takahama university of tokushima, tokushima, japan, university of tokyo, tokyo, japan, rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th and th antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd + t cells or functional deficits that selectively interfere with th or germinal centre responses. in this talk i will present data from some of the first strains that have been identified including the first strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn c-kit high (etp), dn and dn thymocyte populations was hybridised to affymetrix mouse a- . genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors ( out of genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included signal transducers (out of genes) such as acvr , bmpr , fzd , chemokine receptors cx cr , cxcr and integrins a , a , a , ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata- , tcf- , notch- , rag- , rag- and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks out of of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by - days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd + cd ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged month to years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd + cd precursors with recombinant wnt a ( ng/ml) or with licl ( mm) for hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab ( e ) under conditions of phosphatase activity inhibition. wnt a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il- and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st cells suplemented with il- and flt l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il- and flt l and modify the transcription factor profiles of cd + cd thymocytes mainly increasing hes- and id expression levels. human th clones and circulating th cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th and th clones or circulating th oriented t cells, respectively. accordingly, human th cells exhibited lower expression of clusterin, and higher bcl- expression and reduced apoptosis in the presence of tgf-beta, in comparison with th cells. umbilical cord blood naï ve cd (+)cd (+) t cells, which contain the precursors of human th cells, differentiated into il- a-producing cells only in response to il- beta plus il- , even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd (+) t cells but it increased the relative proportions of cd (+) t cells differentiating into th cells in response to il- beta plus il- , whereas under the same conditions it inhibited both t-bet expression and th development. these data suggest that tgf-beta is not critical for the differentiation of human th cells, but indirectly favors their expansion because th cells are poorly susceptible to its suppressive effects. m. irla , w. reith university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd (+) or cd (+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd (+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd on mtecs by cd l induced on the positively selected cd (+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd (+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu , i. ravens , g. bernhardt hannover medical school, institute of immunology, hannover, germany cd is originally identified as human poliovirus receptor (pvr) and as rodent tage , which is overexpressed in rodent colon carcinoma. cd is also known as necl- , a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd is overexpressed in transformed cells and promotes the cell cycle. thus, cd seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd and cd ; the dn subset is further subdivided into four stages (dn - ) by differential expression of cd and cd . thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd + sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd lineage. here we show that the frequency of terminally matured cd + sp cells but not that of cd + sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd , a selective deficiency of mature cd + sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd + sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd + t cells. in cd deficient animals, a shift in the tcr repertoire displayed by the pool of cd + sp cells was found demonstrating that cd is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. , , . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht , propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd + ter + erythroid and cd + cd b + myeloid cells are simultaneously cycling (s/g /m) in the post-gastrulation mouse embryo (e - ). the peak of lymphohematopoietic cell proliferation occurs at e in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence - hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e - that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd -cd interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd , we hypothesised a role for cd -cd interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd -/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd on reconstitution of b cell numbers following depletion. we show that cd is expressed by pro-b cells, and these cells proliferate in response to cd signalling in vitro. pcr identified a source of cd , negative for cd eta, in the bm of wt mice showing this cd is not provided by activated re-circulating t cells. we have shown that when cd -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn (cd -/cd -/cd + /cd -) to the dp (cd + /cd + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn stage on. in the bone marrow we found yfp + /b + and yfp + /b populations. thus these pta expression analyses show closely similar pattern to those observed with hucd preta-reporter transgenic mice (gounari f. et al. , martin et al. . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. , - ( ) martin c. h. et al., nat. immunol. , - ( . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz , g. klein zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm- which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x kda.mmp- , a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm- . in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp- . by western blotting the zymogen and the activated form of mmp- can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm- and mmp- can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp- which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp- , timp- and timp- , are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp- plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of - % compared to only % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd /cd expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd +cd + double positive (dp) tcrab low cells entering selection, and their cd +cd + dp tcrab-immediate precedents followed by underrepresentation of the selected cd +cd + dp tcrab high and the most mature cd -cd + and, particularly, cd +cd -single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd -cd -double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd +cd -/cd -cd + sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd / cd /tcrab), in adult wistar rats subjected to -day-long treatment with a -ar blocker urapidil ( . mg/kg body weight/day s. c.). the a -ar immunoreactivity was found in both thymocytes (mainly less mature cd and cd low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed -postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd / cd /tcrab expression were observed. these changes comprised of an increase in the percentage of cd + + tcrabthymocytes, which was accompanied by the reduction in that of cd + + tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd + -tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd - + tcrab high was reduced. in addition, the percentage of cd + t regulatory and cd +tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c bl/ mice infected with m. avium ( cfus, iv) were sacrificed at different time points after infection ( , and weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il- ) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il- in this organ. in the spleen, ifn-g reaches a peak of expression earlier ( weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm and ai from nih and grant i- from the robert a. welch foundation to wtg, and by grants hl and hl from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. ) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c bl/ x balb/c)f mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski , s. lang , m. stein , t. winkler friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h ac) or methylation of h on lysine (h k me / ). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h ac and h k me / ) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar # ) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 'rlm race at the ivar # element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo locus, is thought to be accessible only during the double-negative (dn) and thymocyte stages based on mrna expression, implying that translocations between lmo and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx (hox ), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed ) to evaluate lmo and tlx breakpoint-site accessibility during thymocyte development; ) to determine in which stage of development there is an increased chance for lmo or tlx translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo and tlx loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn and dn development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn , dn , dn and immature single positive (isp) stages for both lmo and tlx . conclusion: our findings show that both the lmo and tlx loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn and dn stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that to successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e - ), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd cell depletion. however, cd depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd t cells has important implications for vaccine development against neonatal infections. ( ) showed that irf knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs . conclusions: although recent findings indicated that irf function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez , t. boehm max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd and cd t cells from tuberculosis patients highly express pd- when compared to healthy uninfected individuals. in addition, analysis of pd- expression in lung biopsies from tuberculosis patients revealed that pd- is expressed on cd and cd t cells confined to lung granulomatous lesions. finally, blocking of the pd- /pd-l axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd- /pd-l pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p to p . among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th and th cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd + t cells, nfatc and c are predominantly expressed. nfatc is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc /a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc /c. as demonstrated by y h screen and co-ips, nfatc /c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc /c -but not the unsumoylated nfatc /a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc target gene interleukin- . other lymphokines like ifng and il are reversely regulated. interestingly, ntreg cells which do not express il exerted only nfatc /c, but no nfatc /a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc function. therefore, especially ntreg cells and anergized cd + t cells might be regulated by the long sumoylatable isoform nfatc /c. lnk/sh b and aps/sh b , two members of the lnk/sh b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b- cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il- signalling in pre-b cells overexpression of lnk dramatically inhibits il- -dependent growth demostrating that lnk negatively regulates il- pathways. furthermore, we showed that il- stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav . to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh or sh domains known to mediate protein-protein interactions are key players in these processes. sly (sh domain protein expressed in lymphocytes ) was identified as a putative adaptor protein containing a sh and a sam domain as well as a bipartite nls. sly belongs to a family of three molecules: sly , sly and sash .in humans, the sly gene is located on chromosome , in mice on chromosome . sly is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin -associated polypeptide p (sap ) as a putative interaction partner of sly . sap is a conserved member of the sin a-hdac corepressor complex that contains histone deacetylase (hdac ) and histone deacetylase (hdac ) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected t cells. in addition, we could show a direct interaction between sly and hdac . to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly increases the activity of hdac in whole cell lysates and, more precisely, in nuclear extracts of t cells. the interaction of sly with sap and hdac indicates a transcriptional function of this protein. within the sin a-hdac corepressor complex sly might act as a switch for the activity of hdac . cd -cyt and cyt are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd (b cell marker) fused to the transmembrane and intracellular domain of cd -cyt or cyt in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt coengagement controlled il- secretion, while cyt coligation inhibited ifng production. moreover, our preliminary data suggest that cd -cyt inhibits the phosphorylation of several molecules known to be activated by cd stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh domain and three sh domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut cells with gst fusion proteins containing full length nck, the three sh domains or the individual sh and sh domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp and cd epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam ) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g , results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g ) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g ) degranulation as measured by cd a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose- -phosphate receptor which exhibits structural and functional similarity to the vps p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose- -phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla- (sctla- ) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla- in immune responses. using a specific anti-human soluble ctla- monoclonal antibody, jmw- b that selectively binds the soluble isoform but not membrane bound ctla- , or cleaved fragments of it, we demonstrate that sctla- plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla- , secreting increased amounts of cytokines including interferon-g, il- and tnf-a, but lower amounts of il- . soluble ctla- was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla- induced secretion of the immunoregulatory cytokine il- by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine , dioxygenase enzyme cascade was also initiated by sctla- . it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla- it is crucial to t cell inhibition. membrane-bound ctla- exists as a homo-dimer on t cells but sctla- is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b ligands on antigen presenting cells. a third important observation from this study is that sctla- exists both in serum and culture supernatants as a natural kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla- , concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il- dependent regulation is most critical, boosting sctla- secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin- /efhd- , in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin- /efhd is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin- /efhd- in the immature murine b cell line wehi enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin- /efhd- impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g cell cycle arrest. to understand how swiprosin- /efhd enhances pro-apoptotic bcr signals, we analyzed whether swiprosin- /efhd is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin- /efhd enhanced bcr-induced calcium flux in wehi cells, whereas shrna-mediated down-regulation of swiprosin- / efhd impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin- /efhd interacts with phospholipase cg (plcg ) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg and swiprosin- /efhd was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin- /efhd silenced wehi cells with swiprosin- /efhd- was inhibited by the syk inhibitor bay - . in analogy, swiprosin- /efhd regulated syk activity positively. moreover, swiprosin- /efhd re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg and of syk tyrosine residue , which is involved in syk activation. finally, reconstitution of swiprosin- /efhd knock-down cells with swiprosin- /efhd mutants revealed that the n-terminal putative sh -binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi cells. interestingly, swiprosin- /efhd re-expression in swiprosin- /efhd -silenced cells induced already in unstimulated cells raft partitioning of syk, plcg and the bcr, which was reversed after min of bcr stimulation. in summary, swiprosin- /efhd is an accelerator of proximal bcr signalling and acts through syk and plcg by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p mice die within hours after a second challenge with p peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e ligase dead mice can spontaneously reject tc tumors. conclusion: cbl-b e ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd + cd ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal ) and co-stimulatory signals (signal ), provided by beads coated with anti-cd /cd antibodies. gene expression patterns were compared for cells stimulated with anti-cd /cd beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip , itac). the enhanced expression of granzyme-b, ifn-g, trail and ip were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd -coated p cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal- to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd + foxp + and cd + cd high t cells. moreover, t cell receptor activation with combined anti-cd and anti-cd stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk- , but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv- infection leads to immune dysfunction owing to a successive loss of the cd + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv- virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk / . this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk / directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap- , nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk / is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal , t. brdicka , v. horejsi institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd are key regulators of src-family kinases in leukocytes. while cd is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh domain of csk binds phosphotyrosine of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd -csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie- kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa- integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi , s. parusso , b. frossi , g. gri , c. pucillo university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il- -/-mcs and anti-il- receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il- derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd on naï ve b cells and the interaction of cd on b cell surface and cd l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd l e cd on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves , n. bercovici , a. caignard inserm u , paris, france inhibitory killer ig-like receptors (kir dl - / ) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd + t cell subsets. these receptors suppress cd + t cell activation through recruitment of the src homology domain-containing protein tyrosine phosphatase (shp- ). to further analyse the yet largely unclear role of inhibitory kir receptors on cd +t cells, kir dl transfectants were obtained from a cd + t cell line and primary cells. the transfection of cd + t cells with kir dl dramatically increased the t cell receptor (tcr)-induced production of il- independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir dl -itim phosphorylation, shp- recruitment, zap- and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp- and p-pkc-v but not of shp- . in contrast, the kir dl /hla-cw interaction led to a strong synaptic accumulation of kir dl and the recruitment of shp- / , inhibiting tcr-induced il- production. kir dl may induce two opposite signaling outputs in cd + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir dl receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg antibody during these conditions. the observed igg switching behaviour mimics that of b cells responding to lps and il- , but is mediated by a different, stat -independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana , c. schwindling , m. pasche , c. junker , c. kummerow , u. becherer , e.c. schwarz , j. rettig , m. hoth saarland university, biophysics, homburg, germany, saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of days. under tolerogenic conditions (ova alone), cd + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, - bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd + t cell expansion and contraction kinetics in the early phase of the t cell response (days - ). in the late phase of the primary response (days - ), under immunizing conditions, the large majority of transgenic cd + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il- , ifn-g, and il- a, and only few ova-specific foxp + regulatory t cells ( x %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells ( %) was substantially increased. on day , both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells ( %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il- a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin- , interferon-g and macrophage inflammatory protein- by cd tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog - protein. our results provide direct evidence that mc contribute to cd -specific priming in eae and show that the tc proliferation failure is specific for cd tc from mog - -immunized w/w sh mice. the role of mc-cd tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla- , but not via any conventional motifs in this region. overexpression of trim augments ctla- surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla- localisation, mainly restricted to the tgn. ctla- vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla- trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla- expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla- transport to the cell surface. it is imperative to reveal the mechanisms by which ctla- is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd molecules, nkt cells are activated and release cytokines, including ifn-g, il- and il- . nkt cells are efficiently recruited to the liver via cxcr -dependent chemotaxis toward cxcl and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd t cell tolerisation via interaction with lsec. to this end we analysed cd d expression on lsec and their ability to activate nkt cells by presentation of the cd d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd d, as agalcerpresenting-lsec were capable to induce tnf-a, il- , il- and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd and b -h on lsec. as naï ve cd t cell tolerisation by lsec critically depends on b -h , we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg , jg . , and vd gene products. they recognize nonpeptide antigens like (e)- hydroxy- -methylbut- -enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd- . one of the inhibitory receptors, pd- , is a member of cd /ctla- family and contains a single ig v-like domain in its extracellular region. pd- can bind to two b homologue molecules, pd-l and pd-l . it has been reported that interaction of pd- with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd- is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with -methyl- -butenyl- -pyrophosphate plus il- to obtain gd t cells. pd-l + and pd-l -human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l mabs, the pd-l extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd- in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd- /pd-l interaction. results: gd t cells expressed pd- upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l . we first examined whether or not the engagement of pd- receptor could modulate the cytotoxic activity of gd t cells. pd-l -expressing tumor cells tempered cytotoxic activity of pd- + gd t cells, and cytokine production such as tnf-a was down-regulated by pd- engagement. in addition, inclusion of anti-pd-l mab reversed cytotoxic activity and cytokine production when pd-l -expressing tumor cells were challenged by pd- -expressing gd t cells. conclusion: pd- delivers inhibitory signals in gd t cells upon engagement with pd-l . peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il- production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova - peptide)-specific t cells from do . tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler , p. aichele immh, university freiburg, immunology, freiburg, germany interleukin (il- ) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il- has a direct influence on cd + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal ) and costimulation (signal ). we analysed direct il- signaling to cd t - signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd t cells lacking il- signaling failed to up-regulate klrg and to down-regulate cd in the context of listeria but not viral infections. thus direct il- signaling to cd t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd + t cells are tightly controlled in their effector functions by cd (ctla- ). we demonstrate that signals induced by cd reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd + t cells. for this novel function cd specifically represses the transcription factor eomes, but not t-bet. a cd mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd -mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd + t cells differentiated in the absence of cd signaling could be demonstrated in vivo. the novel insights that cd -mediated signal transduction in vivo indeed alters cd + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p receptors. p x - receptors open to non-selective ion channels, whereas p y , , , , - are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p x antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca( +)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek , a. brouckova , d. filipp institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih t cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y flck. comparative -d gel analyses followed by ms/maldi identified rack as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih t cells of ha-tagged rack with either a wild type lck or constitutively active y flck revealed a significantly enhanced complex formation between y flck and rack compared to that of wtlck. ectopic expression of y flck with its domain-inactivating mutations showed that lck-rack interaction depends on functional sh , sh and the c-terminal tail sequence of lck. lck-rack interaction is readily detectable also in primary cd + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack in the context of lck translocation to lr is further strengthen by the observation that rack is associated with elements of cytoskeleton. these results are the first to characterize rack as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin (il- ) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd +cd +cd -cd -igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th response to alumova inducing large early germinal centres and massive plasma cell formation with more than % of these switching to igg . the plasma cells up-regulate cxcr , but not cxcr , a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th -associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚ %), igg a (˚ %), igg b (˚ %) or igg (˚ %). in addition to cxcr , some % of these plasma cells strongly express cxcr . the induction of cxcr in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg a switching during th responses. this is functionally significant for oti-dependent cxcr expression, as well as induction of switching to igg a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg b. t-bet is known to be induced in b cells exposed to ifng or tlr stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd . objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd ) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd + t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd + t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla- pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa- and cd in the psmac of untransformed human t-cells. in marked contrast, tcr/cd accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl- regulates interclonal t cell competition during acute and chronic immune activation. we found p -independent noxa gene induction and mcl- downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl- , which was delayed in noxa -/cells. using ot- cells and altered peptide ligands we observed that the level of mcl- downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl- -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np ) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl- axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen (ebag ) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag , which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g -adaptin in t cells. both interactions suggested an involvement of ebag in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag -/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag -deficient ctls. these data imply a role for ebag in regulating the formation of mature ctl granules and identify ebag as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag -related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd / and il- . in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd /acd or pma/ionomycin could not significantly activate cd and cd t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca + influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena , s. carrasco , i. merida centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp -ras-erk signal intensity is critical to determine the final cell outcome. rasgrp is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. . analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. . develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd cells expressing gfp-c domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h , cd ), a cd -like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th biased responses and high production of the anti-inflammatory cytokine il- , and was essential to the development of germinal centres. however, icos can also help in the il- -dependent differentiation of inflammatory th cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p subunit of class ia pi- kinases. these can complex with one of the three kda catalytic subunits (p a, p b, and p d) expressed by leukocytes that generate pip affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi -k regulatory (p a, p b, p a) and catalytic (p a, p b, and p d) pi -kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p a g p a g g p b and p a g p d g g p b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi -kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd -induced secretion of il- and il- . during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap , a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap construct that acts as a dominant negative, or sirna knockdown of endogenous akap expression in t cells prevents the correct organization of cd z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa- localization was also analyzed to assess p-smac architecture and, interestingly, confocal d reconstruction revealed that lfa- ring was not clear in the akap -disrupted cells. moreover, akap was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap decrease the phosphorylation of molecules such as lat, plcg and pkcv. these defective activation events as reflected in a reduction of il- production. together, our results underscore a key role for akap in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin /cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca + mobilization and cell activation involving the pi k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc d c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr ) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as ) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. ) we then studied the phenotype of carabin kd (shrna expressing) a b cells after bcr stimulation. ) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t tot b cells and to follicular mature b cells. ) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. ) bcr simulation, but not lps stimulation, of carabin kd a b cells shows an acceleration of ras target erk / phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk / pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor (tlr ) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd + t cells express tlr and respond to the well characterized synthetic tlr ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr . tlr stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor (irf ) as revealed by realtime-rcr analysis of ifn b and irf , whose transcription depends on the activity of irf . combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd + t cells. this study was supported by dfg spp "innate immunity" (ka / - ). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd e cytoplasmic domain (cd e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd e cd and a fluorescent membrane dye (r ). with this assay, we show that the cd e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd e cd to lipid bicells. membrane binding by the cd e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia , y. ge , u. quitsch , p. beckhove german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd + t cell help is believed to contribute to optimal cd + memory expansion via cd l on cd + t cells binding cd on dendritic cells. however, a few reports suggest that cd l-cd engagement may mediate direct cell-cell contacts between cd + and cd + t cells. in this study, we investigated the importance of cd -cd co-operation and cd l-cd interactions for t em proliferation. methods: we isolated human cd + and cd + t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd + and cd + populations were activated in vitro using anti-cd /cd beads. proliferation was measured by [ h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd or cd l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd + or cd + t em cells, demonstrating that optimal t em expansion requires direct cd -cd interactions. surprisingly, not only cd + but also cd + t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd + t em proliferation depends on signals from cd + t em cells. activation induced the expression of cd on both populations and cd l on subsets of cd + and cd + t cells. blocking of cd l on cd + t em cells impaired significantly cd + t em proliferation, which confirms that the improved expansive potential of cd + t em cells in mixed populations depends on cd l co-stimulation by the cd t em subset. conclusions: our data demonstrate for the first time that activated cd + t em cells deliver help to the cd + t em subset via cd l-cd signalling and may play an important role for cd + t em expansion upon stimulation. the t cell surface glycoprotein cd , a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd localization upon t cell:apc conjugation. we have questioned which domains of cd mediate the localization within the is, and for this we have expressed cd mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd depends on sequences within the cytoplasmic domain, as a cd deletion mutant lacking most of the cytoplasmic tail, cd .k stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd translocation was mapped within amino acids glu and his since the cd .h stop mutant, just short of aa is still able to translocate to the is, whereas cd .e stop , that lacks important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu -his ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna- is missing, but lmp- is still expressed. using a hl derived cell line, we have shown that the cytokine il- can induce lmp- expression in vitro and can replace ebna- . we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is , or . a high affinity stat binding site is spaced by nucleotides. we found three potential stat binding sites in the lmp- promoter, which we named lrs, tr and edl . they were spaced by , and nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il- -treated or non-treated kmh -ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat binding site, or lrs-stat . a stat complex binding to the gl-epsilon promoter and lrs-stat was induced by il- . the specificity of the stat complex was shown by supershift experiments with anti-stat , but not anti-stat antibodies. when gl-epsilon or lrs-stat was used as cold competitors in a -fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat contains a functional stat binding site. oligonucleotides, corresponding to lrs in which the stat site had been mutated, could not compete for stat binding. interestingly, the unlabeled lrs-tr with nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by nucleotides (lrs-edl ), it could not compete. thus, expression the transforming protein lmp- can be induced directly by the t cell derived cytokine il- in a stat dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp- and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji (ji . : . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd + t cells primarily affect the proportion of cd + t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd , cd and cd on parameters of cd + t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd , a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd leads to an enhancement of ig and cytokine production. the current dogma postulates that these signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd l (rmcd l) can increase proliferation induced by tlr (poly ic) and tlr (lps) agonists. by contrast, we never observed any synergy between rmcd l and tlr / (pam csk ) or tlr / (pam csk ) agonists. to go further in the study of cd l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd l, named mini-cd ls, based on a c -symmetry core holding cd -binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd ls bind to immobilized human cd and compete with the binding of cd l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd l, mini-cd ls synergize tlr (lps), tlr (poly ic) and tlr / (r ) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd ls and tlr / (pam csk ), tlr / (pam csk ) and tlr (odn ) agonists. synergy between cd l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd + t cell tolerance. we have defined a phenotypic profile for cd + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd and cd , and high levels of expression of cd l and ly c. whereas, cd + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd and cd , and loss of cd l and ly c expression. ly c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly c, expressed on naïve cd + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to days after infection. results: daily injection of paraoxon induced g % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining weeks of treatment. mice exposed to paraoxon exhibited g % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with % of mice surviving the infection compared to % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th and th cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide -kinases (pi k) constitute a family of enzymes that generate -phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi k are divided into two types: class ia p /p heterodimers, which are activated by tyr kinases, and the class ib p g (p gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p g deletion affects tcr-induced t cell stimulation. mice lacking p g show a partial defect in t cell differentiation, activation and survival. p g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd +/cd + t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi kg-specific inhibitor causes a reduction in the number of cd + memory t cells that mediate renal injury. similarly, pi kg deletion in p pi k transgenic mice also reduces the numbers of cd + memory t cells. there is therefore evidence that pi kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi kg regulates this process is not well understood. we studied the specific role of p g in t cell activation. methods: we studied whether the tcr activates p g and the consequences of interfering with p g expression or function on t cell activation. results: we found that after tcr engagement, p g interacts with and forms a complex with ga q/ , lck and zap . tcr stimulation activates p g, which affects -phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p g controls rac activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p g-/-t cells. our observations clarify the activation mechanism and mode of action of p g in the control of t cell activation, confirming a crucial role for p g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a b and a , expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c bl/ j mice. they were stained with fluorescently labeled igm-, cd -or cd specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by h-thymidine incorporation upon stimulation with anti-cd and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a or b nachr subunits inhibited binding of igm-and cd -specific antibodies but facilitated that of cd -specific antibody. in contrast, antibody against a subunit prevented binding of anti-cd but not of anti-igm or anti-cd suggesting that a nachrs are located close to cd , while a b ones are close to bcr/cd . consequently, anti-cd -induced b lymphocyte proliferation was increased by mla much stronger than by a b -specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine- . in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd -mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd + t h -lymphocytes to antigen presenting cells or of cd + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim and trpc ), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about %. down-regulation of stim was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, ) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be - pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein (hsp ) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd + cytotoxic t-lymphocyte (ctl) and cd + t-helper cell (th ) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k cells as well as targetindependent cytotoxicity. cd + cells exhibited a strong increase in proliferation after stimulation with hsp , with rates of up to %. in the presence of target cells, a -fold up-regulation of granzyme b mrna was observed after stimulation of cd + t-helper cells with hsp in combination with il- , - and - . the target cell-independent secretion of granzyme b by cd + cells was greatly augmented after stimulation with hsp plus il- or il- , - and - . in this study, we have shown that hsp is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd + and cd + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd , which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk overexpression in a model system was achieved by transfection. pjnk / expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk , but not jnk activation. cd ligation on primary tonsillar b cells also resulted in jnk activation. however, bcr crosslinking did not affect the level of jnk / phosphorylation. cd -mediated jnk activation was independent from sh d a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase (hpk ) was associated with cd in primary b cells as well as in b cell lines. cd -hpk association was independent from cd tyrosine phosphorylation and sh d a expression. overexpression of hpk in a model system significantly enhanced cd mediated jnk phosphorylation. it is known that tnf family receptors such as cd , cd , rank trigger survival signals in hrs cells. we observed the expression of pjnk / in hrs cells of primary classical hl. cd could be involved in sustained jnk activation in primary hrs cells, and this may reflect the role of cd receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk is activated via cd in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk is involved in cd -mediated jnk activation. objectives: cd has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd are involved in c-cbl phosphorylation and association. methods: el thymoma cell line was stably transfected with wild-type human cd or hcd cytoplasmic tail mutants: cd .k stop (maintaining only a pseudo itim); cd .h stop (lacking the distal s and y in the carboxy-terminal region); cd . ¿ e -l stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd in combination or not with anti-human cd biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py ) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x . ). in murine thymocytes, co-crosslinking of cd with cd induces an increase in c-cbl phosphorylation compared to cd alone. analysis of the el- transfectants showed that mutants cd .k stop and cd .h stop lost the ability to costimulate cd -mediated phosphorylation of c-cbl. in contrast, cd . ¿ e -l stop mutant, was able to efficiently costimulate cd -mediated c-cbl phosphorylation, similarly to the hcd wt. our results indicate that the absence of the pseudo itam in cd does not interfere with c-cbl phosphorylation in response to cd plus cd crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd cytosplasmic tail, but rather, may indirectly associate with cd through the interaction with other sh -sh domain-containing molecules, that may be recruited to cd through its carboxy-terminal region. l. kolly , s. narayan , j. tschopp , a. so , n. busso chuv, rheumatology, lausanne, switzerland, unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase- into inflammasomes and thus plays a key role in regulating capase- -dependent il- b and il- production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd + and cd + t cells were activated in vitro through anti-cd stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd activated asc -/-t cells predominantly displayed a more th phenotype, producing more il- ( vs. pg/ml; asc +/+ vs. asc -/-t cells respectively; p= . ) and less ifn-g ( , vs. , pg/ml; asc +/+ vs. asc -/-t cells respectively; p = . ). when asc +/+ and asc -/-t cells were purified into cd + and cd + t cell fractions and activated individually using anti-cd , no inhibition in proliferation was observed amongst activated asc -/-cd + and cd + t cells. interestingly, the activated asc -/-cd + t cell fraction produced significantly more il- when compared to activated asc -/-cd + t cells and asc +/+ cd + and cd + t cells (asc -/-cd + t cells = pg/ml il- ; asc -/-cd + t cells = undetectable il- ; asc +/+ cd + t cells = pg/ml il- ; asc +/+ cd + t cells = undetectable il- ). cd + and cd + t cell mixing experiments revealed that asc -/-cd + t cells are able to inhibit the proliferative ability of asc -/-cd + t cells, asc +/+ cd + and cd + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd + t cells. collectively, these results demonstrate that the absence of asc drives cd + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge and pge suppress some t-cell functions including proliferation, activation and cytokine production. pge signals through four types of gpcrs called the ep receptors. at low concentrations, pge is believed to be necessary for t cell function, whereas at higher concentrations, pge inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd + t cells with ep receptor antagonists was found to impair cell surface expression of cd , cd , cd and ox but not cd . suppression of t cell proliferation by pge has already been widely studied. however, blocking ep receptors in cd + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd + t cells to the chemokine sdf- b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd + cd + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd + t cells. our results also suggest that considering pge -mediated camp signaling in cd + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd + human t-cells was examined. oxidation affects several ca + signalling pathways by altering the activity of ip receptors, trp channels and store operated ca + channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca + signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd knockout mouse possess enhanced mixed lymphocyte reactions and cd monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd in jurkat t cells augments the secretion of the t cell growth-factor interleukin- (il- ) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il- promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd , we identified the immunomodulatory sub-domain of cd . supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd and il (simulates t cell-dependent activation). microarray assays identified about mirnas in unstimulated b cells. of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor- (irf- ). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf- transcripts differs from that observed for irf- protein abundance after b cell stimulation. further analysis identified the irf- transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk and the dfg forschergruppe for . objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey , f. hauck , i. berberich , f. berberich-siebelt , gk -immunomodulation institute for virology and immunobiology, university of wuerzburg, würzburg, germany, university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd + t lymphocytes, the transcription factor is predominantly expressed in t helper (th ) compared to t helper (th ) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp- encoded by prdm is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp- is expressed in differentiated effector t cells where it is higher in th than th cells. the regulation of the blimp expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm promoter and activates blimp- expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp- in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp- in b cells using cre recombinase. moreover we found a new putative blimp- isoform lacking exon . currently, we analyze the expression of c/ebpb and blimp- in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd + t cells but not transitional b cells or cd + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein- a (mip- a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy revealed constitutively high background levels in cd low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas , v. courtois , k. de luca , r. sodoyer sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il- , il- or il- could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il- , il- and il- . furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a- - - ). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen- (ctla- ) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla- during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il ) in the tissue and the released eggs and first stage larvae (l ) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla- during the infection, a neutralysing antibody (a-ctla- ; f ) was administered intraperitoneally ( mg) two hours before subcutaneous infection with s. ratti il . the in vivo neutralisation of ctla- -signalling by applying a-ctla- during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th cytokines, such as il- and il- and a reduction of the proinflammatory cytokines ifn-g and il- . the investigation of the humoral response showed a remarked increase of the igg -titer in the serum during secondary infection in mice that had been treated with a-ctla- during primary infection. furthermore, the blockade of ctla- resulted in a diminished worm burden as indicated by reduced release of l in the faeces. these results suggest that the blockade of ctla- during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg during secondary infection also reflects the induction of a potent th response. objectives: cd is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd expression. cd knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd ko compared to heterozygous mice. since reduced levels of cd are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd ko mice. after one injection of apoptotic cells, cd ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd on b-cells are involved in setting this threshold. conclusion: our data suggest that cd is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd acts as a major check-point of immune responses, but the mechanism by which cd controls peripheral t cell responses is unknown. the consequences of cd signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd in th cells reduced migration towards ccl , cxcl and ccl . crosslinking of cd together with cd and cd stimulation on activated th cells increased expression of the chemokine receptors ccr and ccr , which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd expression and cd -mediated migration-enhancing signals. importantly, migration of cd positive th lymphocytes in in vivo experiments increased, as compared to cd negative counterparts, showing that indeed cd orchestrates specific migration of selected th cells to sites of inflammation and antigenic challenge in vivo. these data show that cd signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd adds to the already significant role of cd in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd signaling on the longevity of cd null t cells. using a sensitive staining method for cd , we show that human cd + cd null and cd + cd null t cells rapidly express surface cd . serological inactivation of cd using specific fab fragments or blockade of cd ligands using ctla ig in cd + cd null and cd + cd null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd crosslinking on activated cd null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd is mediated by pi 'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd act directly on activated cd null t lymphocytes and, due to its exclusive expression as a receptor for cd /cd on cd null t cells, prevention of cd mediated signaling is likely a major target mechanism taking place during therapy with ctla ig. objectives: cd is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd (ptprj, dep- ) which acts as a positive regulator of sfk in cd -/-b cells and macrophages and can compensate for cd deficiency in these cells. indeed, combined deficiency of cd and cd in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd -/mice were reported so far. however, in human t cells the role of cd may be different since naive human t cells express cd at a level comparable to b cells. using cd -/-/cd -/human t cell line (jurkat-derived js- cells) we tested the ability of cd to complement cd deficiency in t cells. we used retroviral transduction to express human cd or cd in js- cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js- cells. expression of wild type cd or cd in js- cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js- cells expressing cd when compared to control cells. finally, cd substrate trapping mutant expressed in js- cells interacted with lck in vivo suggesting that lck is a direct substrate of cd in js- cells. the results suggest a level of redundancy between cd and cd in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam . ) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr and tyr we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca + release-activated ca +) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa- (leukocyte function-associated antigen ) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa- prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al. ) we describe a non-viral vector based approach (vockerodt et al. ) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd signaling cascade was conducted. after activating this particular signaling cascade (cd ) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. ). a rat thymic epithelial cell (tec) line (r-tnc. ) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc. cell line in vitro. it was found that a number of adhesion molecules, such as cd , cd , cd , cd a, cd , cd , cd was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc. line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il- activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc. cells. we found that both the adhesion ( min and h) of activated thymoytes were partially blocked by mab to cd and cd molecules (ifn-g unstimulated and ifn-g stimulated r-tnc. cells). early adhesion was inhibited by mab to cd , abtcr, mhc class i molecule (ifn-g stimulated r-tnc. cells) and cd molecule (ifn-g unstimulated r-tnc. cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc. cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay ( h), namely mab to cd , cd , cd , cd molecule (ifn-g unstimulated and ifn-g stimulated r-tnc. cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc. cells). our results also suggest the involvement of cd a/cd dependent -cd independent pathway in adhesion and cd a/cd dependent -cd dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc. cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin- (il- ), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il- favours naï ve cd t cell differentiation into th cells to the detriment of th or th differentiation. the il- receptor (il- r) is a heterodimer composed of tccr, which confers ligand specificity, and gp , a signal transducing chain that is utilized by several other cytokines. il- has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd t cells, but the potential impact of il- on human cd t cells has not been elucidated. our goal is to investigate the impact of il- on human cd t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il- r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd than cd t cells expressing the complete surface il- r (gp +tccr). however, we detected high amounts of intracellular tccr in both, cd and cd t cells, but only polyclonal activation (anti-cd ) of cd t cells led to an actual increase of il- r surface expression. purified cd t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il- activated stat and stat signalling with rapid kinetics in both cd and cd t cells, indicating the capacity of il- to signal through these molecules. addition of il- to anti-cd activated cd t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il- on human cd t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd- /pd-l pathway is associated with production of the immunoregulatory cytokine il- , the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l and pd , as well as myelin basic protein (mbp)-stimulated il- production, pakt inhibition, and apoptosis (annexin v), in ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); had a diagnosis of stable disease (sms). results showed that: ) pd-l -expressing cd + and cd + cells are reduced in ams compared to sms individuals (p= . ); and ) pd expression is increased in cd + t cells of sms individuals and is comparable on cd + t cells of ams and sms patients. this is associated with a significant decrease in il- production by mbp-stimulated cd + and cd + cells of ams patients (p= . ). additionally, cd + anexin v+ (av+) cells were diminished and cd + pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd +av+ and cd + pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l /pd- pathway seen in ams patients result in a reduced mbp-specific il- production by cd + and cd + cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd + t. the pd /pdl pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti- - bb in cd cells. this difference could be due to down regulation of cd by activated lymphocytes and possible preferential response of cd cells to anti- - bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin (stx ) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx . rna interference technique was also used to down regulate stx expression in primary human ctls. results: we identified stx in ctls by pcr and immunocytochemistry. stx accumulates at the is after ctl/target cell contact. when stx function was blocked by overexpression of a dominant negative stx mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the years history of mouse t h and t h subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h , t h or t h phenotypes, based on their cytokine production (il- , ifng or il- ). a comparative analysis of t-bet, ifng and il- mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca +response, membrane raft expression/organization, k + -and ca + -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h g t h g t h , although the membrane cholesterol level (detected with anti-cholesterol ab, ac ) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h cells upon activation than in t h cells. t h cells produced a more sustained calcium response with higher amplitude than t h cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav . and kv . ion channels, major functional determinants of the sustained calcium influx. t h cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd -dynabeads. cd + isolated cells were facs sorted into cd + cd naïve b or cd + cd + memory b cells. dna synthesis was measured by h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd + cd naï ve b cells, of which bmp- and - were most efficient ( % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd + cd + memory b cells by - %. to induce differentiation, both naï ve and memory b cells were stimulated with cd l and il- . this increased the production of igm, iga and igg - -fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad / / in cd + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd l/il- -induced production of igs in mature human b cells. s. gutenberger , k. warnatz university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases and (erk / ). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of hd and cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk / phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk / . to increase the signal intracellular phosphatases were inhibited by h o . as markers of activation and initiation of proliferation, cd and ki expression were measured after days of in vitro stimulation. k. theil , p. aichele immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p cd t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b -recipient mice and compared their expansion with wild-type (wt) p t cells after viral infection. we could demonstrate a severe impairment in the capacity of p t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p t cells into h mice. h mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp - . therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h mice are infected with lcmv . . this is a lcmv variant that has got a point mutation in gp - and consequently cannot be recognized by the p t cells. s. frischbutter , r. baumgrass deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap which are important for expression of cytokines such as il- , ifng and il . it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl- was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl- phosphorylation but rather an inhibition of bcl- dephosphorylation. furthermore, calcineurin and bcl- co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl- /malt signaling complex and dephosphorylates bcl- and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop , j. lamoureux , c. fortin université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla- has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla- expression was higher after stimulation in t cells of elderly. there was differences between cd and cd t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh and ww domain proteins. since fasl surface expression is regulated by adam -mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh domain proteins that potentially interact with the riped fasl prd, we used a sh domain phage display library containing all sh domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conse- optimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b family members on antigen-presenting cells with cd on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound , with classical gcs regarding their suppressive effect on cd -costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd and anti-cd , and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd -costimulated human memory/effector cd + t cells by compound than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound than prednisolone. when evaluating possible mechanism for the increased activity of compound in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound which was not prevented by the partial gc receptor antagonist, ru- , in vitro. moreover, in vivo we observed less induction of il- beta and tnf-alpha by pre-treatment with compound than with prednisolone. our data indicate that the non-steroidal segra, compound , may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b mice elicits robust cd + t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il- + cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa -specific cells also express tnfa, but only about half of the ifng+ np -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd + t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd + t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova - peptide shows a close correlation with division in vivo. early after antigen encounter ( - divisions) the vast majority of cells express only tnfa. after - divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions ( - divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd + t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd + t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd + t cells, expansion plays a very important role in the regulation of cd + t cell effector function. in addition to its chemo-attractant function, sdf- a (stromal-cell derived factor- a, cxcl ) has been described to costimulate cd + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd + t cells was increased by immobilized sdf- a to a level similar to that obtained with the costimulatory molecule cd . as visualized by real time confocal microscopy, t cells entering in contact with sdf- a formed a tether and displayed an active scanning activity. since sdf- a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd mabs, it is conceivable that the sdf- a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf- a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf- a in the context of cd + t activation by antigen-presenting cells secreting sdf- a. this study should help us to better define how sdf- a modulates cd + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd and anti-cd antibodies and cape for days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il- production and lymphoproliferation in cd + t cells stimulated by anti-cd /cd . cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd t cells express a variety of molecules on their surface, such as mhc-class ii, cd , cd , cd , whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd t cells might induce t cell proliferation and differentiation from cd resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd , cd , and cd . analysis of the cytokine profile of these cells revealed the differentiation of il- -and ifn-g-double-producing cells in response to contact with th effector cells, and il- -producing cells in response to contact with th effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd /cd stimulation. whereas neutralization of ifn-g or il- during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd , cd , and cd could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to - %. conclusion: interaction of cd memory t cells with activated t cells resulted in the production of the cytokines il- , il- , and ifn-g. given the immunomodulatory capacity of il- and il- , these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir , s. thompson , j.j. murphy king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il- and il- and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp l by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp l has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl differentiation was observed to be associated with downregulation of zfp l protein. in an attempt to determine whether zfp l downregulation was directly linked to bcl differentiation, a zfp l shrna expressing lentivirus (psicor) was employed to knockdown zfp l expression. this reagent downregulated zfp l expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp l shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp l downregulation in promoting bcl plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd (fas, apo- ) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd /cd -triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il- and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk / phosphorylation, the expression of various activation markers, the il- production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb ) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb , galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd + and cd + t cells. in addition, mitogenic stimulus increase -fold the all binding to cd + t cells. previous studies in human pbmc showed that all binds to human cd + t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd + t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd -dependent activation of purified cd + t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd expression, but enhanced the anti-cd -dependent proliferation and cd expression of purified cd + t cells. analisis of calcium influx showed that all enhanced anti-cd dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd + t cells by up-regulating t cell activation mediated by anti-cd stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in ) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd receptor. it has been previously demonstrated that cd ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp- , slap- and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il- r, il- r, il- r, il- r, il- r, il- r, cd , cd , cdw ( - bb), cd (fas) and cd (fasl) on hpbls in different cultured time, i. e. d, d, d, d, d, d, d and d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. . the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. ) the expressions of membrane immune molecules before cultured. ) expressions of the membrane molecules on hpbls during culturing. % fbs rpmi group ( group), il- group, pha group... ( ) mtt assay. ( ) proliferative times and growth curves of hpbls... . during cultured in vitro, there are expression changes of the il- rs (il- ra, il- ra, il- rg, il- r, il- r, il- r, il- r), co-stimulatory molecules (cd , cd , - bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in group, il- group and pha group, but the rests are different. . our data also suggest that the hpbls cultured in cd mcab+cd mcab+il +il a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. . celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun , r. tauber zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p lck and the ras/rac signalling pathway ( ) followed by mitogen-activated protein kinases ( ) and c-jun n-terminal kinase ( ), which leads to an enhanced binding of l-selectin to soluble ligands ( ). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues ( ). here we show that the protein phosphatase a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer , e. glasmacher helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago - genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm and rck or expression of dominant-negative gw . interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd + t-cell activation and differentiation. naïve cd l hi cd lo cd + t cells were sorted and stimulated by anti-cd and anti-cd antibodies. results: firstly, we show that the activation and differentiation of cd + t cells require il- provided by activated cd + t cells at the initial priming stage after stimulation. secondly, this critical il- signal is delivered through il rbg of cd + cells and is independent of il- ra. besides promoting cell proliferation, il- stimulation increases the amount of ifng and granzyme b produced by cd + t cells. conclusion: therefore, our studies demonstrate that a full cd + t-cell response is elicited by a critical temporal function of il- released from cd + t cells, providing mechanistic insights into the regulation of cd + t cell activation and differentiation. most antigenic peptides recognized by cd t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a - is a tumor antigenic peptide presented by hla-a and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a - we setup an in vitro digestion assay using a -mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a - by tumor cells. by electroporating hla-a cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart- /melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart- /melan-a - specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart- /melan-a - ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart- expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart- /melan-a - epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart- /melan-a - ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev : - , ) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h -m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity : - , ) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide - , whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from - peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the - epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, ), we found, after an initial induction at hrs p. i., a strong inhibition of tapasin transcription at hrs p. i. furthermore, also reduction of tap and tap transcription was observed contrasting to the elevated levels of erp and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß , ß and ß , which are replaced by their ifng-inducible counterparts ß i, ß i and ß i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß i is replaced by a thymus-specific subunit ß t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß i, ß i, ß i and ß in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß ,ß and ß i (single intermediate proteasome), and the other comprises ß i, ß and ß i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent - % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about % of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a* ) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a* binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a* from tapasin, but only ttp-ha dissociated hla-a* from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl- . -a showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a* from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a* . m. basler , , c. lauer , m. groettrup , biotechnology institute thurgau, kreuzlingen, switzerland, university of constance, division of immunology, department of biology, konstanz, germany two lmp -dependent antigens have been described that relied on the 'structural presence' of lmp in the proteasome but not on the activity of lmp . here we have investigated processing of the h- d b -restricted uty - epitope of the male minor antigen uty reported to be lmp -dependent. using splenocytes from lmp -/-, lmp -/and mecl- -/mice we found that the uty - epitope requires lmp and lmp but not mecl- . curiously, a selective lmp inhibitor did not interfere with uty - presentation. objective: we investigated why the deletion but not the inhibition of lmp interferes with uty - presentation. we hypothesized that the 'structural' requirement for lmp is based on replacement of the caspase-like activity of b in the proteasome. methods: it was determined if t a mutants of lmp and/or b can rescue the uty - epitope. we used a b -selective inhibitor to determine if the inhibition of the caspase-like activity of b preserves the epitope. finally we determined by mass spectrometry if the uty - epitope embedded within a mer precursor peptide is differentially cleaved by lmp -deficient and proficient immunoproteasomes in vitro. results: we found that t a mutants of lmp and b rescue presentation of uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . also inhibition of cells with a b -selective inhibitor preserves uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] presentation. an aspartate in position of the uty - sequence wmhhnmdli is preferentially used as a cleavage site by lmp -deficient but not half as frequently by lmp -proficient immunoproteasomes. the generation of the uty - epitope relies on the replacement of the caspase-like activity of b by lmp because the b activity destroys the uty - epitope. this is the first example for the 'structural' requirement of lmp for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp and perhaps also lmp and mecl- exert their function in antigen processing. a. linnemann , a. musiol , r. lindner hannover medical school, cell biology, hannover, germany, hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf -regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf ) were overexpressed in t fibroblasts. we show that antibody-mediated oligomerization of mhc i in t fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf : endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf -regulated to a novel, arf -independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv virus and since sv binding triggers mhc i oligomerization, this novel pathway may be involved in sv uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab b, c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta -microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab b and rab c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab b or c positive vesicles. furthermore, the rab b, c positive compartment were colocalizd with a fraction of rab a at a juxtaposition of phagosomes. together these data demonstrate that rab b and rab c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd + t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf a and bglf have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf , has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens ). this represents a novel function for a virally-encoded gpcr. bilf is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf can hinder the recognition of virally-infected cells by cytotoxic cd + t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe natural ligands, including two peptides derived from semenogelin- , a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg is transcribed in thymus from both male and female individuals. finally, we detected the semg mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta , beta , beta ) and immuno-subunits (beta i, beta i, beta i). deficiency in beta i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta i-deficient cells could be restored by treatment with d t, which increases the amount of proteasomes independent of beta i via induction of mixed proteasomes containing beta i, beta i and beta . consequently, not the lack of the specific proteolytic activity of beta i or immunoproteasomes, but the reduced proteasome quantity in beta i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr , and , which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd + t cells. the presence of exogenous tlr and tlr ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james , i. bailey , t. elliott university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct colon carcinoma. this response is long lasting and mediated by both cd and cd t cells. importantly, the treg depleted ct specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a , c , bcl , renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw , which is h -d d restricted. analysis of the generation of gsw in ct revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct cells substantially increased the amount of gsw present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct and immunised balb/c mice. greater than % of mice injected with eraap kd ct were found to reject the tumour. analysis of t cell responses revealed the presence of gsw -specific t cells, however, these responses were small ( . - %). this compared to a much larger response to ct (˚ %). preliminary results indicate that the majority of the t cell responses (non-gsw -specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [ ] . the di-allelic hla-a restricted minor histocompatibility antigen ha- locus codes for the highly immunogenic ha- his and the non-immunogenic ha- arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha- arg immunogenicity was the estimated numbers of cell surface presented copies i. e. /cell for ha- his and less than / cell for ha- arg [ ] . as ha- his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha- his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha- mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha- allelic peptides, we investigated the impact of the ha- his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha- his and ha- arg peptides were performed. moreover, the crystal structures of hla-a in complex with either ha- his , ha- arg or a ha- variant with a citrulline residue at position were determined in order to obtain atomic level insights into the conformation of the hla-a /ha- peptide complexes. our results exclude a role for antigen processing in preventing ha- arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha- arg allele in the hla-a peptide binding groove [ ] . they provide a rationale for the lack of ha- arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd t cell response. on the other hand, immunosubunit expression is not essential for development of cd t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp (ib ) and mecl- (ib ) develop a variety of autoimmune responses, including a latent form of t d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd but not cd t cells. a cd t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose- -phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr and dr . analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type (hsv- ) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd + and cd + t cells in hsv- immunity have yet to be clearly elucidated. to better understand the role of hsv- -specific cd + t cells in immune control we have identified a amino acid epitope derived from glycoprotein d of hsv- . following flank infection, gd-specific cd + t cells were first detected in the draining brachial and axillary lymph nodes (ln) -days post-infection (pi), peaking at day and declining thereafter. gd-specific cd + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day pi and peaked at day . while hsv-specific t cells were first observed in the draining ln at day pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as days pi, with peak activity days pi before declining to background by day . however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd + t cells was observed up to days post-infection in the brachial ln. ex vivo analyses suggest that only cd c + cells were involved in gd antigen presentation at days , and post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd a + dcs can present the gd antigen to cd + t cells at day pi, whereas by day pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv- infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap and erap unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap /erap in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp . upon assembly of the heavy chain with b m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp . both crt and erp are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp point mutant that fails to bind to cnx or crt was just as effective as wild type erp in normalizing rates of disulfide formation. we conclude that erp does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp point mutant, class i heavy chains, crt and the tapasin-erp disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp have no effect on plc stability or class i surface expression, suggesting that erp plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies - % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb * /dqb * haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr* transgenic ab nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd + and cd + t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr* transgenic mice (cd + t cell competent) develop aip even unprovoked, similar to ab nod mice (cd + t cell deficient), while hla-dr* , hla-dq or hla-dr* /dq (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr* fails to protect from aip, likely due to defects in the induction of cd + regulatory t cells. our results identify hla-dr* as a prominent risk factor for aip on the hla-drb * /dqb * haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan , c. britten , h. overkleeft , g. van der marel , k. melief , d. filippov , f. ossendorp leiden university medical center, section tumorimmunology, leiden, netherlands, leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd and cd t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi , , x. hu , , a. kawana- tachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping -mer and -mer epitopes (rypltfgwcf (nef - ) and rypltfgw (nef - )) were found to be presented by hla-a* and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y f, y w, t c, f l, w r, f r, f y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef - (wild type) and its four mostly common immune escape mutants (y f, t c, y f&t c, f l), and also nef - (wild type). we found that there was little difference between the nef - (wild type) and nef - (y f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y or f . interestingly the central bulge region of the peptide was becoming very flexible for the nef - (t c) and nef - (y f&t c), which may affect the binding of peptide and the recognition of t cell receptor. for nef - (f l), the side chain of l was more flexible compared to the nef - (wild type). alignment of the nef - and nef - showed that the nef - became flat and the side chain of f was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef - was featured, while the peptide nef - was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef - was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd + and cd + t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a restricted cmvpp derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a - log reduction of activation efficiency. this pattern was seen for / epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to - log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b restricted cmvpp rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv -mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd + t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp , and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor- (fgf- ) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf- peptide was produced in vitro after incubation of proteasomes with a -amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf- spliced peptide by cells transfected with mutant constructs encoding fgf- proteins where the intervening segment was shortened from amino acids to , or residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b* , are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b* , appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b* and b* are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us protein. to address this hypothesis, us was stably coexpressed in b lymphoblastoid cell lines expressing hla-b* or hla-b* . in the presence of us , the surface expression of hla-b* was substantially reduced whereas hla-b* expression was relatively unaffected. although us was able to form complexes with both hla class i allotypes, only hla-b* was retained intracellularly in an immature form whereas hla-b* was transported to the cell surface. accordingly, in the presence of us , hla-b* , but not hla-b* , constitutively presented a hla-b restricted alloantigen to reporter t cells, suggesting that us binds hla-b* without interfering with peptide loading. us has been reported by others to bind the plc but surprisingly we have not detected such us -plc complexes in our system. rather, in the presence of us we identified a pool of class i molecules distinct from the plc and only present in us expressing cells, implying that us may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa , b. seliger martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il p . since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for h with the gold standard of maturation (tnfa, il b, il and pge ) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase- (lap ) had a more than -fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap and erap and of the immunoproteasome subunits lmp and lmp was enhanced between and -fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr ligand mpla in comparison to the tlr- and / ligand polyi:c and r . these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il- induced surface expression of mature mhc class ii molecules and cd . when ifn-g/il- primed pcmc were used as antigen presenting cells for cd + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type -diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b - to specific cd + t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd + t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd + tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd + t cell clones, faure, , eur j immunol ( ): - ). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd + t cells after their in vitro differentiation into dc, days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv- genome contains arfs within gag, pol and env genes encoding for a panel of hla-b* restricted epitopes. qprsdthvf (q vf/ d) is one such epitope but its parental epitope qprsnthvf (q vf/ n) has a significant higher frequency among hiv- isolates. strikingly, q vf/ d-or q vf/ n-specific ctls recognize apcs infected with hiv strains encoding for q vf/ d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad ) encoding for q vf/ n do not activate ctl responses raising the possibility that q vf/ n epitope is not presented by infected cells. we asked whether introducing mutations within q vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q vf/ n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q vf mhc-i presentation. we performed in vitro proteasomal digestions of mer peptides encompassing q vf/ d or q vf/ n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q vf/ n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q vf/ n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q vf/ d or q vf/ n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q vf/ d peptide. we cloned and sequenced hiv- genomes from the three donors. surprisingly, out of hiv proviral genomes isolated from pbmcs of q vf/ d reactive donors, we could not find any virus bearing the q vf/ d sequence. the isolated hiv sequences either encoded for q vf/ n or had a stop codon within the epitope. in contrast, viruses encoding for q vf/ d were isolated from pbmcs of the q vf/ d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv- seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch , y. wang , d.c. tscharke the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h- bxd f mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f progeny, with some peptides being almost equally immunogenic in f and inbred mice, while others elicit responses that are reduced by more than % in f mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd + t cells with a restricted vb usage in f mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr agonist, s-[ , -bispalmitoyiloxy-( r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp- ), as an adjuvant for cross-priming against cellular and soluble antigens. malp- has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd + and cd -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr / results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap ) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, and % as compared to control rma cells, were injected s. c. in the flank of c bl/ syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to days after injection. all mice injected with rma clone with a % level of eraap expression developed a tumor and died within days after injection. surprisingly, any animal injected with rma clone with a % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd + t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor ( bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor ( . bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb * -restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with -mer synthetic fviii peptides to stain epitope-specific cd + cells.cd + t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a domain: fviii - , fviii - and fviii - , as well as the c domain peptide fviii - , but not any c domain peptides. the c domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost. : - , ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni , s. albini , m.z. limongi , l. cifaldi , d. fruci ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap and erap , we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap , erap and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap and erap . this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p nf-kb subunit, we demonstrated that nf-kb is involved in erap and erap expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap and erap and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap , erap and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch , s. tenzer , h. schild , e. von stebut-borschitz uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c bl/ mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th immunity, whereas ifng secretion of both cd + th and cd + tc cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il- -dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm (lmp -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c bl/ mice. in addition, ex vivo co-cultures with infected dc from either lmp -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc cell ifng secretion and the dc restimulatory capacity of cd + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd + t cells, isolated from low dose infected c bl/ wildtype or lmp -/mice, were not detected. in summary, our data indicate that despite the fact that cd responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd + t cells against l. major is independent of the lmp subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a , generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd + t cell epitope ( - ) from the g domain of aggrecan , times more efficiently than non-specific b cells and over times more efficiently than the macrophage line j . however, despite this highly efficient aggrecan capture, processing and presentation of the - epitope took at least hours, comparable to the time required for presentation of aggrecan by j . treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the - epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions , and at the peptide binding groove. position also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b* , peptide loading is relatively independent of this chaperone. we analyzed the effect of b subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b subtypes. the association of b heavy chain with tapasin was analyzed in c r cells transfected with hla-b subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta- , which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me monoclonal antibody that recognizes b properly folded b /peptide complexes. the formation of fully assembled b molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc , which recognizes mhc class i free heavy chains (hc), or with me . maturation was analyzed by pulse-chase analysis, immunoprecipitation with me and treatment with endoglycosidase h (endo h). hla-b polymorphic positions other than , both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c bl/ mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately % of vacv global presentation in the context of h- b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd + and cd -dc, which differ in their mhci antigen presentation capacities. cd + dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd -dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd + and cd -dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd + and cd -dc was determined. surprisingly, cd + dc exhibit rapid surface mhci turnover compared to cd -dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd + and cd -dc were stabilized and no longer underwent rapid turnover. this suggests that cd + and cd -dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd + and cd -dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h- kb loaded with the ova-derived peptide, siinfekl. cd + and cd -dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd + dc, but not the cd -dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd +, and not cd -dc. this data further validates the role for cd + dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp- cytosol using gelatinase b/mmp- as a model enzyme. in the first dimension, ion exchange chromatography separated the thp- proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp- . in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp- on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp- cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp- -cleaved or intact thp- cytosol. proliferation was assessed by measuring incorporated hthymidine. results: - mmp- candidate substrates were isolated, of which were identified, revealing many novel mmp- (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about / of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp- decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e - k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue in hla-a and residue in e - k are highly critical for association of both proteins. this defines a putative interaction surface between e - k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e - k and the class i antigen presentation pathway. moreover, because soluble e - k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap and tap . tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap and tap were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap on tap was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap mutants suggested that the lack of tap protein expression is associated with a strong reduction of tap protein levels, which could be restored by tap gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap on tap different shrna plasmids specifically targeting tap or tap , respectively, were stably transfected into constitutively tap and tap expressing hacat keratinocytes and colo melanoma cells. in both cell types the shrna-mediated tap and tap inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap caused not only an almost complete loss of tap , but also a strong decrease of tap protein expression. in contrast, the tap knock down exhibited no influence on the tap expression. specific inhibition of the proteasome prevented the degradation of tap in the tap knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap on tap protein expression is not restricted to rare tap mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of healthy volunteers was exposed twice to . minimal erythema dose of uv. blister roofs were then collected before and , and days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd + cd + and of a macrophagic cd -cd + cell subsets that emerge and days post exposition, respectively. more importantly whereas classical cd a hi cd + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd a low cd and cd a low cd + that emerged and days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd and hladr. finally, days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey , e. reeves , t. elliott , e. james university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd + cd + regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct , stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct -derived cross-protective antigen, gsw , which was found to be encoded within the ectotropic murine leukaemia virus (emv- ) envelope protein, gp . this protein has previously been shown to encode ct -specific cd and cd antigens, implicating it as a 'hot-spot' for ct tumour antigens. interestingly, we have identified a truncated version of gp which may be responsible for generation of gsw . expression studies have revealed increased gp expression in ct compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw ) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p , p and p tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp - , bound to hla-dp with high affinity ( - nm). binding studies of mbp - derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f a, f a and k a) were affected, and only by a - fold reduction in affinity for hla-dp . the observation that none of the substitutions resulted in a complete loss of binding to hla-dp indicates that ) hla-dp binding to peptides does not depend on a large hydrophobic residue accomodated in p , or ) mbp - can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp molecule binds the immunodominant epitope in ms, mbp - , possibly in more than one register. additional studies are required to resolve the hla-dp peptide binding properties, and to determine whether expression of hla-dp affects the disease course in ms patients. results: depletion of mncs for cd + cells abrogated the tg-induced cytokine production and proliferation of cd + t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd + t cell proliferation was significantly reduced in cd /cd -depleted mnc cultures, as compared to cultures depleted for cd + monocytes alone. the same applied to the production of il- , il- and tnf-a. production of ifn-g and il- was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa , , p. pala , g. miiro , j. todd , p. kaleebu , , n. imami , f. gotch , mrc uganda, basic science, entebbe, uganda, imperial college, london, united kingdom, mrc uganda, entebbe, uganda background: hiv- -specific t-cell responses are preserved in hiv- infected individuals with non-progressing hiv- disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv- for g years, maintaining cd + t-cell counts g , and with minimal cd + decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: art naï ve hiv- infected patients from the entebbe cohort in uganda were recruited and stratified by cd + t-cell count, cd + decline slopes, and time of enrolment, into groups - ltnp and rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd t-cell count, and ifn-g, il- and il- elispot responses to pools of to peptides ( -mers overlapping by aa). peptides were based on consensus sequences of gag and nef from hiv- clades a , a and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il- responses were significantly higher in the ltnp than in rp (p= . , ifn-g responses to gaga pool ; p= . , il- responses to gaga pool ). il- responses were low and not significantly different between ltnp and rp. there was a positive correlation between il- responses to gaga pool and cd t-cell counts (r = . , p= . ), but no correlation between either il- or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv- specific responses were higher in ltnps than in rps confirming previous results. non-specific il- responses were high possibly reflecting baseline th responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her /neuand her /neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using -aad for dna analysis and specific antibodies directed against the proliferation marker ki- , the m-phase specific phistone h as well as for the h- l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her /neuversus her /neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g /g -, s-and g /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g /g -phase, which continuously raised and peaked within the s-phase. however, h- l d surface antigen expression was not altered in her /neucells during cell cycle progression. in contrast to her /neufibroblasts the her /neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h- l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd ) and ccr were cultured from cd + stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd + derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il- -dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mØ). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mØ than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mØ have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, ) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, , ) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x , ), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd +, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for of them. for of these patients hla-drb data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p= . ). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb- a preparations, whereas no differences were shown for ifnb- b. an association between hla-drb * and nab-negativity was detected (p= . ). the known associations between hla-drb * and csf-positive ms and hla-drb * and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb- a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb- b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart- and gp more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni , s. lorenzi , f. mattei , f. spadaro , l. gabriele istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg thymoma line, we show that type i ifn promote the ability of cd a + dc to capture apoptotic eg cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd a + dc and the persistence of apoptotic eg -cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg -derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd a + dc. as a result, eg -loaded dc become competent at inducing ot-i cd t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. ( ). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd + dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam csk , and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd + dc but not cd -dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm , which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a human lung cancer cell line. methods: dm induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a cells. we examined the morphological change using optical microscope. and gfp-lc punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm was showed high cytotoxicity on various cancer cell line, especially a cells. dm treated a cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc and beclin- protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc was increased concentration and time-dependently. beclin- also was increased. and then, we examined gfp-lc punctation on a / gfp-lc stable cells using confocal. a cells were formed gfp punctuation after dm treatment. to confirm these data, we measured cell death ratio using -methyladenine ( -ma), an autophagocytosis inhibitor. after -ma treatment, dm induced cell death was decreased comparing with dm treatment. conclusion: dm did not induce apoptotic cell death. but dm showed several characteristics of autophagic cell death. taken together, dm induced autophagocytosis on a cells. these finding indicated that therapeutic potential of dm by triggering autophagic cell death. s. b. rasmussen , s. r. paludan university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr) , which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv- infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv- , we found that tlr dependent ifn regulatory factor activation was abrogated. in addition, inhibition by -methyladenine of phosphoinositol -kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr dependent recognition. in the ongoing project we are examining how hsv- triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko , i. berberich , gk -immunomodulation universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl- family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl- family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a and its human homologue bfl- are anti-apoptotic members of the bcl- family. a is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a . unless the c-terminal ends of other bcl- proteins the tail of a does not function as a strong membrane anchor. nevertheless, the last amino acids of a are important for the protein. in fact, the c-terminus of a serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl- factor bimel results in increased stability of a . this is due to reduced ubiquitination of a after binding of bimel. we conclude that the cterminus of a /bfl- serves as a docking site for e ubiquitin ligase(s) that control the stability of a by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e ubiquitin ligase that interacted with a in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs- in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs- is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs- significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs- was manifest not only by the suppression of jaks acting upstream of p mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak / activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs- overexpressing cells. the results strongly suggest that socs- acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl /sdf- a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u- , and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p and fas-l expression was carried out by western-blot. results: cxcl induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p -ser levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after h of tnf-a treatment. conclusions: cxcl induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p function/levels. s. y. demiroglu , r. dressel universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp does not protect against cell death mediated by ctl. acute overexpression of hsp can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res : ) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp on granzyme b-induced apoptosis. methods: hsp was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type . results: hsp did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase- were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g peak measurements (p= . ). in contrast, a partial protection of ge-tet cells was observed after acute hsp overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood : ) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk and dr / - . the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr and tnfr , both belonging to the tnf receptor superfamily. tnfr -mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr -associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr , resulting in depletion of traf from the cytoplasm. here we confirm that tnfr induced depletion of cytosolic traf by the use of tnfr -and tnfr -specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf in the alternative nfkb pathway, we show that tnfr induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr and membrane tnf. j. c. morales , d. ruiz-magaña , d. carranza , c. ruiz-ruiz universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase- . in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp l is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp l may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl- protein is an important cell survival protein at different stages of b cell development. bcl- mrna also contains are regions that could possibly be targeted by zfp l protein. in the present study, we have tested the ability of zfp l protein to bind to a bcl- mrna are probe and to degrade bcl- mrna. recombinant bacterially expressed zfp l protein was shown to bind specifically to a bcl- are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp l also provided evidence that endogenous zfp l in b cells could bind to the bcl- are. in order to examine whether zfp l binding to bcl- are resulted in bcl- mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp l knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl- mrna was expressed in both wild-type and knockout mefs. the half-life of bcl- mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp l does play a role in degradation of bcl- mrna. overall, our data are consistent with a role for zfp l in degradation of bcl- mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp . since lmp is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi -kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te ) and lymphocytic (nc ) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes or tes (transforming effectors site) derived from the c-terminal intracellular part of lmp , in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp 's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase- . using this nc tumorigenic model in scid mice, we showed that induction of the dn lmp -tes prevent development of tumours and mouse death. these dominant negative derived from lmp could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs and socs differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p mapk activities, while socs promoted apoptosis with an increase in p activities. in contrast, both socs and socs display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs or socs induced a slight decrease in g or s phase cells and a prominent increase in g /m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g /s transition and g /m arrest induced by socs or socs . socs promoted dna damage-induced p induction and g /m cyclin b expression, while socs induced decrease in g cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein (hsp ) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp is released during necrotic cell death and proinflammatory effects of extracellular hsp have been observed. thus, we asked whether apoptozing cells cleave hsp during apoptosis or how hsp is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp protein content. we observed that hsp is degraded during apoptosis resulting in the formation of a fragment of about kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp (hsp alpha and hsp beta) we could show that the kda fragment is formed after degradation of the alpha isoform of hsp . further, we were able to show, that hsp cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp was inhibited or if exogenous hsp was added. these results demonstrate that cleavage of hsp represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc -i is processed to lc -ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf- gfp-lc cells were therefore incubated in starvation medium for hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl- /beclin- control of autophagy. recently, jnk was shown to be necessary for beclin- upregulation, and jnk-mediated phosphorylation of bcl- is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf- cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol -angelate (pep ), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb turned their response to pep from an increased to decreased rate of apoptosis. furthermore, our data show that pep inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl- and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p , trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch and analysed by flow cytometry. expression of the caspase inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases and . diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase- abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor (tnf-r ), and tumor necrosis factor (tnf)-a receptor (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid -o-octadecyl- -o-methyl-rac-glycero- -phosphocholine (et- -och ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd independent of its ligand fasl/cd l. fas/cd is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin- , an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl -family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment ( -fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo , sw , lovo, caco- , ht- ) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki- , pcna, p , bcl- ) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl- , bax, caspase , , , nfkb, parp / kda, tnfr /cd a and cd /fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl- , bax, nfkb, parp was significantly lower and expression of caspases, tnfr as well as percentage of cd cells significantly higher as compared with control group. caspase expression was significantly higher as compared with nfkb, parp and tnfr . in comparison to the standard nutrition preoperative immunonutrition increased bcl- and nfkb expressions and decreased caspases and parp expressions. in addition, we found a significant down-regulation of bcl- expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega- fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x . ). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x . ). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with healthy volunteers receiving either la ( cfu/day) or placebo, during days prior to uv ( × . med). blister roofs, liquid and skin biopsies were collected , and days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p ). while a similar decrease of lc for both groups was observed on day after uv exposure compared to placebo, la- group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd a low cd -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il- and a tendency to inhibit il- was observed in la- group compared to placebo. on day , la- group presented a greater recruitment of early lc precursors and a trend to increase cd a low cd + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il- , tnfa, il- , and il- ) was observed in la group compared to placebo. we show that la limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la supplementation for photoprotection at a lower dose ( log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn appears to be expressed in all epithelial cells of the early thymic rudiment starting around e . . previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn in a nude (foxn -deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn . if true, it should be possible to target this cell type by use of foxn -promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either or glutamine residues under the control of foxn promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei , , y. toriumi , k. kimura european university viadrina, frankfurt (oder), germany, shimane institute of health science, izumo, japan, shimane university faculty of medicine, department of pediatrics, izumo, japan, japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a -minute-rest period, followed by a -minute yoga exercise called asana, a -minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a -minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp ). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r= . , p x . ), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r= . , p x . ) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd (r= . , p= . ). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd , within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd + cd + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox l expressed on mcs and the constitutive expression of ox (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca + responses and degranulation. the cross-talk between t reg cells and mcs through ox -ox l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th or th effector cells and the induction of antigen specific foxp + regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th and treg subset, gata and foxp respectively, counter regulate each other (mantel y et al., ) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp + tregs and high gata + th cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost million allergic patients are sensitized to the major birch pollen allergen bet v , which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g %) of patients' ige binding to bet v was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa - (p ) and aa - (p ) of bet v . cross-reactive ige epitopes between bet v and related pollen allergens and plant food allergens involved primarily p . p and p are not adjacent peptides in the bet v sequence but define a surface-exposed patch on the three-dimensional structure of bet v . as determined by surface plasmon resonance, monoclonal antibody mab specific for p and mab specific for p showed high affinity, i. e., dissociation constants, k(d) = . e- m and k(d) = . e- m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p and anti-p antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th cells as well as tc cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th -mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th /tc cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd + and cd + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd (gk . ) or anti-cd ( . . ) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr- monoclonal antibody rb - c or monoclonal antibody nimp-r . one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd + t cells, cd + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd + or cd + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th and tc cells operative in the elicitation of airway inflammation. conclusions: robust type immune responses, although highly effective in the counter-regulation of local th -mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h . the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p , we were able to show that the major allergen of phleum pratense, phl p , although sharing molecular similarities with der p , does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p . chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il- and il- from nci-h cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p . in contrast to hdm extract grass pollen allergens induce the release of mcp- from respiratory epithelial cells, as well as moderate levels of il- . detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr ) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): . ± . ) and of il- (mean±sd: ± pg/ml), and reduced il- (mean±sd of il- -spot forming units/ x cells (sfu): ± ), compared to healthy subjects ( . ± . si, p x . ; ± pg/ml, p x . ; ± sfu, for proliferation, il- and il- , respectively); children with non-ige mediated cma had a significant reduction of il- ( ± pg/ml), compared to ige patients (p x . ) and an increased, although not statistically significant, production of ifn-g ( . ± . sfu) compared to control ( . ± . sfu) and to ige-allergic patients ( . ± . sfu). finally, tolerant patients showed reduced il- ( ± pg/ ml, p x . ) and proliferation ( . ± . si), compared to acute ige-cma children. interestingly, the high level of il- observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il- was undetectable in all patients even blocking the il -receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th -like response with il- and proliferation is dominant in ige-mediated cma patients; by contrast a th -skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang , b. chua , f.c. lew , a. ho , k. wong , k.l. wong , d. m. kemeny national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd th t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd t cells inhibits the induction of the th response. here we have investigated the effect of cd t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd + t cells ( . %± . % to . %± . %). when ifn-gamma -/-ot-i cd t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced ( . %± . %), suggesting an important role for cd t cell ifn-gamma. cd c + cd + cd blung dcs from cd transferred mice secreted higher levels ( pg/ml) of il- p following ex-vivo stimulation as compared with animals that were not given cd t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd t cells can subsequently divert the local lung environment to one that favors th immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in % of patients with hiv infection. this reaction is strongly associated with hla-b* and appears to be mediated by cd + t cells producing inflammatory cytokines. we show that cd +t cell responses can be primed in vitro, in normal blood donors who are hla-b* +, but not in non-b* + donors. cd t cells, but not cd t cells, are expanded by abacavir pulsed autologous apc over a -day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b* . similar responses were detected in abacavirhypersensitive hlab* + patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b* since they were undetectable using apc expressing closely related hla-b or b allotypes. responses to apc expressing mutants of hla-b* demonstrated a crucial role for residue . isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b* triggering cd t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b* may be relevant to the role of other disease-associated class i allotypes such as hla-b and b . a. jenckel , s. bulfone-paus , n. föger research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin a (coro a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro a during mast cell activation. cell fractionation experiments demonstrated that the association of coro a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro a phosphorylation. a functional correlation between coro a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro a. thus, coro a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin a (coro a) deficient mice have demonstrated that coro a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin b (coro b) is a closely related homolog of coro a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro b deficient mice and crossed them with coro a deficient mice to obtain coro a/coro b double deficient mice. analysis of t lymphocytes from coro a/coro b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro a/coro b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro a/ b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro a/ b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn (also known as hacs or sly ) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology (sh ) domain and a predicted bipartite nuclear localization signal. the samsn gene is located on mouse chromosome and encodes a well conserved protein with amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn in all tested hematopoietic cell types. the highest expression level of samsn mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd + and cd + t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly (hacs ) and sly (sash ) -were expressed only at a very low level in mast cells. the high level of samsn mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn deficient mast cells. in additional studies we are now investigating the effects of samsn deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., ). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st siai-v; st galnac i-iv, st gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg abs. results: we observed that the expression of st gal i, iii and v coding genes was similar in both hybridomas, while the st gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg . in addiction, the expression levels of st sia and st galnac genes in the hybridoma producer of anaphylactic igg were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg . conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il- and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il- and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf mediates the differentiation into th cells by activating multiple genes which are independently crucial for the development of naive t cells into th cells. because irf is expressed in many different tissues, it can be considered as a master switch factor for th cell differentiation. methods: here, we tested mice deficient in irf in the murine acute asthma model to evaluate its importance in this th cell-mediated disease. the protocol setup was the following: sensitizations s. c. with ova, followed by challenges via ova inhalation and adoptively transferred wildtype cd + t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf deficient mice with the help of adoptively transferred cd + t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by . fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. ) were also significantly higher in irf deficient mice. conclusion: interferon regulatory factor plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd + t cells. mechanistically, a potential counterregulation of asthma by th cells is not available in irf knockout mice. together with our previous report that irf represents a susceptibility gene for allergy in the human, our data highlight irf as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c bl/ mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk -receptor and ppt mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd c expressing apc substets characterized by cd and cd expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th- cytokine profile and increased levels of il- mrna. further the number of cd + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: ) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase , ) asthma and ee are prototypic th diseases in which the cytokines il- and il- play a principal role through the activation of the transcription factor stat , and ) we have recently demonstrated that some chloromethyl ketones can downregulate stat by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat , we analyzed its effect in the activation of stat by il- and il- . we found that treatment of cells with ppis inhibited the ability of il- and il- to signal stat activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd and cd high (treg)), that might inhibit the development of a th response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd or cd at hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h r. the level of intracellular ccl production in human lc was reduced after stimulation with h r agonists and basal production could be restored when h r was blocked with the specific antagonist jnj . moreover histamine and a h r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h r in allergic skin diseases and encourage further exploration of the h r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g p , an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g p was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g p which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th to chronic th -predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received weekly intravenous infusions of rituximab at a dose mg/m body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g , ; g , ; g , mg/dl, respectively). all patients underwent prior treatment with omalizumab for months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the nd week after initiation of rituximab therapy up to year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up year), ige levels decreased from , to , mg/dl. the other two patients are in the and -months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) and th cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either %dncb, %tma or to vehicle alone for . - h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il- (mean= pg/ml, n= , p x . ), il- (mean= pg/ml, n= , p x . ) and il- a ( pg/ml, n= , p x . ) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of ng/ear of murine recombinant il- or of the known regulator of lc migration; il- b. interleukin- b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after ( %) and h ( %) (n= , p x . ). in contrast, il- or control injections were without effect. however, il- administration caused an increase in cutaneous il- production ( pg/ml, n= , p x . ) compared with control injection and naï ve tissue ( and pg/ml, n= , ns). in addition, systemic treatment with anti-il- antibody failed to impact on lc migration provoked by dncb ( % reduction; n= , p x . ). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il- a ( pg/ml, n= , p x . ) and il- ( pg/ml, n= , p x . ) expression was reduced to baseline levels by anti-il- treatment. these data demonstrate that il- is not involved in the regulation of lc migration, unlike il- b and tnf-a. however, il- is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il- may influence lc and dermal dc maturation, via the expression of il- a and il- . allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey ), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p -mapk-pathway related protein (p prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct- - , www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il- receptor antagonist (il- ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il- b and of il- -like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il- -related genes by real-time pcr on mrna from blood cd + monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il- , il- bp, and caspase- , both before and after therapy with kineret. il- b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il- b, tnfa, il- ) nor anti-inflammatory cytokines (il- , tgfb) were detected. as expected, il- ra was only detectable after therapy. il- was detectable in schnitzler's sera at higher levels than in controls ( . vs. . pm) and decreased after therapy ( . pm). the circulating il- inhibitor il- bp was lower than in controls and not affected by therapy. thus, free il- levels were increased in schnitzler's patients as compared to controls ( . pm vs. . pm in controls) and decreased after therapy ( . pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il- -related cytokines (il- b, il- ), nor of the il- /il- -converting enzyme caspase- in blood monocytes. however, the high circulating levels of il- suggest an increased activity of caspase- , as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr signal via myd to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c bl/ myd -deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd -/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg -titers in ova-treated myd -/-mice compared to wildtype mice, although the nacl-treated myd -/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over , species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies ( mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = ) or from non-infested contra-lateral sites. expression of mip- a, igf- , mcp- and ip- genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd , cd , cd , cd , mhc class ii, and p than that of holsteins. lymphocytes expressing wc and cd antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x . ). infested skin of nelores contained significantly more message for mip- a, igf- , mcp- and ip- than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd + t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il- plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa- in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam- /lfa- binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th than a th local response pattern by virtue of il- secretion and icam- /lfa- interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th like micromilieu in acute towards a th dominated milieu in chronic lesions. the genotyping of ccl l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi / ). psoriasis is an inflammatory dermatosis with % prevalence among caucasians. hla-cw allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in - and - positions. strong association was found between polymorphisms in the - region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b , and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to years of age) were selected. hla-a -b -c -dr -dq alleles and tnf- and - snp were differentiated by pcr/ssp. analyzing the total group, patients ( . %) were male, ( . %) were female. in the male group, the mean age at disease onset was , years. severe forms were seen in this group (psoriatic arthritis in cases and erythroderma in ). seven patients ( , %) had a favorable evolution of the disease, but ( , %) developed extensive psoriasis, covering over % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw* allele was positive in cases ( , %), tnf g/a genotype was found in ( , %) and tnf g/a in ( , %). in the female group, the mean age at disease onset was , years, one case of psoriatic arthritis and one of erythroderma. twenty-nine ( , %) had a favorable evolution of the disease and ( , %) an unfavorable evolution. cw* allele was positive in cases ( , %), tnf g/a genotype was presented in ( , %) and tnf g/a in ( , %). severe disease was seen in male patients. there was no difference in frequency of cw* allele between male and female groups, but there was a tendency of significant difference in tnf g/a genotype. we found that c bl/ mice were more susceptible than balb/c and dba/ mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il- and mrp / , among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: cases, which were studied retrospectively, included children ( boys and girls), aged - years, who had an anaphylactic reaction, out of the that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food ( %-particularly sea food and dried fruit), drugs ( %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites ( %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema ( %), and gastrointestinal symptoms ( %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g was found in out of the severest cases ( , %), in which the adequate control was held, while in their vast majority ( out of ) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in , % ( cases) there was a hereditary family history of atopy, while in children ( %) there was also a personal history of asthma. finally, at a great percentage ( %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that )the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. ) in particular, in many cases it is proved that there is a personal but also a family history of atopy. ) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd we could observe dose dependent upregulation of programmed death ligand (pdl- ) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since years. in march a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters ( mg) every second day. in april the patient presented himself in the consult with multiple livid papules with a diameter of mm in the area of the auricle. the histological examination showed an hhv- positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs , socs and foxp in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n: ), the patients who showed recurrence or progression after treatment (non-responders, n: ) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n: ) were evaluated for socs , socs ve foxp mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd , cd , cd , foxp , cd + cd high , cd + foxp + . expression of socs and foxp- mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd , foxp , cd +cd high , cd + foxp + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs , socs and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs and socs molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of silicosis patients, with mild ( ), moderate ( ) and severe ( ) silicosis, coal workers, exposed to inorganic dust containing fcs (exposed), and healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than . was considered significant.neopterin level was significantly elevated in exposed ( , ± , ng/ml) compared to controls ( , ± , ng/ml; p= , ). moreover, the neopterin level in exposed was similar to silicosis patients ( , ± , ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls ( , ± , au vs , ± , au p= , ) and to silicosis patients ( , ± , au p= , ). in contrast, igmcic was significantly elevated in silicosis than in exposed ( , ± , au vs , ± , au; p= , ).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p= , and , respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a* , b* , drb * (these of good prognosis) * /* /* /* ) or tbc (drb * /* /* /* and associations with dqa * /* /* ) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a* , b* and drb * (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a* , drb * . as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd + , cd ++ , foxp + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein (hhsp ). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il- , il- , il- , tgf-b and ifn-g production prevailed, pointing to a th /th weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp . importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th /th cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices ( ) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c -complement, myeloid related protein and two new proteins, integrin-ß and fibrinogen. data from more than patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than % and %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least mice per group with distinct cytokine pattern: th (c bl/ , c bl/ ) and th (balb/c). muscular injury was performed by injection of bupivacaine. both th -dominant strains presented more areas with regenerating myofibers and macrophages at dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at dpi. balb/c mice showed increased collagen expression and decrease of mmp- activity associated with more mrna for tgf-b . this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th cytokines) or myonecrosis and collagen deposition (th cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß -adrenergic receptor (anti-ß ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß ec ii -specific rat monoclonal antibody (clone f ), and showed by elisa that it binds to the linearized ß ec ii peptide. additionally, we confirmed with flow cytometry that f also binds the ß ec ii in its native conformation, i. e. directly labeled circular ß ec ii (dyl -ß ec ii ) peptide. moreover, we demonstrated activation of the ß -adrenoreceptor by f using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß ec ii -specific peptide-based therapy, by intravenously applying a circular ß ec ii peptide in the dcm lewis rat model to neutralize the anti-ß ec ii antibodies. while the peptide therapy strongly reduced the anti-ß ec ii titers in the serum by up to % and consecutively lead to clinical remission, elispot assays for the detection of ß ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca - epitope elicits autoreactive cd + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca - peptide. in a first step, hybridoma cells were generated by fusing bw tcra -cd lymphoma cells with myhca - -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca - -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle , a. schwarting , p.r. galle university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase (pr ), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr -positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il- . pr -mrna from the murine cancer cell line wehi- was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr -promoter in the second intron of the mouse pr gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr transcript and its promoter indicates that the murine pr may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n= ) or pbs (n= ) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr activation, in the presence/ absence of och. subsequently we transferred . x mdcs (n= ) or och-primed mdcs (n= ) ( times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a . % reduction in plaque size compared to mice treated with mdcs (p x . ). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant . % (p x . ) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a to -fold increase in these cells was detected days after the last treatment with och-primed dcs (p x . ). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il- . discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c bl/ background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c bl/ and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg c were isolated only from fcgriib deficient mice, but not from c bl/ control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box protein (hmgb ), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb . we investigated if hmgb -containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb remains associated with ncs released from late apoptotic cells in vitro. hmgb -ncs complexes were detected also in the blood of patients with sle. hmgb containing ncs from apoptotic cells induced secretion of il-b, il- , il- , and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd and toll-like receptor . neither hmgb -free ncs from living cells nor from apoptotic hmgb -or hmgb / -deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il- release by macrophages. immunizations with hmgb -containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr -dependent manner. in conclusion, hmgb in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher , k.p. hoefig , e. kremmer , v. heissmeyer stretches and helps in the selection of the correct splicing borders. a allele of (r h) creates a strong binding site for a splicing enhancer protein srp according to bioinformatics. our findings indicate that, the putative branch point, r h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank . finally, we believe that bank delta protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta protein may contribute to the observed reduction in sle susceptibility. s. beermann , r. seifert , d. neumann hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h receptor (h r), h r, h r, and h r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or -methylhistamine, a h r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and -methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c bl/ mice induced by either a-cd antibodies or cpg-odn. this histamine effect was completely inhibited by the h r-specific antagonist famotidine, while h r-, h r-, and h /h r-selective antagonists had no or only moderate effects. interestingly, the h r-selective reagent jnj , which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h r, jnj may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h r and, to a much lesser extend, the h r. using this assay system, cells obtained from control c bl/ mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega- fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of ng/ml resolvin e (rve ) on the release of tgfb, il- and il- in the culture of peripheral mononuclear cells ( x /ml) stimulated by phorbol ester (pma) ( nm), and the combination of pma and ionomycine ( mg/ml) for hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from healthy subjects and from patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = . ) and sjögren's (p= . ) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p= . ) or pma+ionomycin (p= . ), however rve was ineffective at reducing tgfb release in the sle and ss patients. rve caused a non-significant decrease in il- release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il- was not significantly modified by rve in any of the groups tested. the release of tgfb by ng/ml of rve can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve tested, il- and il- release were not significantly affected in healthy or autoimmune patients. omega- fatty acid derived rve may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis , a.k. tsirogianni , m. herrmann , h. m. moutsopoulos , m.n. manoussakis university of athens, dpt pathophysiology, athens, greece, university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included with primary ss (american-european criteria ) and with sle (acr criteria ). age-and sex-matched healthy blood donors to the ss and sle groups ( donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, ) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma ( patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd -staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p= . ). in ss patients, defective mb-phagocytosis involved only monocytes (p x . ) and significantly correlated with the presence of extraglandular manifestations (p= . ). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x . ) and of ss (p= . ). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: healthy persons have been included in our study and sle patients ( women and men) with various clinical signs ( persons had st degree of disease activity, persons - nd degree of pathological process activity). women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in , %, aphl of igg class -in , % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x = , ; p x , ). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x = , ; p x , ). the increased levels of anti-ada were revealed in of women with hnp, and the combination of anti-ada and aphl ( / ) was found out more often than isolated anti-ada ( / , x = , ; p x , ) or isolated aphl ( / , x = , ; p x , ). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc and mdc , and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc , mdc and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from pss patients fulfilling the american european consensus group criteria (aecc) and gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p= , ) and mdc (p x , ) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd + b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin (il- ) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd + b cells and increased il- levels. we therefore hypothesized that il- may have similar biological effects on cd + b cells in autoimmune diseases. here we demonstrate that the amount of il- in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to % of cd + b cells in sle individuals expressed grb. in-vitro experiments revealed that il- was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor . importantly, il- significantly decreased the cd +/cd -b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd + b cell death. these results suggest that il- -induced grb may play a regulatory role for cd + b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il- and grb levels in sle and showing that il- reduces the cd +/ cd -b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il- may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il- in sle and other autoimmune diseases. r. de palma , e. d'aiuto , s. vettori , g. abbate , g. valentini second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd + t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il- beta, il- and il- , cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed igg antibodies from single facs purified cd +cd +cd +cd + bone marrow plasma cells of patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran , e. moazemi godarzi , e. kamali sarvestani , e. aflaki shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin (il- ) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il- gene at positions - g/c and - g/c have been described that are key regulators of il- gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il- gene polymorphism at - position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the - position significantly differed in sle patients and controls. at this position gg genotype was observed in . % of patients compared to . % in the control group (p x . ). the frequency of - g allele in patients ( . %) was also higher than in controls ( . %) (p= . ). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. - polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x . ). at - polymorphism, a significant difference with regard to photosensitivity in male patients (p= . ) was found. in conclusion, results of this study showed that - polymorphism plays an important role in susceptibility to sle and that - polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained patients with ibd ( with cd, with uc, with gluten sensitive enteropathy, gse) and healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd -ve cd b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd (+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling- (rgs- ) protein, we have now found substantial over-expression ( - fold) of rgs- in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs- decreases primary t cell responses to cxcl and ccl , strongly implying that it may regulate t cell localisation. thus, rgs- may be a novel target for modulating t cells in ibd, consistent with which snps in rgs- have been associated with both coeliac disease and type diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c bl/ j mice were divided in control (c) and experimental (e) groups. c -c received peanut protein immunization, animals of the control groups c were sham immunized, and control group c received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a day peanut containing cd, the experimental group was divided into groups (e -e ). ova feeding began days prior cd (e ) on day (e ), (e ), (e ) and (e ) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e ) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase and secretion of bioactive il -b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il -b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying % destrane sulphate sodium (dss) in the drinking water for days to wildtype mice. when ultrafine nanoparticles were administered on day and by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl , n. schneiderhan-marra , m. blum , g. stein , m. schmolz , t. joos nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [ ] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco- cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of -well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h -associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h -like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il- production and t h polarization and decreases recruitment of cd + cd + foxp + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen , , g. van assche , p. rutgeerts , a. liston , j. l. ceuppens k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, k. u. leuven, experimental immunology lab, leuven, belgium, k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp- allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced types of colitis dss (th /th ), tnbs (th ) and oxazolone (th ) in hp ko mice. neutralizing anti-il- mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th /th cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. ) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; ) in dss but not in oxa colitis mice, il- , ifn-g, tgf-b and il- were significantly increased (p x . , dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x . , ko vs wt). in tnbs colitis, we found elevated il- and ifn-g (p x . , tnbs vs control). although not significant, il- was also somewhat upregulated; ) in dss colitis we observed that il- enhanced differentiated th cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il- and il- , more mln-t cells from hpko mice differentiated into th cells; ) anti-il- mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il- showed reduced il- , il- and ifn-g in both mln and lp (p x . , anti-il- vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il- , supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann , g. talabér , g. süt" o , p. németh , t. berki university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that . million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd +cd hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th and th cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd + and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd + inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells ( . ± . %, mean ± sem), while cd + inkt cells ratio was moderate ( . ± . %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: . ± . %; cd: . ± . %, mean ± sem), while the percentage of cd + inkt cells was elevated (uc: . ± . %; cd: . ± . %, mean ± sem) in both disease groups. proportions of foxp + treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b adenovirus (tgf-b -exo). the t cell inhibitory function of tgf-b -exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b -exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b -exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd + foxp + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b -exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b -exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd + foxp + regulatory t cells (treg) revealed that the relative numbers of cd + foxp + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd + foxp + treg. tgf-b -exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd + t-cells was similar in the four vaccinees as observed by ifng, il- production-profiles. however, the killing capacity of expanded cd + t-cells was distinct as observed by the competence to kill ns -peptide presenting transfectants in vitro. as depicted in figure , cd + t-cells cells from both vac (cleared ) and vac (chronic) produced il- and ifng after stimulation with ns -peptide . however, specific killing of the peptide loaded transfectants was only observed in vac , who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure ] killing of ns peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd + t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr -expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns or eda-ns under the control of cmv promoter were prepared. recombinant ns and the fusion protein eda-ns were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns . results: immunization of mice with the plasmids expressing eda-ns , but not ns alone, induced strong t cell responses against the main hla-a restricted cytotoxic t cell determinants from ns . the recombinant eda-ns fusion protein, but not ns , was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp- monocyte cell line. immunization of hhd mice with eda-ns fusion protein induced both cd + and cd + t cell responses against ns and, when immunized with poly(i:c) and anti-cd antibodies, was able to down-regulate the intrahepatic expression of hcv-ns rna. the recombinant eda-ns fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd +cd a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd + responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd + t cells by mature dcs transduced with melan-a-recombinant human coronavirus e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin (il ) and interleukin (il ) will be involved, and fms-like tyrosin kinase ligand (flt l) will be expressed to modulate dendritic cells. il is known to enhance early t cell expansion and limits t cell overshoot, whereaes il guarantees survival of high affinity t cells during memory phase. on the other hand flt l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik , ag waisman uniklinik mainz, . med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il- production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il- produced by b cells by conditional deletion of the il- gene in these cells. we found that b cell secreted il- has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for days in the presence of il- and il- with exposure to overlapping peptide pools of latent ebna- and lytic bzlf- antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd , cd , cd , ccr , cd ra, il- , tnfa, ifng and cd a. the majority of ex vivo ebv-reactive cd + t cells as well as ebna- -reactive cd + t cells were il- and tnfa-producing memory cells, the later being more frequent in bone marrow (cd +, median, ebna- : bm . %;pb . %; bzlf- : bm . %;pb . %, p= . ). after in vitro expansion a major subset of ebv-specific cd + and cd +t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd + and cd + t cells retained, however, a tnfa single, tnfa/il- or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd + and cd + t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr /cd ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il- delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd + and cd + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd + and cd + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd + and cd + t cells in peripheral blood with a hexon protein-spanning pool of synthetic -mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to - % in the t cell lines and the absolute numbers of both hexon-specific cd + and cd + t cells were to log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd + and cd + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype (ad ) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad vaccine vectors expressing whv core protein was first examinated in c bl mice. groups of mice were immunized with a dna prime-ad boost regimen or with dna and ad alone. ad was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd + t cell responses against peptide pools and in spleens of dna and dna-ad immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd + t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad very weak cd + t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad level of antibodies did not change. those antibodies were only igg a subclass what indicates th t helper type of response. ad -immunized mice had over -fold lower level of anti-whc: both igg a and igg subtypes were detected. the weak response induced by ad may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal ) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m e serum ab titers against peptide and native m e. this correlated with a large number of m -specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h n ), which contains a variant m e-sequence different in amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x (h n ) and the highly pathogenic pr/ (h n ) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m e as a potential "universal" influenza vaccine. this research is supported by a nih t fellowship ca - , the nih grant ai and a grant from the commonwealth of pa. l. yu zhejiang university, zhangzhou, china interleukin- (il- ) is a cytokine produced by stimulated mononuclear macrophage system. in this report, -day-old chicken embryos were vaccined with the plasmid dna (pci-chil- ) encoding chicken interleukin- and the copy numbers of chil- in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil- in -day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil- could accelerate high concentrations of chil- in nonage peripheral blood, accelerate high expression of chil- in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil- could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil- (p x . ). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on volunteers and a phase i study on volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv- neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) and the transmembrane protein gp . two sites on gp and gp are attractive targets for vaccine design: the epitope in the third hypervariable region (v ) is recognized by the human monoclonal antibody - d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies e and f . in order to elicit anti-hiv- neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp -v region or the gp -mper. the vlps are based on the acyltransferase component (e chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e chain self-assembles into a nm protein scaffold resembling a vlp and that contains copies of e . efficient display and refolding of the v and mper regions in e vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the ' of the gene coding for e . the priming and boosting with a combination of vlps and specific hiv- envelope dna were used to immunize mice and rabbits. results: the v -e and mper-e vlps were purified as stable mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of - d, e and f antibodies to hiv-e monomers was confirmed by western blot. we obtained high titers of hiv- gp -specific antibodies in mice immunized with a combination of vlps plus dna (hiv- sf gp ). these antibodies generated a low ( %- %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e vlps plus dna elicited a low titer of hiv- gp specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e vlps are able to display antigenic determinants of hiv and to induce high titers of hiv- -specific antibodies. the e vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h n whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h n influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [ ] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h n whole virus wild-type vaccine, could induce an immune response and protect mice against h n influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h n antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h n virus. the protective efficacy of the trivalent vaccine derived mainly from the h n component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h n virus, suggesting that antibodies are the main contributor to protection. h n specific serum did not inhibit neuraminidase activity of h n virus suggesting that protection was not mediated by neuraminidase n -specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h n whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h n vaccine enhanced anti-h n antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h n vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h n candidate vaccine. [ ] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of children in the first grade of primary school in the area of central and west macedonia. there were greek and foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following ) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to %. ) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). ) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. ) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om- , a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om- . cytokine secretion, activation and proliferation of b cells and foxp + tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr , tlr or the myd adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om- . two synthetic tlr agonists used as adjuvant in human (om- -dp and om- -mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om- -induced protection of diabetes required natural tregs, as nod-cd -/mice were not protected. remarkably, om- activated b cells and not t cells, promoting their proliferation and il- secretion, two phenomena that were tlr -and myd -dependent. om- -dp and om- -mp-ac two synthetic murine tlr agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd + cd + foxp + t cells and the proliferation of il- secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr signaling in the protective effect of om- on development of diabetes and show that two other tlr agonists induce proliferation of b cells and their secretion of il- as well as stimulation of regulatory cd + cd + foxp + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit , a. legat , s. thomas , m. van mechelen , p. hermand , m. goldman institute for medical immunology/université libre de bruxelles, gosselies, belgium, glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide -phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor (tlr ). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd (scd ) for its tlr ligand activity, we investigated the involvement of both forms of cd in the responses elicited by crx- , a prototypical agp. first, we found that crx- efficiently induces nf-kb and irf activation in hek cells transfected with tlr and md- genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd . likewise, crx- efficiently induces the synthesis of nf-kb and irf- dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd respond to crx- but not to lps in serum-free medium. the addition of the soluble form of cd did not modify the levels of il and tnf produced by crx- stimulated dc but increased the levels of interferon-b (ifn-b). when scd was added to hek cells expressing tlr /md- , nf-kb activity was not modified but irf activity was increased in a dose-dependent manner in response to crx- . we will further compare the responses induced by crx- in wild-type and cd deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h- d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled and days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin- gene polymorph isms (c t, g a, c a and a t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [ ] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n= ) have higher amount of both nonactivated (subtype infg+/cd -, p= . ) and activated (subtype infg+/cd +, p= . ) cbmc, producing ifn-g, as compared with newborns from urban mothers (n= ) exposure to pets and the risk of allergic symptoms during the first years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc / to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity ( - %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: months - years (n= ), - years (n= ) and - (n= ) tb has the highest diagnostic value in children n years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp- antigen is more sensitive for detection of tb-specific t cells compared to esat- antigen. . in children with tst - mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr and tlr in the development of spontaneous lupus disease by creating tlr or tlr deficient c bl/ lpr/lpr mice. methods: tlr and tlr deficient lupus prone mice have been generated by crossing c /bl -tlr -/-or c /bl -tlr -/-mice with c /bl lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr or tlr deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr or tlr in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr deficient mice suggesting an important role of tlr in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr pc / expression of full length mcl- and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl- , an anti-apoptotic protein. a splice variant of mcl- arises by removal of exon and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl- (mcl- l) and its splice variant (mcl- s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl- . method: neutrophils were isolated from children (diagnosed x years) with jsle (n= ) and non-inflammatory conditions (control, n= ) and incubated with control serum, jsle serum alone or with jsle serum plus pg/ml gm-csf. quantitative real time pcr was used to assess mcl- l and mcl- s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl- s to mcl- l was also higher in jsle patients compared to controls (p x . ). the addition of gm-csf to jsle serum was associated with an increase in mcl- l ( . ± . ) and a decrease in mcl- s ( . ± . ) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl- compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl- s, and less anti-apoptotic full length mcl- cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex / mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd + cyld ex / t cells were transferred into rag -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex / cd + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex / mice are capable to control inflammatory responses. for this purpose cd + cd + cells were co-transferred with naïve wt cd + t cells into rag -/-recipients. interestingly, rag -/-recipients of cyld ex / t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc / the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a* in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb * , * and * alleles, and a decreased allele frequency of drb * in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb * is associated with the development of severe disease and positive association of cd with drb * and drb * . indeed, drb * was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb * frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb * in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb * were strongly increased with respect to cd and controls. however, the frequency of drb * was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb * is associated with uc in european, north american, japanese and korean populations methods: a total of children were studied, ( boys and girls), up to years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, positive cases of children were found ( %) with active infection : boys ( x years of age, - years of age and g years of age) and girls ( x years of age, - years of age and g years of age). pharyngitis was present in children ( , %), had fever ( , %) and had lymphadenitis ( %). the lab tests revealed leukocytosis up to . leukocytes in cases ( , %) and leukocytosis g . in cases ( , %). the most frequent complication documented was streptococcal superinfection in children ( , %) and thrombocytopenia in children ( , %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and ) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il- ra , previously believed to be a decoy for il- only, is able to transmit a signal via il- . our results support this and may suggest that il- / il- ra signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il- ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il- or il- ra and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd + cd -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il- and il- b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd + cd -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi- , l. acidophilus ncfm tm and b. bifidum bi- ) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi- strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il- or il- b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed pediatric cd patients ( active, remission), pediatric uc patients ( active, remission), and age-matched non-ibd controls. nkg d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x . was considered significant. results: nkg d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd +cd + nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg d ligands on circulating monocytes is also observed. the dramatic increase of nkg d+ lymphocytes, and the strong upregulation of nkg d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc / peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp + t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd + t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec- antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa - ) or hcv-ns (aa - ) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns and core we successfully optimized culture and protein purification conditions. briefly, ns was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec- to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec- / hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd / mva-nef vaccination induces polyfunctional cd t-cells and increases the proliferative capacity of cd t-cells in hiv- infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv- protein nef (mva-nef) was safe in hiv- infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il- and mip- b, the expression of the activation marker cd and the differentiation marker cd ra in nef-specific cd and cd t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il- and mip- b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd t-cell response and a significant increase of polyfunctional nef-specific cd t-cells, simultaneously expressing ifn-g, il- and cd . using the standard ics no increase of nef-specific cd t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il- and cd expressing cd t-cells and the increase of proliferating cd t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd t-cells in hiv- infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv- mediated membrane fusion and p -detected replication. db was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd + and cd + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns ( - aa) and rns a ( - aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/ j mice were immunized intraperitoneally times at a month interval with different doses ( . - mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns protein. immunization with rns a-immunomax conjugate and rns in cfa ( . mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd / degree of cross-genotype reactivity of hcv-specific cd t cells directed against ns the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd t cell response targeting hcv genotype (gt ) and genotype (gt ) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: subjects ( with gt , with gt and anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns from gt or gt . individual reactive peptides and the degree of cross-reactivity between the gt and gt variants were determined by ics. complete ns is sequenced from all viremic patients pd / anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes , , or suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes , or , but not led to a significant reduction in viral loads. decreased splenic viral load after ifna treatment correlated with an expansion of activated fv-specific cd + t cells and nk cells in the spleen, whereas in ifna -and ifna -treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna , and are under investigation pd / elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a* -or k b -restricted cd t cell responses by these dna vaccines differed. k b /ova - -and k b /s - -specific cd t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd + and cd + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least healthy, randomly chosen blood donors were cultured for days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. out of tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il- -exposed cd + t cells showed at least five-fold increase of survival rate in vivo than il- -exposed cd + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il- -exposed cd + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il- and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x . ) . there was a significant elevation of t ada and ada activities in iga deficient patients as compared to healthy individuals (p x . ) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd / hiv- sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after - weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in out of patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, -day-old ross broiler chickens divided randomly into groups, experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/ strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/ and h strains, via the drinking water at days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, & days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd t cells from patients with active tb and patients with successfully treated tb were analysed for simultaneous expression of ifng and il at the single cell level using multi-colour flow-cytometry after hours stimulation with ppd. moreover, cells were stimulated with esat- and cfp- receiver operator characteristics analysis revealed that a percentage of ifng /il dual positive cells x % served as an accurate marker for active tb patients (specificity %, sensitivity %), while frequencies g % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd / necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately inhabitants) is . to . among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, out of children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently . to inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of to led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd / novel analogues of thalidomide inhibit cd expression and production of tnf-a, il- , ifn-g, cxcl- this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta -dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta r k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd and cd . these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il- a and il- b, while il- levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il- b production. using bmdc from nlrp -/-mice we examined il- b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il- b production is dependent on nlrp . collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho- expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from % in wild type (hmox +/+ ) mice to % in ho deficient (hmox -/-) mice. hmox -/-but not hmox +/+ mice developed end-stage multiorgan failure. mortality of hmox -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h o or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb . similarly, circulating and cytoplasmic hmgb was increased in hmox -/-relative to hmox +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b- , -linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at - years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and - weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were . and . iu/ml and those of dtwp-pasteur were . and . iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were . eu/ml for dtwp-local and . eu/ml for dtwp-pasteur vaccines, respectively (p x . ). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd / united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b: , a gram-negative coccobacillus. jrmt , an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of cfu of jrmt . a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b: on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for h before adding concanavalin-a (cona) pd -vaccination and immunotherapy against parasitic diseases pd / evaluation of simian adenoviral vector adch expressing msp- as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype (adhu ) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp- and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho- expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho- is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp expression was confirmed by sds-page and elisa using monoclonal antibody against gp . discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a - recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a - recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at - °c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r= . , p= . ) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il- also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il- on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il- to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il- and il- -related inhibition of cfu-e growth were dependent on p mapk activity, since the p mapk inhibitor, sb , markedly downregulated il- -induced activation of nos and reversed the growth inhibitory effects of il- on cfu-e. the in vivo stimulating effect of il- on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il- effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il- acts on bone marrow cells and also revealed a link between the il- , no and hematopoiesis. further studies on il- -mediated induction of both inos and enos methods: a total of blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of samples ( , %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of iu/l anti-hbs in all the blood units, which also goes for our study pd / neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen , national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa- a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa- a iggs we monitored children ( boys and girls) in ages from . to . years with average age of . years. in of them all was diagnosed for the first time. subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest ( subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc h gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. , nature , - ). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 'utr of icos, in which a bp sequence, containing a possible mir- binding site, was sufficient (yu et al. , nature, , - ). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. , j biol chem , - ). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia- -and ago -deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r ai . we have recently reported that -hydroxyl- -methylindole- -acetonitrile ( -hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw . macrophages. in this report, we investigated the effect of -hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin- (il- ), monocyte chemotactic protein- (mcp- ) in ht- human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed -hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated -hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. -hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p . in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of -hma significantly reduced the mrna levels of il- and mcp- stimulated by tumor necrosis factor-a (tnf-a) in the ht- cells. pretreatment of -hma also significantly blocked the ixba degradation and nf-xb p nuclear translocation stimulated by tnf-a in the ht- cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of -hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, -hma could be new potential therapeutic agent for inflammatory bowel disease.cd serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd igg immune response in hiv- -exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp -binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp , reacted with the external domains of soluble human cd , in the absence of the target cells expressing the co-receptor ccr . the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd -gp complex and trigger the membrane fusion between effector (gp expressing) cells and target (ccr expressing) cells. thus, in the absence of ccr we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv- infections. a conventional protocol for the generation of monoclonal antibodies was used. db- (igg , x), one of the anti-cd antibodies obtained, recognized preferentially cd complexed to gp , as compared to cd alone, not competed for the gp binding site on cd and was specific for the second extracellular domain of cd . g. röder , l. geironson , a. darabi , m. harndahl , c. schafer-nielsen , k. skjödt , s. buus , k. paulsson copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, lund university, immunology bmc d , lund, sweden, lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, schafer-n, copenhagen, denmark, department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a* . to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn - / , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn / was demonstrated to be located on tapasin [ ] [ ] [ ] [ ] [ ] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd + t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region - of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b* -restricted np-flu epitope (aa - ) was replaced by hla-b or hla-a -restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard cr release assay and through the ifn-g production. results: using hla-b or hla-a restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b as a remarkable hla-class i molecule that, differently from hla-a , can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv- )-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv . consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p -gag and p region was determined in vitro. mer peptides were digested with i s and c proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv- p -and p -gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers , g. koster , o. landsverk , f. skjeldal , o. bakke university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi kinases and thus ptdins( )p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier , l. visvabharathy , j. fu university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e - k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e - k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type e - k and class i molecules.results: we showed that e - k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a molecules, with the mature form being more tightly associated. we also provided evidence that e - k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e - k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e - k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il- , il- , tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands and , but also with the t-celldependent stimulus cd l (cd ) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd , cd , and cd was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p and erk- / . strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd + t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th (il- +) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th / th balance and to identify potential t cell target antigens (ags), we analyzed circulating cd + t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il- expression in peripheral blood cd +cd + lymphocytes were measured in ccs patients and healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il- /ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il- and lower ifn-g intracellular expression were detected in cd + t cells from css patients, as compared to hs (il- : . ± . % vs. . ± . %, p x . ; ifn-g: . ± . % vs. . ± . %, p x . ). similar results were obtained by elisa (il- : . ± . vs. . ± . pg/ml, p x . ; ifn-g: . ± . vs. . ± . pg/ml, p x . ). elispot counts of hexavalent vaccine-stimulated cd + cells were positive for il- in / ( %) css patients and in / ( %) hs, and for ifn-g in / ( %) css patients and / ( %) hs. mpo stimulation determined significant ifn-g release in / ( %) css patients, but not in hs ( / ) no il- response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd + cells from css patients show a th -polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd + t cells, and responses towards it are instead th -polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd + t cells in css. a. voigt , e. opitz , k. savvatis , k. klingel , k. stangl , u. kuckelkorn , p.-m. kloetzel charité -universitätsmedizin berlin campus mitte, berlin, germany, charite -campus benjamin franklin, berlin, germany, universität tübingen, tübingen, germanymurine models of coxsackievirus b (cvb )-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp of the stress-induced ip, were infected with x e pfu cvb nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp as early as day p. i. in lmp -deficient mice. however, lmp -deficiency was linked to less severe myocarditis at day p. i. (he stain of cardiac tissue sections: wt . ± . vs. lmp -deficiency . ± . (grade of myocarditis; scale - ; p x . ). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x . ), there was no difference in lmp -deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb infection in lmp -deficient mice. likewise, diastolic function was preserved in lmp -deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp -deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp -immunosubunit function in regulatory processes of viral replication. absence of lmp confers host protection in enterovirus myocarditis. h. w. liao , j. xu , j.q. huang sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype (den- ) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr and tnfr - were constantly very low whereas trailr - decreased after den- infection. fasl was expressed at similar levels on huvecs throughout den- infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase , caspase were also observed by western blot after by den- infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for days into wis rat paw web tissue in saline containing . x cells. group ii. s.epidermis was injected as in group after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day .they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors , and , respectively (p x . ). immunohistochemical pictures showed increase in percentage of ox (migrating dendritic cell), mhc ii, his (granulocytes), ox (stem cells) and cd (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed (macrophages) and ox cells. popliteal lymph nodes contained evidently less of ox , his and mhcii cells than in group i (p x . ). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega- fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega- fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega- fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega - fatty acids consumption. the results indicate that omega- fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega- fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega- fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density . and , g, top % layer contained lipoproteins only and - % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin- . objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e f mutation, and other affecting b cell apoptosis control, such as bcl- over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c bl/ (b ) genetic background. e f -/-bcl- tg were obtained backcrossing e f -/-and e f +/+hbcl- tg mice. e f -/-bcl- tg, e f -/-, e f +/+hbcl- tg and control mice (e f +/+) were followed up to mo-old. serologic studies: serum samples obtained at , and month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of , and mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h ]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl- tg in b lymphocytes of e f -/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e f -/-bcl- tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e f -/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e f or the overexpression of a bcl- tg in the b genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor (tlr ) abolishes the generation of antinucleosome igg a and igg b autoantibodies but exacerbates lupus disease. however, the tlr -dependent tolerance mechanism is unknown. here we show that loss of tlr in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th and th t cells. transfer of a synthesized monoclonal polyreactive igm to tlr deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr -dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n= ) (vitali et al. ) . ninety-seven of ( %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g (fs+), while biopsies from / ( %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in / ( %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x . ). interleukin (il) , il- ra, il- beta, il- p , il- , macrophage inflammatory protein (mip) alpha, mip- beta, eotaxin, interferon (ifn) alpha, and il- levels were significantly increased in the gc+ patients (n= ) compared with the gc-patients (n= ). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il- found in these patients (gao et al. ) . increased titers of th -associated cytokines, il- , il- beta and the il- subunit il- p , may indicate a higher activity of these cells in gc+ patients (nguyen et al. ) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il- . the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg . production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd -dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd -dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/- baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il- . objectives: increased levels of il- , an innate and inflammatory cytokine of the il- family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il- and other genes of the il- /il- r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from patients and healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il- and il- bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il- are higher than in controls. serum il- correlates with disease activity and decreases upon remission. monocyte expression of the receptor il- rb is increased and correlates with disease severity, while expression of tir /sigirr (a down-regulatory receptor of the il- r/il- r family) is reduced. in mrl lpr/lpr mice, expression of il- , caspase- and il- rb genes precedes disease onset in lymph-nodes. in other organs, changes in il- -related genes (il- and tigirr- up-regulation, tir /sigirr down-regulation) occur after disease onset. free il- levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il- levels correlate with autoimmune lupus both in mice and humans. free il- may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il- rb is a marker of pathology, suggesting increased il- -dependent activation. both in mouse organs and human monocytes, tir /sigirr expression decreases with disease, suggesting impaired control of il- r activation. thus, il- may be involved in autoimmune lupus pathology, and il- -related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f (amino acid (aa) - ) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f was recognized by all patients. fragment f (aa - ) was recognized by of dcssc patients. fragment f (aa - ) was recognized by of sle patients. analysis of clinical data revealed a significant difference between the f negative and f positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f and f could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril- . by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd + t cells enriched in tregs were co-cultured with activated cd + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd + t cells anergy was also evaluated based on cbl-b, grail and foxp mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b- cells through targeting p d by shrnas strategy. methods: we used the drugs, ly and wortmannin, pan-specific inhibitors against pi ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p d. we then introduced either pan-specific inhibitors against all pi ks or p d-targeting shrnas into an sle-prone animal model, nzb/w f mice, for therapeutic purposes. the results suggested that pi ks are not only important for the development of b- cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p d. either pan-specific inhibitors against pi ks or p d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f mice. one inhibitor, ly , and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi ks are critical for the maintenance of b- cell populations might shed light on future treating other diseases associated with b- cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool , m. alarcón-riquelme , s. kozyrev institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs , r h). we identified that bank gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon (delta ). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs ), which is in complete ld with r h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia , which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a -year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti- gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow cnrs , strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed patients, with minor clinical and/or biological manifestations of their disease, for at least monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below . b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd surface protein for all patients. this cd lower expression is associated with cd lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf lacking an exon in the c -domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf in t-cells. smurf expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf expression was upregulated by tgf-beta stimulation in t-cells and smurf was markedly upregulated in tumor infiltrating cd + lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day , smurf transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf showed an upregulation of the tgfbrii and an activation of smad and as compared to wild-type t-lymphocytes, which were previously described as typical smurf targets for degradation. in addition the transfection of smurf and a caga-luc plasmid into cos-cells for smad -promotor studies yielded the same effect as shown by upregulation of the smad activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf has beneficial effects on mucosal inflammation and tumor development. smurf thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat has important functions in cytokine signalling in a variety of tissues. however, the role of stat in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat activation in dss colitis is dependent on il- rather than il- . il- was secreted by colonic cd c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat activity were highly susceptible to experimental colitis, indicating that epithelial stat regulates gut homeostasis. stat iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il- and epithelial stat was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat activation regulates immune homeostasis in the gut by promoting il- -dependent mucosal wound healing. stat seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd and tlr expression at day and day after infection. mycobacterial infection did not result in differential tlr expression as compared to uninfected cells. cd is involved in stimulating th pro-inflammatory responses, although map may interfere with cd signalling ( ) . tlr signalling elicits anti-inflammatory responses, which can contribute to bacterial replication ( ) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd and tlr receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd t cells by exogenous antigen leads to colitis. methods: eight million naïve cd + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h -kb, were transferred i. v. into b mice. at day and , mice were treated intra-rectally (i. r.) with % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day after the injection of ot-i cells. the phenotype of effector cells was evaluated at day by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd , the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd + t cells. our study demonstrates that the local activation of antigen-specific cd t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of patients, ( male and female), with age average , years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x and t-test programmes. results: in patients ( , %,with age average , years of age) no antibodies were detected. on the contrary, in the remaining , male and female, ( , %, with age average , years of age), antibodies were detected. out of them, in cases the results were strong positive ( male and female) and weak positive in cases ( male and female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: ) helicobacter pylori infection is relatively common in the general population ( , %). ) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women). ) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova , t. ulmannova , k. stechova , k. tesarova-flajsmanova , v. stavikova , j. nevoral charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of mothers whose infants were diagnosed with allergic colitis and mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis ( - weeks, average . weeks of infant's age) or at the age of weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il- , il- , il- , il- , il- , ifn-gamma, tgf-beta , egf and eotaxin. man-whitney u test was used for statistical analysis, p x . was considered statistically significant.results: il- as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x . ) in breast milk of mothers whose infants were suffering from allergic colitis (range - . pg/ml, mean . pg/ml) than in control group (range - . pg/ml, mean . pg/ml). higher concentrations of il- and lower concentration of tgf-beta were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. - . background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il- (murine homologs kc and mip- ) and its receptor cxcr are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at , , and h post-transfer. anti-kc antibody or its isotype control was administered at mg/mouse one hour before transfer followed by whole body and organ imaging hours post-transfer. results: facs analysis revealed % neutrophil purity, % of which were cxcr + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model ( . % dss in drinking water for week, followed by pure drinking water for week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell ), treatment of commensal-depleted mice with the tlr ligand lps in drinking water protected against the lethality of % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of . % they may become pathogenic and drive an intestinal inflammation. at % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by . % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il- production which is an important pathological factor. neutralizing il- or il- prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th cytokines in colitis remain undefined, we used mice deficient in il- /il- or the key receptor through which they signal, il- ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il- ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il- /il- double deficient mice which were protected from colitis. removing il- production from il- ra -/mice, by using il- ra/il- double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il- mediates susceptibility in an il- ra independent manor. recent evidence pc / introduction: the activation of cd + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th -like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th cytokines, such as il- ,- and il- . however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc was found in uc and cd colonic tissue compared to control specimen. transmitted to the th -mediated oxazolone-induced colitis model, nfatc -production is significantly increased in both diseases, too. nfatc deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc deficient mice compared to control mice, which can be observed by tunel assays, caspase and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl- and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il- , ifn-gamma, il- and il- by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc regulates il- /il- in an indirect way. last, administration of il- blocked the protective effects of the nfatc deficiency in experimental colitis, suggesting that nfatc through il- signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc in colitis by controlling mucosal t cell activation in an il- dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd cd rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd cd rb high t cells also regulatory cd cd rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at different time points during colitis progression. after weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at weeks. microrna was isolated from colons of mice in different stages of colitis progression ( , and weeks) and control mice that do not induce colitis (n= for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from micrornas that demonstrated an induction during the development of disease we selected micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd cd rb high transfer model. objective: the purpose of this clinical trial (id: nct of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in groups: group a (n= ) receiving haart+ gh (for months) + tt+hva/b vaccines (at month post gh adminsitration); group b (n= ) receiving haart+gh but not vaccines; and group c (hiv control group, n= ) with haart+vaccines (at month ) but without gh. all patients are followed up months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after weeks of administaring hormone the absolute numbers of cd incresase from ± to ± cells per mm (mean and sem; p x . ). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd absolute numbers from ± to ± cells per mm (p x . ). viral load remained undetectable in all patients. despite the increase in cd counts the percentage of recent thymic emigrants (as assessed by the expression of cd ) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv ( , - , ), hepatitis c virus (hcv; . - . million) and hepatitis b virus (hbv; - million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv- infected patients with g cd + t-cell counts and plasma hiv-rna levels of x . log copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g and when the responses were g times the standard deviation above the mean of replicate negative controls (mock electroporated dc). / patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in / patients before and / patients after vaccination. for rev, / patients showed a pre-existing rev specific response and / patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already / patients responding before and / patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in out of patients before vaccination and / patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus (ev ) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev inactivated virus. methods: mice were immunized intraperitoneally with mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after , , and weeks. blood samples were collected at , , , and days. the total serum anti-ev igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il- from splenocytes was also measured. results: immunization with ev /ps-g showed that the anti-ev igg levels were significantly increased compared with ev alone or ev /cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev /ps-g group compared to ev alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev /ps-g-immunized mice compared to those of ev or ev /cfa/ifa-immunized mice, indicating a th cells response elicited by heat-inactivated ev vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd + t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd + t-cell responses against hcv-ns , studying induction of il- , ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il- and ifng production by cd + t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv- -recognizing cd + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv- replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd + t cells by electroporation, and chose tcrs which were able to recognize the hla-a restricted hivpol-peptide iv , or the hivgag-peptide sl . results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il- , tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd , after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt l pretreated balb/c mice were incubated for hrs with hcv ns -coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g % dcs and was used for immunization. dc expression of the maturation markers cd , cd and cd was determined before and after ingestion of ns -coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns expressing sp / cells). results: in immunized animals, the ctl activity was increased -fold compared to mock immunized mice. accordingly, tumor challenge with ns expressing tumor cells showed a significant reduction in tumor growth. the number of cd + ifn-g + cells was increased g -fold and the number of cd + il- + increased g fold in the dc-ns -bead immunization group. these results paralleled the proliferative response of splenocytes to ns protein obtained from immunized animals with the most significant response in the cd + population of dc-ns -bead immunized animals. the use of ns coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec- antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec- induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec- promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec- antibody can be developed. we screened the anti-dec- sequence computationally for putative hla dr -restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec- antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec- as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih r ai a live oral vaccine based on human adenovirus (ad) has proved safe and effective in us military recruits for nearly years. in these experiments, we have investigated whether replication-deficient ad can be an efficient potential vaccine carrier for oral vaccination. ad vectors were used throughout to provide a benchmark for efficacy. we generated novel ad and ad vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad than ad . ad routinely infected and provided transgene expression in˚ % of human cd + and cd + t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to - % of cd + t cells present, showing that ad infected a surprisingly large proportion of t cells. in comparison, ad provided egfp expression in x % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad and ad vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad and ad induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day after dosing, and transgene expression being reduced below detectable levels by day . interestingly, when delivered together ad and ad vectors targeted the same subset of cells. together, these data show that ad is a viable alternative to ad -based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies ( recently commercialized; g in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that -treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, ; gros et al, ; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp ) of rhdv at two different locations: ) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp- ), and ) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp- ). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp- ) was more immunogenic than insertion at position for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp- at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr- ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd + t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd + t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np - specific cd + t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr- targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd +, cd +, cd +, cd + t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by , - -fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns and ns a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h n influenza virus inhibits the ifn-g synthesis (mibayashi et al., ) and causes a decrease in cd + and cd + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., ) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions - (tat'kov c. i. et al., ) . deltaferon was i. p. administered to male non-inbred mice in doses of - * iu once or twice at an interval of hours, alone or in combination with double-stranded yeast rna preparation ( mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., ) . results: when deltaferon was administered in doses of - * iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of * iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon ( * iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd responses (tetramer+ hiv- gag p cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd responses after pla-p immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas , c. prego , s. vicente , m.j. alonso , a. gonzález-fernández university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer , with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with or immunizations to balb/c female mice ( weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day to post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease ( miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigen- background: infection with human immunodeficiency virus type (hiv- ) is characterized by dysfunction of hiv- -specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p antigen from hiv- and the tlr ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p microcapsules containing p and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p peptides. intracellular staining of interferon-gamma (ifn-g), interleukin- (il- ) and cd a after p stimulation was also performed. results: mddc from hiv(-) subjects, incubated for hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il- , upon p peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd b between our model cytokine il- and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il- dependent proliferation assays of il- variants. results: murine il- attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il- dependent cell line ht- . we found that the membrane anchors comprising one and four ig-like domains (il- :: iggpi and il- :: iggpi) resulted in severely reduced vlp production by producer cells and despite of an increased targeting of il- :: iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il- fused to the minimal gpi-anchor acceptor sequence of hcd b (il- ::gpi). il- :: iggpi, however, showed comparable particle production and biological activity in vitro when compared to il- ::gpi. furthermore, il- fused to ig::gpi showed an increased capacity to co-stimulate primary p tcr transgenic t-cells specific for lcmv-gp - in the context of h -d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il- ::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f -b of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild , interdisciplinary transplant laboratory charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects - % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of - months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from , month to days. t cell lines are composed of cd and cd cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker , m. assenmacher , a. richter miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp -specific cd + and cd + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd ro -cd -) cd + and cd + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp peptide pool and cd -depleted autologous pbmc as feeder cells in the presence of il- , il- , and il- . already - days after primary activation pp - /a -tetramer + cd + t cells were detectable for hla-a + blood donors. to analyze cd + t cells having other specificities than for the peptide pp - as well as probably primed cd + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to . % of the cd + t cells and up to . % of the cd + t cells after restimulation with pp peptide pool, but not with either irrelevant ie- peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for days led to a - fold expansion of pp -specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd and cd only if restimulated with the pp peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il- , indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd + and cd + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf , k. mozdzanowska , l. otvos , j. erikson the wistar institute, philadelphia, united states, temple university, philadelphia, united statesthe influenza virus a matrix protein ectodomain (m e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine ; virology journal ), we generated a novel peptide and investigated its efficacy in inducing an anti-m e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd + t cells, have been considered. clinical data confirm a crucial role for antiviral cd + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a* and hla-b* previously. considering other frequent hla alleles cmv specific cd + t cells were monitored longitudinally in hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a* /pp ( - ) ( . x cells/l) and hla-b* /pp ( - ) ( . x cells/l) specific cd + t cells are significantly lower than those for hla-a* /pp ( - ) ( . x cells/l) and hla-a* /pp ( - ) ( . x cells/l) specific cd + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. ( , - mcg) . no adverse effects were indicated during trials (up to month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after th immunization with mcg of vichrepol.no differences were detected in cd + and cd + t cell counts and cd +/cd + ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp or gp alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb f hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb f mice with adenoviral nanoparticles expressing fusion proteins containing gp resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted - % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf did not show any increase in their levels of ifn-gamma. conclusion: caf enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from children aged - years old ( boys and girls) -group , and children ( boys and girls) - years old -group were isolated on a gradient of density before vaccination ( or revaccination) with priorix, week, and months after and incubated with cfse. then million/ ml pbl were incubated in rpmi- supplemented with % fcs (the negative control), at presence of mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing % cÎ at °c within day. intensity of a fluorescence estimated on fl by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control % pbl in both groups did not enter mitosis. in the positive control % of cells have passed one and more mitoses. in group measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in months - % of lymphocytes demonstrated antigen-specific proliferation. in group , on the contrary, before the vaccination the most part of cells ( - %) has not entered division, but - % of cells have passed and more mitoses. in a week specific lymphocyte proliferation decreased and in months it was increased up to - %. production of the interleukin (il) , ifn-g, tnf-a, il- , il- was more informative than il- , il- , il- , il- . measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp cd -binding site (cd bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv- neutralizing polyclonal iggs directed against the cd bs. the mabs were shown to react with an anti-cd bs human neutralizing mab (b ), to elicit antibodies that recognize the gp molecule and an anti-hiv- neutralizing response in rabbits, confirming them as cd bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p and p ) reacted in elisa only with the cd bs-directed igg fraction. the clones were also recognized in elisa by b . p and p -immunized rabbit sera showed a strong anti-gp titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb strain glycoproteins. in particular, / rabbits in the p group and / in the p group showed an % hiv neutralization at dilutions ranging from : to : . the immunoenzymatic assay used, allowed to detect a p and p reactivity in hiv-positive sera and was able to detect a b concentration equal to ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines ( d and dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a -year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following d- vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd , cd and cd ) along with increased levels of nkt-cells (cd + cd +/-cd +/-/cd + ratio) and activated t-cells (cd + and cd + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il- , il- , il- , il- and il- ) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il- + ), cd + t-cells (il- + and il- + ) and b-lymphocytes (tnf-a + , il- + and il- + ). the analysis of cd + t-cells revealed a complex profile with increased frequency of il- + and ifn-g + and decreased percentage of tnf-a + , il- + and il- + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester , h.-g. rammensee , s. stevanović eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd t cell epitopes for the frequent mhc class i alleles a* , a* , and a* from the proteins pii (hexon), pviii, and e a of adv strains ad and ad by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a -day recall stimulation of pbmcs from at least healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd t cells. we could identify new peptides eliciting ifn-g responses, several of which were confirmed as novel cd t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle ( ara criteria) was diagnosed in a -year-old african female patient with hiv- (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd t-cell count x cells/mm . initiation of mg mmf bid was associated with biological and clinical remission of sle and cd t-cell increase. no opportunistic infections or cancers were noted during a -year follow-up and the patient remained always naive to art. hiv- -specific cd and cd t-cell responses were analyzed after months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il- production following stimulation with a panel of hiv- -derived optimal epitopes ( / -mers) covering various hiv regions and a pool of hiv- -derived peptides ( -mers overlapping by aminoacids) encompassing the entire gag protein. all peptides are derived from hiv- consensus strain iiib. results: highly polyfunctional hiv- specific cd and cd t-cell responses against gag were detected. epitope-specific cd t-cell responses were identified: except for one response restricted by hla a* and another one by hla cw* , all the others were restricted by hla-b alleles and mostly by b* (n = ). seven out of responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il- +) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv- specific cd and cd t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv- t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of dl /ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf- of - g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt- , cofaa and cgpi- . . state of vaccine-induced measles immunity was determined by means of elisa in - , - and - years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in - years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p . ) measles immunity and antibody level much lower (p p . ) than among children with mt conversion. in - years the comparison group kept decreased (p p . ) measles immunity, the majority ( ± . %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p . ) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p . ), the majority ( . ± . %) of persons lost protected antibody level; among children with mt conversion in - years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p . ) to low values; among children with hyperergic mt igg level decreased (p p . ) and reached low (in - years), minimal protected (in - years) and lower than protected (in - years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il- producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein (dnahsp ). methods: balb/c female mice were infected by intra-tracheal route with h rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp genetic vaccine was done at days , , and post-infection. each dose consisted of micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd +, cd + and gamma-delta t cells from lungs were determined and days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x . were considered significant (t test). results: at day after the end of the immunotherapy, dnahsp treated mice exhibit increased numbers of absolute cd + and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il- producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp treated mice showed more ifn-gamma producing cells in both cd + and cd + cell populations. at day after the end of the therapy, the main observation in mice which received dnahsp treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd + and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd + cells, together with a more frequency of gamma-delta t cells producing il- . finally, the immunotherapeutic effects of dnahsp vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd + cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il- , are the main effects of dnahsp immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il- and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of - log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput ( -well format), requires only ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to % in pb, % in rx and % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd l, a co-stimulatory molecule preferentially expressed on activated cd + t cells, is the ligand of cd . cd -cd l interaction induces the production of il- and the initiation of a th -type immune response. several studies show that cd l is required for the activation of macrophages and the maturation of dcs. moreover, cd l enhances the capacity of cd + t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd l. we have constructed the recombinant bcg strain expressing cd l (rbcg ) by electroporation of bcg with pgfm /signalsequenceag b-cd lec and an another recombinant strain with empty vector pgfm (rbcg ) as a control. the expression of cd l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd l weeks after vaccination but not at and weeks. rbcg seems to be more protective against paratuberculosis than rbcg , but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il- a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x . ) of tnfa, ifng and il- a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x . ) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd mab and ag a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd binding on cd transfected l fibroblasts. the conjugates were tested in vivo in wild type and cd + cell-depleted mice for the induction of specific anti-ag a serum antibodies. splenocytes were challenged ex vivo with ag a and were examined for their ability to produce th -related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il- . we developed a method to successfully crosslink anti-cd mab to ag a. serum antibodies against ag a were detected after immunisation with this conjugate vaccine in both wild type and cd + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag a alone. production of two other th -related cytokines, tnfa and il , was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer , r. burger robert koch-institute, cellular immunology, berlin, germany, robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd -positive and cd -positive t-lymphocytes. cd -positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd + t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd + t-lymphocytes in vivo. similarly, the cd -positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd + and the cd + subpopulation depended on the presence of apc. stimulation oft cd + cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd -positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target (esat- ) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, iranian and afghan adults ( patients with sputum smear and culture positive tuberculosis, recovered patients during months after full course of chemical treatment and healthy individuals) were recruited to quantify the frequency of esat- and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of spot forming unit ( g spots per million), we found detectable response to esat- in almost % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and % of these individuals had detectable esat- specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in %, % and % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th immune response, against esat- , in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal , p.k. upadhyay national institute of immunology, pdc- , delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c bl/ mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: . % alginate, . % pva concentration gives particles with size of - micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with % trehelose and . % pvp (poly vinyl pyrollidone). there was fold increase in proliferation index of spleenocytes and releases pico-gram of interferon gamma after week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by % increase in cd and . % in ccr expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b s. by cell cycle analysis we determined that b s is able to induce apoptosis in up to % of jurkat and molt- t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase is the first to be cleaved, followed by caspases and , thus suggesting that b s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd , cd and cd , and of the inflammatory cytokines il- , il- and mcp- . using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th and th /th skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd t cell responses. microcapsule based vaccination resulted in a marked induction of il- secreting th cells, without inducing strong th (cfa) or th (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg , igg c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd , cd and cd and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il- and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr -dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr -defective c h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il- b secretion by dc, through its effects on caspase- processing, which is also tlr -independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc (st ) or lactobacillus rhamnosus ncc (lpr), each at x cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week to week of life. fresh feces were collected at , and weeks of life for determination of iga levels (assessed by elisa). pups were bled and weeks after immunization for determination of measles-specific igg and igg a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st -fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, ( - )- -amino- -deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at : ration and washed times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw . by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of extracts from euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc /propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that up to euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd + cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il- and ige serum levels were increased on animals from group i when compared to control.the enhancement on th immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd + ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd +t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg a and igg b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren , , n. almqvist , a. lönnqvist , s. Östman , c. rask , e. telemo , a.e. wold university of göteborg, department of clinical bacteriology, göteborg, sweden, university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; . mg/ml) in the drinking water for days and, days later, mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin- and interleukin- . examination of gut sections from sea treated donor mice revealed increased density of cd a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp , proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna , a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp , or groel together with cpg-dna reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp together with cpg-dna . the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp microspheres induced protective ifng-secreting cd + t-cells and raised pulmonary pmp -specific iga levels in vivo. also, pmp microspheres caused lower il- serum levels upon administration than the injection of pmp together with cpg-dna , indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in cases of s. aureus bacteraemia in iv drug users and cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen ).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day ) and four weeks thereafter (day ). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter ) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st (spa-type t , agr , and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p= . ).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at and years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in and ds children, respectively. samples were taken before and - weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg , igg and igg ). at years, ds children had lower postvaccination geometric mean igg anti-tt-titers only. post-vaccination igg -anti-tt avidity levels were decreased in / and / ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. kda; , kda; , kda; , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda and , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an -month period. sera were obtained from healthy subjects from the same community ( months to years of age). the highest incidence of salmonella-associated diarrhea, / , occurred in children under years of age. the lowest incidence, / , was observed in the population aged to . whereas serum from individuals ranging - years of age showed maximum igg , igg and iga anti-s. typhimurium titres, children less than years-old did not show detectable igg and igg titres and had weak igg , iga and igm antibody levels; only their igg levels were comparable to those detected in adults. moreover, the levels of igg and igg antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs - , a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses ( %), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a- , linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after -fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková , s. bystrický institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd , expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age - years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg , igg and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after to days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers : ). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones ( : vs. : ). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was . fold of normal control). subcutaneous immunization gave a weaker stimulation: . fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv - and vega / / grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a- , -linked d-mannopyranose units and many branches composed of a- , a- , and/or b- , -linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant ( mg oligosaccharide per one conjugate dose) two times in days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs ( g- g) were treated orally with mg respivax five consecutive days. after the last application, on days , , , , and six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on mm thick serial sections, stained with hemalauneosin. the populations of cd , cd and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd positive cells, which number reached maximum at the end of the second week. on day b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd and cd positive cells. on days and the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta , s. majumdar bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein- (ip- ) and interleukin- (il- ) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip- mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase (inos ) expression and was linked to the mapk signaling pathway via antagonistic regulation of p mapk and erk / . further, ip- was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th cytokines and no. thus this study strongly demonstrates that ip- , like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th /th cytokines mediated by cd + t cells and activating cd + t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x . ) following immunization and after challenge with leishmania major. il- values were increased in all groups, but there was no statistical difference between the groups(p g . ) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x . ) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x . ) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x . ). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at week intervals. three weeks after booster injection, × stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg a/igg was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin (il- ), and cellular expression of cd and cd . results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd + lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with , infective n. brasiliensis larvae on day post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg and igg a). this was independent of level of me supply. feeding regime did not affect levels of the type- cytokines il- and il- . conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp of l.major strain mrho/ir/ /er. methods: l.major promastigotes were grown in rpmi supplemented with % fcs. l.major rna extraction and cdna synthesis were carried out. gp gene segment was amplified by specific primers and cloned into ptz r to construct ptz r/gp . the presence of gp into ptz r was confirmed by pcr. then, ptz r/gp was sent to determine the sequence of its nucleotides. after that the gp gene segment was sub-cloned into pet a (+) expression vector and transformed into e.coli bl (de ) plyss and gp protein was expressed in presence of mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, ) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including volunteers. male volunteers ( and years) for the adult group, and children of both sexes ( - years) were enrolled. subjects received virosomal formulations containing mg of ama -c (pev t), an apical membrane antigen- derived synthetic phospatidylethanolamine (pe)-peptide conjugate and ug of uk (pev t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days , and . results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling % of the treated mice to survive till days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only . % of the amount fed and it remained in circulation in the blood only for minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to . % ( of the amount fed but it's circulation was sustained till hrs post feeding. under such conditions when mgm of curcumin bound to mgm of chitosan nano particles were fed one time daily for days post infection to plasmodium yoelii infected mice % of mice were cured and survived atleast for days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma vdelta t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il- responses, to epitopes on the dominant rbc autoantigen, anion exchanger- (ae- , or band ) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla- + regulatory t cells or soluble forms of ctla- , may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =- . ) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b (r medium =- . ) and il- (r medium =- . ) in isolated splenic hscs ex vivo. analysis of effector cd + t cells in spleen showed decrease of t h cells quantity and simultaneous t h cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd + cd + ctla- + -and cd + cd -il- + il- regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h , t h and regulatory t cells were correlated (r th =- . , r th = . and r th = . and r tr = . ) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th = . and r tr = . ) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b -and il- -produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p , jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin- , t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il- , as compared to non-infected controls. the infection also altered the effect of il- on mapk activation by preventing its stimulating effect on p mapk. moreover, in s.obvelata-infected animals il- markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il- induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. .-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from ⁄ to / with a normal serum pool. .-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r g . . the analytical range was from g/l to g/l. the coefficient of variation (cv) was x % for [mc] n g/dl, and x % for [mc] x g/dl. this procedure was successfully implemented to quantify the mc in serum samples between march and february, . among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: . we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. . the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from day and reached plateau level after day of teratocarcinoma insertion. moreover, cd + cd cells showed in spleen and main lymph nodes from day and achieved plateau level after day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at day and had a maximum pick at day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than , times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b , il- , pge and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number = . and r activity = . ) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd -ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd l is cleaved to soluble (s)cd l. we sought to examine the levels of scd l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd l -along with other products -were assayed by quantitative elisa. levels of il , cd p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l . to examine if scd l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il- secretion. the il- concentration was consistently below - pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il- . the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il- production over control (p x . ) at d after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l (p x . ). pre-incubation of b cells with cd -blocking antibodies substantially abrogated il- secretion, unlike isotype-matched control. the partial blocking of cd binding on cd + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd l (under investigation) and indicates a sustained role for pc-derived scd l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule- (dnam- ) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from aml patients before specific anti-leukemia therapy and healthy donors. all results were analyzed statistically using spss version . results: aml patients under years showed a significant reduction in the expression of dnam- , nkp and nkp compared with age-matched controls. both healthy individuals and aml patients older than years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam- on nk cells and its ligand cd on aml blasts has been found in aml patients under years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam- expression on aml patients and confirm previous reports showing a significant decrease of nkp and nkp on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel , e. gorska , u. demkow , m. wasik medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for or hours with or without dexamethasone in concentration - m. analysis of: p-gp surface expression, p-gp function (rhodamine test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was , %± , . after hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine efflux, which characterized p-gp activity, was , %± , . after hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine ( , %± , , p= , ). average phi in lymphoblasts was , ± , . acidification of cells incubated hours with dexamethasone was seen in - % percentage of cells ) . rest of lymphoblasts showed alkalization (phi - , ).the percentage of lymphoblasts in early stage of apoptosis after hours incubation with dexamethasone (annexin-v test) was higher than in control cells ( , % vs , %; p= , ). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd d/cd integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd and anti-cd (clon huts ) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures ( hours). results: cd active form was expressed in the majority of normal and tumoral bm pc from healthy subjects ( . ± . , n= ) and mm patients in the early stages of the disease ( . ± . , n= ). in these cells, huts epitope was clearly upregulated by mn + . in contrast, circulating pc were almost all huts negative, and levels did not significantly augment when these cells were exposed to mn + . moreover, not only pb but also bm malignant cells from pcl patients were also huts negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index ( . ± . brdu+ cells, n= ) in comparison to pc from mm patients in the first stages of disease ( . ± . brdu+ cells, n= ). these results suggest that the active form of cd must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either % fetal bovine serum (fbs) with and without fgf ,or % human platelet lysate (hpl), %hpl, ( % fbs + % hpl), ( % fbs + % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day ). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: ) ten cases with fa diagnosed on the basis of dna breakage analysis, ) ten cases with aaa, and ) ten normal control cases. the presence of p dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p dna was demonstrated in bone marrow of % of children with fa, compared to % in children with aaa (p x . ), while, no p dna was seen in normal control. a positive correlation between dna breakage and presence of p dna was seen in bone marrow from fa (p x . , r . ). the presence of p tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord- -pol qm authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: pol qm nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on - march . when, in the mid- s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in provided stateof-the-art information and consensus on factors involved in injury and sepsis. in , the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until or . however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il- to il- , il- and il- and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in , to be dealt with at this symposium and congress? ( ) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. ( ) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. ( ) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. ( ) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. ( ) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. ( ) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~ ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha- macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro -iarg] ]aprotinin (bay ) or the b kinin antagonist, hoe on the hypotensive response to dxs. in the first study, anesthetized miniature pigs ( pigs/group, randomly assigned) were given one of the following treatment protocols: ) dxs ( mg/kg), - ) dxs plus bay ( , , , or rag), or ) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c adesarg and fibrin mom)mer were all increased. bay produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay . llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs ( /group) were given either dxs alone ( mg/kg) or dxs minutes after a bolus injection of hoe ( #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse , d- munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion ( , mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to u/ . in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to u/ . in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs ( ). when very small plasma kailikrein activities ( , u/kg bodyweight) were given intravenously a % decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c -inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin- ( x iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of , or u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion ( - u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c -inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; thrombosis research center, temple university, penn., usa; oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c b which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c b/c and c b/c, and a significant reduction was observed in animals that had received a lethal dose of e. coli together with mab c (treatment group), compared to animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-= antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c a, c , and c a were measured in patients from an internal intensive care unit. patients were clinically septic defined by the criteria of bone et al.(l) . the remaining patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l days period. during the first days blood samples were drawn every h, on day - every h and the last days once daily. mean plasma concentrations of c a within the first h after clinical onset of sepsis were + pg/ml, whereas non-septic-patients exhibited mean values of only +_ p_g/m/. c levels were lower for septic-patients ( + lag/ml) than for non-septic-patients ( _+ lag/ml). the most profound difference between both groups was found, when the c a/c ratio was compared ( . + . for septic-patients and . _+_ . for the control group). no significant differences between both patient groups were observed in c a plasma levels ( . + . ng/ml in septic-patients vs. . _+ . ng/ml in control patients). in of cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n= ) and non-survivors (n=l ) within the septic-group revealed, that the c a/c ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c a and c in only - minutes ( ) and the significant difference of the c ajc ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of amino acids with cysteine residues (mr daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw , behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of . p.g/kgxh thromboplastin for hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to . % and , % respectively, and by an increase of fibrin monomers from . to > . ~tg/ml. we administered rec. hirudin to rabbits in different concentrations ( . , . and . mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin ( . , . and . mg/kg i.v. bolus) as late as hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, marburg, germany $ novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r- ) and its effect on sepns-lnduced organ injury in rat atsuo murata , hitoshi toda , ken'ichi uda , hidewaki nakagawa , takesada mori , hideaki morishita , tom yamakawa , jiro hirese , atsushi ni~ , nariaki matsuura osaka university medical school, osaka, mochida pharmaceutical co. ltd. tokyo, wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je which lacks the membrane lipoprotein. the recombinant second domain (r- ) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r- was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r- significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai., ) . the cathepsin l-like and cathepsin b-like activity were measured in serum of patients with chronic bronchitis ( -with obstructive, -with nonobstructive bronchitis),acute bronchitis ( ) and healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai., ) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls ( - fold) .after treatment there was a tendency to normalization of indices,but the increase was about - % more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis - - -times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/ - diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il- / , ifn-a, il- , il- , il- , paf, pge, ltb and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur ( , ), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/ selectively t cells to produce lymphokines including i - and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/ + t cells accumulate within - min mrna for the ii- , i - , tnfai~ and ifn- ,. parallel in time high concentrations of bloodborne i - and tnf are noted. anti tnf t~// neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within - h distinct levels of tolerance to seb become discernable on ligand reactive v/ t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd , cd , cd accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c h/hej mice. the absence of sensitization to lps in lps d c bl/ sccr mice was identified as being due to their inability to produce interferon- (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c bl/ sccr mice concerns only the ifn response to bacteria, since the response to the t-cell mitogen con a and to cd monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c bl/ sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il- levels, whereas treatment with aztreonam resulted in progressively increasing levels of il- and a significant increase in mortality. the increased mortality in pbp- specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as -to lo -fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf- dgaln-treated mice with e. coli olll:b resulted in an lds of approximately . x cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection ( fold vs fold respectively, p< . ). parallel studies with endotoxin hyporesponsive c h/hej mice indicate significantly greater levels of protection with imipenem (> fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since , when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe ; pollack ) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: ) enhancing of opsonization and phagocytosis(antibactericidai activity) ) synergistic effects with [ - actam antibiotics ) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis ) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il- release in mice (baumgartner , corriveau and danner ) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger (berger , , in animal models (stephan , berger and also in prospective, randomized, controlled clinical trials (schedel , poynton , behre . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg ak) has been selected and chimerized into a human igglk (sdz - ). in elisa and in immunoblots on purified lps both sdz - and wni - show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz - and wni - have biological activity as they inhibit the lal assay and the secretion of monokines (il- and tnf) by mouse and human macrophages. moreover, sdz - and wni - inhibit the release of il- and tnf in vivo. in vivo sdz - as well as wni - neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni - as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch basel, $chweiz $ presentation of lps to cd by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd (mcd ). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd (scd ). these complexes of lps with scd are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd , either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd . these results show that the delivery of lps to scd is catalysed by lbp, i.e., lbp is not included with the lps-scd complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd . the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm , ai , ai , gm , and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm- , n. torrey pines rd. la jolla, ca usa . modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d- borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i _- , and il- , which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound ) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin- and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a % amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd receptors. a recombinant protein (rbpi ), corresponding to the amino terminal kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi will have to successfully compete with relatively high serum levels of lbp ( - ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of . nm for rbpi and nm for rlbp. in a competition assay format rbpi was approximately -fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi at . ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a -fold weight excess of rlbp over rbpi . and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions - , - and - . a single synthetic peptide ( - ) was bactericidal. these results suggest that rbpi contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p and p receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only % of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il- [ -inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered ( kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in % plasma +/-bpi ( ng/ml). pbmc were incubated for hours at °c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n= , mear~+sem, *p< . vs control). control . _+ + bpi . + % plas. . _+ + bpi . _+ pmp (ng/ml) lps (ng/ml) . _+ . _+ . _+ . _+. . +. . _+. . _+. " _+ " . _+ " . _+ " . _+. . + _+ _+. * _+ " . +. " . + -+ . -+ " . _+. " cba, c bl/ , balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection - % of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats ( - g) were studied [no operation (n= ) , sham operation (n= ), and bile duct ligation for days (bdl)(n= )]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n= ), sham op(n= ) or bdl(n=i ). blood was taken at this time (to) and days later(t at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp< . , mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t [p< , ,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp< . , paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than . pglml. polymyxin b was administered at a dose of , u every hours. plasma endotoxin rapidly decreased to the normal range in of the patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. - uchimaru, morioka , japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, n o r w e g i a n b r e d landrace pigs ( - kg) of either sex, pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a / air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e . m g / k g h a n d d i a z e p a m . m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n . x l cfu of e colt intraperitoneally as a bolus in ml saline, the a n t i b o d y g r o u p received in a d d i t i o n m g / k g e a n t i e n d o t o x i n i n t r a v e n o u s l y over h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for hours. results : a t a n d hours, the o x y g e n c o n s u m p t i o n increased by % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po . in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h- szeged~ hunbary involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit des toxines microbiennes and "unit d'immuno-allergie, institut pasteur, , rue du docteur roux - paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il- d and ~, tnf ~ and il- in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il- and il- . however, the most preponderant cytokine was tnf~, which peaked at i ng/ml after stimulation with i ~g eta. comparable amounts of tnfd (ca ng) were induced by .i ~g eta and .i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i, -glucan (ram ) was injected twice and h before the shock i.v. in a dose of ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli bs, mg/kg. aiiofcmg pretreated ruts survived during first haher ¢ndotoxine, while in controi shock group the lethality was %. the concentration of ~col)terin in serum was significantly elevated hafterthc second cmginjection (appare~tly % if compare with the control rats), but didu't chartged rain and s rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i and min after endotoxine onset ( i % trod ~, respectively of initial level) than in the control shock group ( % and % at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; . modify records; . erase records; . searches; . reports; . configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd ) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of organisms and the chance of identifyin organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group ( cases), the total effective rate was , %, in the contral group (combined administration of gentamycin and dexamethasone, cases) the total effective rate was %. however the obviously effective rate in sa group % was significantly higher than in contral group % (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o ). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and -glucuronidase ( -gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c h/hen mice at i and h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n= /grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at h the thymic cell yield from clp mice was significantly decreased (sham: . ! . x l vs clp: . i . x * cells; *,p< . ), the % of cells apoptotic by facs analysis increased from . i . % in sham to . ± . %* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from to patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). patients ( %) out of patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all patients with chronic septic knee joints showed poor results. additionally, in of these patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco- cell line) is grown on a micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco- monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco- system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco- monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. ) caco- cells are actively involved in the transcellular passage of bacteria across the caco- monolayer, since inhibition of caco- microtubule or microfilament function decreases the magnitude of st. ) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco- cell monolayer. ) caco- cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco- bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco- cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers ( . x colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about . % within the first minutes and are totaiy cleared within minutes. % offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about cfu of viable e.eoli per ml blood could still be counted hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with % respectively % of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at (n~ ), g (n ~ ), and (n~ ) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker - % at hour~ with return to % of gut leugth (similar to ~m) at hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at ,; hours with return to sham control at hours ( log cfu/g veraus log cfu/g ia ileum). translocation to mesenteric nodes - , , and percent at , , and fi hours compared to b in sham controls (n=l of ). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il- , soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs ( mmhg mean arterial pressure: map for - h terminated at an accumulated oxygen debt of - ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of - mmhg - rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at min and were still markedly increased at rain postshock. plasma tnf, il- , and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at min following resuscitation in rats subjected to hs for min, similarly at h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at min, while in baboons higher levels up to pg/ml were found at the end of shock and h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the -hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a (txa ) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky- , a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup , an a- receptor antagonist as a posthum treatment modality was evaluated. treatment with dup prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup from % to % (p< . , fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf -dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il- and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus , lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. male swiss (icr) mice were used weighing - g elimination of liver and spleen macrophages was accomplished by intravenous injection of / suspension of ci mdp-liposome suspension. control mice received an i.v. injection with pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either mg/g, . mg/g or . mg/g body weight (n= in each group), signs of st and bacterial translocation bt were measured hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n= in each group) were tested to verify that neither paraffin nor pbs or ci mdpqiposome suspension alone induced bt or st. furthermore survival over a -day period was assessed in a control and macrophage-depleted group with a dose of d mg/g z. results. neither the paraffin nor ci mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: / vs. / at . mg/g zymosan (p~o. ); vs. at mg/g zymosan (p~q ); / vs. / st . mg/g zymosan (p~o.o ).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of . mg/g zymosan ( . -+ . vs. . -+ , p~o. ) and . mg/g zymosan ( . -+ . vs. . -+ . , p<_.o. ) . the day mortality after . mg/g zymosan was % in the control group and % in the macrophage depleted group (p=o. ). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n= ) and two experimental (n= each). a portal vein catheter was placed and remained in situ hrs. a femoral artery and vein catheter were in place hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of . hrs., given more hours of anesthesia, and studied for further hrs. % of the estimated blood volume was withdrawn from each experimental animal over min. these animals were kept in shock for . hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for hrs. samples from portal blood for cultures and measurement of leukotriene b (ltb ), prostaglandin e (pge ), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to hrs. after hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb , pge and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha- a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c hb-iej mice). five days post-transfusion the animals were gavaged with lxl viable escherichia coil and randomized into groups (n= /group) to receive a sham burn (sb group) or a % thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies ( mg/kg) (ha- a group) or a % burn injury and administration of equal volume of sterile saline (c group). all groups were resuscitated by intraperitoneal injection of . ml of saline. the animal survival rate was observed for days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n= /group) were gavaged with viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline . and hr prior to sacrifice, group ih treated mice receiving ip of utinastatin . hr and endotoxin ( mg/kg) hr prior to sacrifice,group ilk sham reated mice receiving ip of saline . hr and endotoxin ( mg/kg) hr prior to sacrifice,group iv: saline control mice receiving ip twice during rain intervals,group v: mice received ip of utinasmtin, and min later they were received ip of endotoxin ( mg/kg), group vi: mice received ip of saline,and min later they received ip of endotoxin ( mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu - ,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (> points at days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range . - . mind) was studied after i.v. injection of mbq rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results . + + liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r= . ) and between l/m and ela (r= . ). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il- release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and , , , min, . the plasma concentrations (,pc) of -keto-pgft,, txb: and-ki- -pgf ~ (stable metabolites of pgi , txa and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il- levels by elisa. data are given as mean+sem (* p< . placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t . + . e[ . + . ] eu/ml). il- pc demonstrated an increase continuously from t to t (t + [ + ] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of -keto-pgf~,, pc up to h after mt in untreated patients with a peak of *[ ] ng/ at tl. also txb: and kh pge pc showed a considerabe increase up to h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il- release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into groups. group received standard laboratory chow feeding ad lib. in group a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group % of the calculated daily required calory intake (drci) ( /kcal/kg) was given by iv-tpn ( % glucose, , % amino acids) and % by limited chow administration. groups and received % and % of the drci by i.v. tpn and % and % respectively by chow feeding. group received iv-tpn only. after days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. , + , , - , , / + ~ " , -+ , , -+ ~ - , / +~ + _+ , + , , - , -+ + , ~ , , -+ ~ conclusions: the administration of % of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal ( %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and % in animals subjected to % bepatectomy and lreatment with fermented oatmeal, while - % and - %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < . vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < . ) in salinetreated animals following % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. _+ " +_ " . " data=mean_+sd, * stats anova p< . vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<. ) in lps gut translocation compared to control and l+co . this correlated with a significant increase in peritoneal inflammatory responses (o -,tnf) above that of the control and l+co groups, while mac- and cr opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin , ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), -keto-prostaglansin, leueotriene c , interleukin and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of patients showed a significant increase of endotoxin plasma levels during colonoscopy (p= . ), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after hours. ~hromboxane b o levels also increased after five min. from to pgyml peaking at min. with pg/ml. -keto-prostaglandin,leucotriene c , ii- and crp remained unchanged. a control group of i volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into groups were subjected to , and minutes of %, % and % of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p . ). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p . ). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: .long term( - days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. .in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. athol. . . .) . there was a increasing infiltration of the mucosa c m~nating at the d to th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. ~in isehemia was followed by a hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at m_in, i, ~ , and hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the th hour in the arterial blood .and at the d and th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for and minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion , and hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr ). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats hours after reperfusion in ischemia- rain group and and hours after reperfusion in the ischemia- rain group. delayed small intestinal transit time was seen from hours and on after intestinal ischemia for both and rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < . ). the distribution of radioactivity in segments and in the group with repeffusion hours after intestinal iscbemia for rain was significantly higher than that noted in the group with repeffusion hours after intestinal ischemia for min (p < . ). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy ( %) in the rat. water-soluble ehec was administered orally and hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a - % translocation rate to mlns or blood and hours after operation, while only - % of rats subjected to sham operation or animals with % hepatectomy and pre-treatment with ehec (p < . ). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal -hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s- lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca ÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca + concentration, [ca *]i, and ca + fluxes were measured by the use of fluorescent ca + chelating dyes (fura- or indo- ) and ca respectively. to assess sepsis-related changes in ca + dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca + i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response ( % ¢) in the liver, and a blunted insulin mediated sugar utilization ( % ) and increased proteolysis ( % ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca +]i by - % was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm ). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca + from intracellular stores. this protein kinase dependent process of [ca+ ] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca + signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn- (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute biochemistry, albert-ludwigs-university, hermann-herder-str. , d- freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a and kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: "-gggpunnf'ypycc- ") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il- b, tnf, [l- , il- , gm-csf etc), cell adhesion molecules (icam- , elam- , vcam- ), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p . upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a / kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g cells showed that lbp transcripts could be induced to ford within hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter . kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il- responsive elements hstf-hsp . and h-apf- rs as well as the transcription factor hnf- and the glucocrticoid-responsive elements oct- -d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g cells by il- , il- and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd .transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd , a kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd , which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd (cho/cd ). stimulation of cho/cdi with as little as ng of lps/ml resulted in the release of h-arachidonic acid ( h- : ) into the culture supernatant. in addition to h- : release, we examined lps-imiuced activation (transl cation) of the transcription factor nf-~b. similar to lps-induced h- : release, these responses were observed only in cho cells lransfected with cd closely resembling the same response in the marine macrophage cell line raw . . maximum nf-~cb expression occurred at minutes. in contrast to lps-induced h- : release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd , suggesting that nf-r,b activation begins early after lps binding to cd and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd , lps-induced tk activity in cho/cd was not observed. although the tk inhibitor herbimycin inhibited the release of h- : in cho/cd and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd -related lps signal transducer in cho/cd is not a tymsine ldnase. dimished cardiac inotropic function and response to -adrenergic receptor ( -ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin ( : gm/kg). to stimulate various components of the transmembrane signal cascade of -ar was isoproterenol (i -ar), acetylcholine (m ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia ( °c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c b and cdb was given to rats at a dose of mg/kg by intravenous bolus, min before reperfusion of the ischemic gut. a control group was given pbs buffer. at h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri ( . ± . to . _+ . ,p< . ) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced ( . ± . to . ± . u/g). c was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced ( . ± . x i to . ± . x , p<o. ). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia ( hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc , interleukin-i ( l-i) and il- plasma levels increased significantly, reaching maximum levels after hours ofreperfusion. polyclonal antibodies to tnfet and l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by % and % respectively and mpo was reduced by % and %. antml- yielded a protection in muscle and lung vascular permeability of . °, and . % respectively whereas muscle mpo was reduced by . % and lung mpo by . °, . these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using mabeled antmcam-i, constititive expression of icam- was evident only in lung but not in muscle. during reperfusion icam- expression increased by . %. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by . °, and . % respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following min of ischemia (mi) and . h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb . to p-selectin in a feline model of mi+r. pb . ( mg/kg) administered rain post-occlusion ( min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp . ) for p-selectin ( + vs + % of area-at-risk, p< . ). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb . compared to nbp . ( + vs + , p< . ). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb . treated cats ( + vs + pmns/mm z, p< . ). also, normal coronary endothelium activated with thrombin ( u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p< . ) blocked by pb . , but not by nbp . . furthermore, p-selectin is markedly upregulated - min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested , , , , , , , and h after transplantation (n= /time point) . snap frozen sections of organs were stained for cd- (w / ), cd- (ox ), t-lymphecytes (tl) (ox- ), macrophages (ed , ed ), neutrophils (pmn) (mom), icam-i (iap ), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w / , ox , ox , ed , ed and mom, staining for icam , lain and fib was assessed visually using a scale (ve= - ). as early as h after transplantation, fibrinogen and laminin expression increased in i, peaking h after transplantation (ve= . ; nc: ve=i. ) . at and h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i. ) but rose again at h, peaking at h (ve= ) and remaining elevated thereafter (ve= ). the high expression at h correlated well with the maximal pmn infiltration at that time (cf= ) in i returning to baseline at h (cf= . ) and thereafter. the second peak at h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il- , tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the minute interval (il-lb: , __+ , to , __+ , pg/ml; il- : . - , to , __+ , pg/ml; tnf-a: , __+ , to , + , pg/ml; p< . ). immediately after the beginning of reperfusion, soluble icam- and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam- , lfa- or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n= /group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within rain, ischemic hearts were stored in cold ringer's solution for hours before revascularization. representative grafts were removed after . , hrs, and days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts ( vs rain). after hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked ( vs c/f in non-ischemic grafts), whereas edl+macrophages ( vs c/f) and tnfe expression peaked by hrs. by hrs t-lymphocytes began to enter ischemic myocardium and icam- was moderately increased. after days cellular infiltration had returned to baseline, and no differences were seen among both groups after days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il- for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd treatment in the same model. in other species similar controversial results with anti-cd therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a -fold increase in neutrophil numbers associated with dispersed lullg tissues hours after lps treatment, while macrophage levels increased by -fold at the hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin- (il- ). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by hours, while mip-i levels were maximal at hours ( , ng/ml), with a second peak at hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a -fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip- . in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by -integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of -integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib and dreg , baseline values were determined immediatley after drawing blood. in addition cells were incubated min at °c to allow for spontaneous regulation of adhesion molecules. blood specimens from septic patients were obtained during the course of their illness. control values were determined in healthy volunteers. results: baseline expression of -integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g -integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to pg/ml tnfc~ or nm fmlp (but not at pg/ml tnfc~ or nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n = , bacteremia = ); group b : sepsis syndrome without septic shock (n = , bacteremia = ). serial plasma samples obtained on day , , , , and , were assayed using elisas method (british biotechnology), normal control levels of soluble icam- and e-selectin, obtained from healthy volunteers, were respectively ± . ng/ml and ± . ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis, ) . compared to control mean values, initial levels of groups a and b were significantly higher for icam- (p < - ) and e-selectin (p < - ). comparisons of group a and [] (* = p< . ; ** = p< . t) soluble icam- levels of group a enhanced significantly (p< . ) during the first hours, and a sustained high levels was of bad prognosis ( % of survivors at day ). the evolution of soluble icam- and e-selectin levels were significantly correlated with murray's score (spearman test : p < . ). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam- and e-selectin are correlated with endothelial lung damage, and loam- seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il- and il- , govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il- , have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam- . such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: ) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, ) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and ) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room ; barnhill drive; indianapolis, in usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd) chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd is expressed on all leukocytes, whereas cd b/cd and cd c/cd . are restricted to cells of myeloid origin. cd i / cd interacts with intracellular adhesion molecule- (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd , however, would affect all leukocytes, as would antibodies to cdlla/cdi . targeting cdllb/cd would affect cells of the myeloid lineage only, which could prove to be beneficial. cd b/cd is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd lb/cd may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt gs r.he, t focus on replacing slslic ~sd ~nd fuc s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c .an does sle:, s=h compounds are m ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the ~peutics for i~fi~anmat ry disease. lr:rng maimai models of acute lung lu ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi or its endothelial ligaud, intercellular adhesion molecule- (icam- , cd ). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two x -mm zones were produced by applying brass probes heated to °c to the animals' backs for sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd r . prior to burn injury (pre-r . ); animals given r . min after burn injury (post-r . ); animals given a monoclonal antibody to icam-i, r . prior to burn (pre-r . ); and animals given the r . min postburn injury (post-r . ). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies min after injury was as effective as preburn administration in preserving blood flow. at hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to . _+ % of baseline (p < . by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy- , a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but patients who died during the first hours were excluded, as this was part of the protocol. cy- was administered as a single intravenous bolus of . mg/kg (n= ), . mg/kg (n= ) or i mg/kg (n= ) mg/kg. the antibody was well tolerated. none of the patients died during the day follow-up period. organ failure was assessed for organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially . ± . , decreased to . ± . at the end of the study (p<o.o ). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin- (il- ) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh /sh domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb- and shc, via direct binding of the shc sh domain to the tyrosine-phosphorylated chain of the tcr/cd complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/ t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~// t cell population further segregates into cd t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a// t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of /$ t cells. moreover, these studies show that the relative contribution of cd or cd t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl- and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl- does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il- , il- and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format -d page, autoradiography of s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at and hrs. following clp. serum il- levels were determined at , , and hours by the tdi bioassay. by hrs. % of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant % elevation (p < . ) in serum il- levels at hrs. there was no difference in il- at and hrs. when compared to the control groups. d page studies examined splenic macrophage total protein distribution and biosynthetic activity at and hrs. post-clp. at hrs. macrophages from clp rats showed a substantial decrease in total protein pattern ( protein spots vs protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< kd) was observed. at hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early ( hrs.) staining with cd and cd mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd antigen (cd ++) and a minor population with weak expression of cd and expression of the cd antigen lcd +/ cd + cells). in healthy control donors(n= ) the latter cells account for + cells/mm and %+ % of the monocytes. in sepsis patients, the cd] +/cd + cells can become a major population with more than % of all monocytes in of patients and with more than cells/mm in of cases. there was no correlation of cd +/ cd + cells to any clinical parameter except for cd +/cd + percentage and body temperature (p= . ). the cd ++ monocytes showed a substanttial decrease in cd antigen density in of patients. three-color-if shows that the cd +/ cd + cells in sepsis patients when compared with the cd ++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd antigens, consistent with a more mature nature of the cd +/cd + cells. levels of il- were increased in sepsis patients: of patients with high numbers of cdi +/cd + cells ( /mm ) had high levels of il- ( u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il- . human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl ) and production of the cytokines il-lb, il- , tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il- production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il- and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il- -production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps ) on mo mediator release with a second endotoxin stimulus (lps ). methods: in vitro cultures of marine peritoneal exudate mo were exposed to hr. of hypoxic or normoxic conditions, then incubated hr under identical normoxic conditions _+ ng/ml of lps pretreatment. during the final hours all m~l were exposed to a range of lps concentrations. m~i supernatants were assayed for tnf, il- , il- , pge , nitric oxide (no), and superoxide release. results: lps markedly inhibited mo tnf release by lps but hypoxia had no effect on lps -triggered tnf release. hypoxia increased mo il- production in the absence of lps , but inhibited lps augmentation seen under normoxic conditions. pretreatment with lps increased no production from lps under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps signifcantly augmented mo release of il-i and pge , but hypoxia had no significant effect (data not shown) superoxide production was increased by lps under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. ( ) . h materials and methods. different groups of icu patients, postoperative (n= ), sepsis (n= ), septick shock (n= ), and healthy control (n= ), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf pl oflps ( ng/ml) to id of whole blood in presence of various concentrations of ptx ( , " , - , " , i " m). after hrs incubation at °c, the tubes were centrifuged and supematants frozen at - °c. tnfa and/l were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h . the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and , , corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being %, ) stimulation by lps. the control group is much more stimulated (> % for il , > % for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (> % for il , > % for tnfa ). the lowest stimulation was observed in patients with septic shock (median = %), some patients being not stimulated at all. )effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at - m (reduction to less than '¼ of the median values), and is almost complete at " m. the septic shock group has a decreased sensitivity to ptx. il production exhibits a lesser reduction at - m (~ 'a to ½ of the median values), further increased at - m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at - m and the inhibition is nearly complete at - m. surprisingly, there is a lesser, but significant suppressive action of ptx on il , not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. ( ) lymphokine research ( ) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp- was induced with phorbol ester ( h) and activated for h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il- and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received ml of either saline or blood from brown norway donors,iv. the animals were killed or days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day ; -+ vs + , p< . ). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure ( + , p< . vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from % in ileum on day to %, p< . ). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb i, hb nijmegen, the netherlands. leukemia cell line, teip- . robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. . adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa , an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa may alter monocyte adhesiveness. we selected the thp- cell line, a human monocytic leukemia cell line to further investigate the effect of txa on adhesion. we tested the hypothesis that txa alters lpsinduced adhesion of thp- cells and that txa exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa mimetic i-bop ( . .tm,) or txa receptor antagonists bms and l ( ~m). cells were allowed to adhere for hours and adherent protein/well was determined. lps-induced a significant (p< . ;n= ) increase in adherence of thp- cells (basal, . + . gg protein/well; lps, . +_ . p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between . - . mm, and is reduced in septic patients up to % ( . - . mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than %. subsequently cells were cultured in phenolred-free rpmi medium with various concentrations of glutamine ( . , . , . , . , . , , mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between and %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam- /cd on monocytes in a dose-dependent manner. the receptor for fc'/rucd as well as the receptors for complement cr /cdllb and cr /cdllc were down-regulated. cr /cd which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi , the receptor for transferrin cd and fc'friii/cd were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up - times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> days) of low hla-dr expression (< %) predicts fatal outcome (mortality > %). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [ of patients recovered from "immunoparalysis" following repeated plasmapheres; of them survived ( %). patients recovered temporarely and patients did not respond (all died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was of ( %; p< , ). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study : in vivo stimulation:cd- mice (n= ) were randomized to receive taurolidine ( mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after hours and were assessed for pm function [superoxide anion generation (o -), nitric oxide (no), tumor necrosis factor (tnf), fc/cr -mediated phagocytic function (phago) study : control pm were harvested and cultured in vitro with taurine ( . mg/ml for hrs), after which time they were assayed for -and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m . release of the inflammatory mediators -and tnf, and fc/cr -mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & , and nitric oxide. but injury and inflammation als cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi + % fetal calf serum and stimulated with i ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced ! fm of et/ cells (vs. unstimulated controls of < ). this calculates to - % of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal , germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il- receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i , which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il- message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il- message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l , which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il- production and the effects of il- on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med : , ) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il- production. macrophages were obtained from peritoneal exudate of thioglycollate treated c heb/fej mice and cultured in rpmi medium containing % fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b ) for hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for hours. the amount of il- in the supernatant was measured by il- dependent cell line (b and td ) proliferation assay. il- was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il- production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il- during the initial incubation with lps and subsequent desensitization. il- , like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il- shared the similar potentiational pathway, but differed by the fact that only il- was inhibited. (supported by r ai and po a .) department of microbiology, molecular genetics and immunology and the cancer center, wahl east, university of kansas medical center, kansas city, ks - . korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, ~g/ml, min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice - days after (peak at nd day for i.v. and th day for i.p. mode of administration of the same dose of mg/ g b.w.).the preliminary injection of cryelan ( mg/ g, or h before) to mice with acute cold stress (- ° c, h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al., ) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium ( - fold); bcg possessed similar effect.cmg in the same concentrations ( /~g/ml) increased release of these enzymes only saightly ( . times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren, ) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased - fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, , , prostaglandin e , and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et- (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. . x ucells/ ml in rpmi + % fcs were incubated i min., & hrs. with - m et-i, - m vic or i ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps ) for hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin- (il-i) in response to a subsequent endotoxin stimulus (lps ). in this study we examined the kinetics of lps inhibition of tnf and augmentation ofil- . methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or ng/ml of lps for intervals of to hours. culture medium was then replaced with , or ng/ml of lps for hrs. tnf and il- in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin- (il- ), interleukin-i (il-i) and prostaglandin e (pge ). we showed that preexposure to lps (lps ) alters the response of murine m~i to subsequent lps stimulation (lps ). we hypothesized that in vitro cytokine release by lps in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n= ), cultured in vitro hrs, then pretreated hr _+ lps -cultures were then stimulated with lps and mediators in mo supernatant measured: tnf, il-i, and il- by specific bioassays, pge by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps = ng/mh the table shows that all mediators were increased, in the absence of lps . pretreatment with lps resulted in complete inhibition of lps -triggered tnf release. in contrast, lps significantly increased mo secretion of il- , il- and pge (data not shown). serum cytokine levels were as follows: tnf _+ , il-i + , and il- . -+ . ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps -triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps . provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing - g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd , cdllb, icam- and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd , and icam- , but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi on activated neutrophils and icam- on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il- and il- as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps hrs following endotoxin infusion followed at hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p integrln (cdlla, b,c/cd ) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~ integrlns, but not cd , is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (> ~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng- p.g/ml) or f'mlp ( " to " m) et oc for mln, in ~; human ab serum, the expression of the ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase ( fold) in the expression of cd b on pmns from burned patients compared to an and fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = and total occlusion of blood flow to the lower extremities was maintained for g mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h production was determined by a previously described ' '-dichlorofluorescein flowcytametric assay. _+ amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < . vs. baseline; "p < . vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h production was increased by . -fold above baseline values by hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at rain. post-ischemia, however after release the ligature a significant reactive hyperemia was observed by mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from burned patients (mean age , +_ . years, mean burn size . % _+ . %) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at °c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and ,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ , s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of . u/ml (recommended by manufacturer is the range of - o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns ( . % of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin- (il- ) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il- and tgf- in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il- and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il- and tgf-i~ on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll- up to u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf- . in conclusion, these data suggest a contribution of il- in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at h followed by return of this parameter to normal levels at h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at h. this hi~her index persisted until the end of experiments ( days). the early ( - h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes ( ). there are reports of nentrophil dysfunction in inflammatory disorders of the skin ( ), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score ± ) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously ( ) . in parallel, granulocytes from healthy individuals were tested. two granulocyte functians were studied in vitro: . migration of the ceils in a boyden chamber through a filter matrix following stimulation with different receptor dependent stimuli (c a, intefleukin- , platelet-activating-factor, leukotrien b , fmlp). . release of glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of -glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure ( / ) and nonsurvivors ( / ), whereas / patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. : - , klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg , kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i ~g/ml) on granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load ( % of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in h of the recovery and cytosol proteolytic activity (ph . ; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was - -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by h of the recovery (the comparison was done with the sedentary rats). in h cytosol proteolytic activity decreased and then increased again by h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate ( - %) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il ,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s mdrome. the presence of interleukin (il)- in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il- in animal models can attenuate lung injury. collectively, evidence to date suggests that il- attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il- is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il- can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, .albumi n leak was quantitated by measuring the counts which crossed the monolayer, using a gamma counter. il- (lpg/ml) was incubated in the culture medium with .albumi n for hrs. the il- dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to .- addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt expression and primes pmn's for subsequent generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd expression and pmn priming via the [ adrenergic signal transduction pathway. methods: normal human pmns were primed with paf ( ng/ml for min) or pre-treated with - m of isoproterenol (i) or forskoklin (f) for rain and then primed with paf. cd expression was measured by flow cytometry (fig.l) and -generation in response to -rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig. holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf x on cytokine-and endotoxin induced expression of intercellular adhesion molecule (icam- ) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo -and lipopolysaccharide (lps)-induced icam- expression on human endothelioma celts (eahy ) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf x significantly reduced icam- expression induced by interferon-y (ifn-?) and interleukin- (il- ). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il- induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il- driven icam- expression on eahy cells as well as the il- induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody ( f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b (ltb , x " tool/l) or intravenous injection of interleuldn- (il- , - iu/kg b.w.) and lipopolysaccharide (lps, mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of mg/kg b.w., superoxid-dismutase (sod, . iu/kg b.w.) was able to decrease the ltb effect, and the immuumodulating drug leflunomide (hwa ) exerted inhibitory effects on il- -induced permeability at a dose of mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb or il- induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam- in neutrophil migration. blocking antibodies to human pecam- , in which the antibodies are crossreactive with rat pecam- , were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam- / integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs ( - kg) were subjected to % arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added ( , , , i.tg/ml) ex vivo. after hr incubation at - °c, %cd (+) pmns were determined with fitc-ib and flow cytometry from mean channel fluorescence histograms. ± # p< . vs baseline * p< . vs sham $p< . vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd expression was depressed for at ]east hr after trauma relative to sham but by hr, was enhanced, relative to sham, and because fentanyl anesthesia at hr had a greater effect on cd expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps ( ng/ml), tnf( ng/ml), and lps/tnf( ng/ ng/ml). cultures of the human ec tine(ecv- ) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for , , , and hrs. ec necrosis was assessed by a cr release cytotoxicity assay. pmn activation was assessed by cd lb receptor expression and respiratory burst activity hr _+ . -+ -+ . _+ _+ . _+ _+ . _+ . hr + . _ _+ . _+ _+ _+ " +_ +-- . " lghr - . _+ +_ - " o:fo , " ~ +- . * hr _+ . - -+ +_ * _+ _+ * _+ _+ " data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p< . vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received ml/ g bw normal saline. at hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, xi - m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at hr post-clp. after pre-contraction of blood vessels in the isolated gut with xl m ne, vascular responses to ach ( x m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, xl - m), were determined. total vascular resistance (tvr, mmhg/mi/min/ g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n= /group) in the aortic rings were: ach lxl i~s, st-in ~ ~ significantly at hr post-clp (i.e., increased *p(o vs. sham; n- per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin- on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. - g) shook was induced by fractionated withdrawl of arterial blood within rain and maintained for h (map at mm hg, cardiac output % of baseline). rats were adequately resuscitated with % of shed blood and twice the volume in ringer's solution additionally. following h of reperfusinn (map > mm hg, co > % of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n= ) received the il-lra as pretreatment beginning min prior to shock induction. in the group t (n= ) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of mg/kg and was followed by continuons administration of mg/kg/h. the control group c (n= ) received equal volumes in nac , %, the sham-operated group s (n= ) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: , % + , %; c: , % _+ , %) was significantly reduced by pretreatment or treatment with il-lra (p: , % -+ , %; p< . , t: , % -+ , %, p< . , anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: iam /s + um /s, p: llm /s + }am /s, t: ams/s -+ lam /s, s: am /s -+ am /s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il- . pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to retool/ asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc ( g/kg bw bolus, followed by . g/ kg-h infusion) on the endotoxin-induced ( . ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p< . at mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p< . at mmol/l asc) and reduced the adhesion dose-dependently (p< . at mmol/l asc); ec damage was also reduced (p< . at retool/ asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p< . ), decreased the protein clearance (p< . ) as well as the zymosan-induced rom production (p< . ), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi on neutrophils and lymphocytes. methods: from blood samples (n= ) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after , , , , and minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at min was observed in wbc-cultures (n/r: min / , min / , min / , min / , min / ). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to min followed by a decrease to normal values at min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl -expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c -delicient mice in a model of zymesan indt~ed mof. materials and methods. c -deficient b d /oid and c -sufficient b d /new mice were used in this study. on day all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of mg/g body weight. between day and , biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score= ). on day all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x ) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day - ) and a late phase (day - ). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c deficient mice (all p< . ). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was / ( %) in c deficient mice and / ( %) jn c sufficient mice (p= . ), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c bl/ mice received zymosan ( mg/g body weight) intraperitoneally. groups of animals were killed after , , , and h and subsequently at each day until day . plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin -ct, and - (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c were measured in plasma and culture fluid by means of a ria (detection limit . ng/ml). interleukin- (il~) levels were assayed using the b hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day , they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within h after zymosan injection and disappeared within h. from day onwards, tnf levels started to rise again. plasma il- behaved almost similarly in the acute phase, but in the mof phase plasma il- remained low. no circulating il- could be detected at any time point. macrophage lps-stimulated production of il-lcq il- ~ and tnf--c~ was suppressed immediately after zymosan injection. production of il- and tnf-~ was normalized within h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day did production of il-i~ reach control values. il- production was higher than control values from day onwards. il production was similar to that of ili-il the production of tnf-ct was strongly elevated between days and and again during days to . the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il- or il- . also, the ability of macrophages to produce excessive amounts of il- and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~ -acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with antisense dna's (as) complementary to the ' region of the il- gpl receptor. the antisense were '-agtttagggatgagg- ' (asl), '-atcttcatcttctgaat- ' (as ), '-aagtgaatgattaaaacact- ' (as ), '-aaacctttataggcg- ' (as ), and '-cgttctacaactgcaacgt- ' (as ). using hepg , the biological effects of these antisense complexes on the high affinity il- receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for h. underivatized asl was less effective and the complex, asgp-pll-as , had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il- (i units) asgp-pll-asi alone showed a dose dependent ( .i- . ~m) inhibition of ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem , israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l female merino-sheep were included (mean weight: kg). day : hemorrhagic shock (mean arterial pressure (map) mmhg for hrs.), closed femoral nailing (ao-technique), day - : bolusinjection of endotoxin (et) ( , ~tg/kgbw) und zymosan-activated plasma (zap) ( ml) every hrs., day - : observation. bronchoalveolar lavage (bal): day , , . the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm- ), pap -putm.art.pressure (mmhg), pap -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov ), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p< ) results: baseline day day day day heart: co , _+ , , _+ , , _+ , , _+ , * , _+ , * svr _+ + _+ +_ " +- " lung: pap , _- , , _+ , " , +- , " , + , " , +- , ' pap , + , , +- , , _+ , , +- , , +_ , * liver: bill , _+ , , _+ , ' , _+ , ' , _+ , " , _+ , " kidney:crcl , +_ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , _+ , , + , , + , histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il- and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg responses, t cell proliferation, and il- synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il- and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after - rain even without cyclooxygenase inhibition although pgi levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated patients undergoing major abdominal surgery as compared to ibuprofen ( rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time , , , , , , , rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of -keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of -k-pgf~,tp~, peaked minutes after m.t. ( _+ , ibu: _+ , to: +i ng/l) and declined monotonously over h ( +_ , ibu: _+ ng/ ). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after rain ( _+ , ibu: + , to: -+ /~u/ml); aldosteronc also presented a maximum after rain ( + , ibu: -+ , to: +- pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum h after m.t. ( +_ , ibu: -+ , to: +_ ~g/ ). adh pc peaked min after m.t. ( + , ibu: -+ , to: +_ pg/ml) and showed analogously to -k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. rats of them received sma clipping and others wee used as control. control and treated rats at , , , hours were perfused and fixed. the brain were sectioned at pm and stained by abc method using c-fos antibody. plasma endotoxin level of rats were measured at , , , , hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of , , , , hours after the treatment were . ± . , . _- - . , . _+ . , . ± . and . ± . pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi ). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed , , , , . tile plasma concentrations (pc) of -keto-pgft, (stable metabolite of pgi ) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < . placebo vs. [ibuprofen] [ ] mmhg (*p< . i). these changes were accompanied by a marked increase of -keto-pgf~ pc up to rain after mt in arterial and mixed venous blood of untreated patients with a peak of *[ ] ng/l tl (*p< . ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao while -keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for -keto-pgft~ was not detectable. a splanchnic vascular source for pgi release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e , ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day , two groups of n= week old aj mice were subjected to either a % scold burn (ti), or were untreated (c) n= . on day , mice (ti+clp) and mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days , and after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il- and adherent splenocytes (as) were cultured with lps for il- , tnf, il- and pge . results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi ) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. ( /ml) at rates increased incrementally from . to . mi/kg/hr over hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n= ); pga (n= ), graded bacteremta plus anti-pgiz antibody, ml/hr iv, beginning at hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po , mmhg, cardiac index (ci), l/min/m , oxygen delivery index (do i) and consumption index (vozi), ml/min/m , and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg -keto f alpha ; in the first instance~ peak values of lt ~ after i~ hrs post infarction were times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets , ~ev , ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical ( patients) and experimental material( rats and dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period ( - days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin ( microgram per kilogram of body mass intrap'eritoneally within days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [ ] . peep can affect rv function by reducing preload and ejection fraction (ef) [ ] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this -year-old patient (apache-ii: ) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [ ] . the therapeutic procedure included volume replacement, vasopressors (dopamine , dobutamine gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf- , od= mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id= ram, bennett a, pressure controlled mode, pm~x= mbar, peep= mbar, i:e=i:i, fio = . ). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [ ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new , dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [ ] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing mg% glucose & gm% bovine albumin and incubated in dubnoff shaking water bath for min at °c the results revealed that there was an enhancement in release of free fatty acids (ffa) ( %) and lactate ( %) and in glucose uptake ( %) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition ( ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by %. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a , but not with a direct vasodilator (nano ). this failure to relax to ach and a reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by weeks. exogenous no donors (e.g., c - or spm- ), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor ( p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at days. rat carotid artery rings relaxed only + % and + % to gm ach in vehicle treated rats and in inactive no donor treated rats respectively days following injury compared with + % in no donor rats (p< . ). relaxation to gm nan was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name ( mm) was markedly reduced by injury, but was protected by no donors (p< . ). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il- , ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin- and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde- -phosphate dehydrogenase (gapdh) and the cytochrome p (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to mmhg for hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, nm) was reduced by % compared to control sma segments (p< . ) while relaxation to sodium nitrite ( gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p< . l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p< . ), attenuated mesenteric mpo (p< . ) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm- (schwarz pharma), following regional myocardial ischemia ( hour) and reperfusion ( . hours) . treatment with spm- ( rim) significantly reduced myocardial necrosis by % (p< . ) compared to an no-deficient analog of spm- , spm- . furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p< . ) reduced following treatment with spm- compared to spm- ( . + . vs. . + . u/ mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps ( mg/kg, n= : mg/kg, n= ; or mg/kg, n= ) and treated with l-nma ( , , , , mg/kg/hr) for hours following a , , or mg/kg loading dose. animals were resuscitated with iv ringers solution ( ml/kg/hr). hemodynamic data were collected at , , , , , and hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p= . ), and systemic vascular resistance index (p= .ol) and decreased cardiac index (p= . ) and oxygen delivery index (p= . ). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p< . ), but this differential effect on the venous and pulmonary circulation occurred, > hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied ( , , , or mg/kg/h) in either the low ( mg/kg) or high dose ( mg/kg) lps-challenge groups. a nonsignificant (p> . ) trend toward a beneficial effect on survival ol low dose l-nma ( mg/kg/h) in animals given the mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study ( mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p< . ). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad . recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [ ]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no ( and ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad [ ] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation ( - ppm) consistently lowered the pap and augmented the pao /f.o for up to days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below . %. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [ ] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately - ppm, the improvement of oxygenation had a maximum at ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad , thus allowing reduction of the f.o . no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd- -phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il- , ifnj and lipopolysacchafide (lps). hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of , -diphosphoglycerate to glyceraldehyde- -phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by _+ %. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only _+ %. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il- , tnf, ifn, and lps for hours. , , and ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no ); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n= .) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont . + . + . # stim . + . + stim+ o tzm nmma . + . + . # stim+ ~m nmma . _..+ . * + . # stim+ pm nmma . + . * + . # stim+ )lm nmma . + . * + . # anova , . (* p < . versus stim, # p < . versus stim; anova with neuman-keuls) gsh in cont+ i~m l-nmma was equivalent to that of cont ( . vs. . ). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c bl/ mice and lew rats underwent a % and % ph, respectively. sc were prepared - days after ph and plated at to x ecells per well. after h incubation at °c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient days aider liver partial resection ( . nmol nitrite/ cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: ) encletoxin and l-name, ) endotoxin, ) naci and l-name, ) nach preliminary results from groups (n= ) and (n= ) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin ( . gg/kg/h) was given as a continous portal infusion over the entire observation period of hrs. l-name ( mg/kg) was given as a bolus after hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by %* and hepatic artery flow (hal e) by %*, while l-name caused a further and lasting reduction in flow (pvf %, haf %)*. transhepatic (portal-hepatic vein) vascular resistance increased to times baseline value during endotoxemia while l-name caused a further marked increase in resistance to times initial value. portal oxygen saturation (so ) decreased by %* during endotoxemia. l-name caused a reduction in portal so by %*. arterial so was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a % reduction in cardiac output (co). the addition of l-name reduced co by a total of %*. *: p < . . conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; mg kg -i s.c., at h prior to lps) or with an il-i receptor antagonist (il-ira; mg/kg bolus and . mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at - min after lps. for instance, endotoxaemia for min resulted in a fall in map from -+ (control) to -+ mmhg (p< . ; n= ). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at min when compared to lps-control: -+ mmeg (n= ) and -+ mmhg (n= ), respectively (p< . ). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, min, x - m). pretreatment of rats with tnfab or il-ira significantly (p< . ) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at min after lps there was an induction of calcium-independent nos activity in the lung ( . -+ . pmol citrulline/mg/min, n= ), which was attenuated by tnfab and !l-ira by -+ % and -+ %, respectively (n= ; p< . ). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with live e. coli, which consistently increased cardiac output - % above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name, mg/kg iv), prevented this increase and elevated map by - %. in the first groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are rains after e. cob. n= - /group. * p< . vs bl by remanova and § p< . vs e. coli alone by anova. ec+l-name -+ - _+ " § - _+ * § - _+ * § - + * - + * § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 'untranslated region (utr) of ferritin mrna and 'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j ) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k cells, where nos was stimulated by increasing the availability of the essential nos cofactor , , , -tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn- or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group , no, ppm, was added to the inhaled gas from the start of dialysis. in group no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group , from minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats ( - g, n= - /group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received rrd/ g bw normal saline. the animals were killed at , , or h post-clp ( h post-clp=early sepsis; - h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, to - m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, /~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at and h after clp. in contrast, l-nmma administration produces an % increase (p< . ) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a -years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of mg of l-nmma, a continuous infusion was installed ( . mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose ( to mmhg}, heart rate stayed stable ( + b/rain), systemic vascular resistance increased ( to dyne.sec/cm ), cardiac output decreased ( to . l/rain), and cardiac index declined ( . to . l/min/m }. before and after minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after matutes. neopterine increased from . to . ng/ml, tumour-necrosis-factor-a increased from . to . pg/ml and intedeukin- increased from . to . pg/ml. immunoglobulines a, g, and m ( . to . , . to . , . to . g/i), complement factor c- c and c- ( . to . , . to . g/i), alpha-l-antitrypsine ( . to . g/i), c-reactive-protein ( . to . rag/i), interleukin- ( pg/ml) and soluble interleukin- ( to units/ml) did not change significantly. pulmonary-shuntvolume decreased from . % to . % within one hour and to . % after minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse , zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. . microbiol.immuno : ) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc ,um, ifn-,/ u/ml, lps fig/m/, ngmonomethyl-l--arginine (mmla) ram. nitrite (no -) content of the supematants was determined by a standard griess reaction, and h release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x- , and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with . ml of a suspension of . ~" s.typhimurium, strain c , and injected with aminoguanidine (ag) mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from % in the control group to % in the treated group, days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom % to %, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no -accumulation, . nmole/well v.s. nmole/well. conversely h production is enhanced from nmole/well up to , nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, % in iron exposed macrophages v.s, % in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity ( % v.s. %) whereas the addition of afc to these macrophagas restores an elevated ( %) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit de microbiologie. bp . la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at °in tcm- / % fcs, with or without additional rat serum. after h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of u cobra venom factor/kg b.w. at days and . significantly higher (p<o.o ) lps-stimulated no-generation was found in spleen cells prepared at day ( . - . nmol nitrite/ cells, n= ) compared to spleen cells from non-treated animals ( . ± . nmol, n= ) complement activation was effective also in vitro. when incubated in % zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli ( qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l bioassay. survival rate (%) qrql/,p n hr hr dav control (normal saline . ml/body i.p.) n (l-name mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to min of partial liver ischemia ( %) and min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i mg/kg). sod, when used, was given as mn-containing preparation at a dose of u. i.v. i min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : . ± . "#/ . ± . / . ± . . alt (u/ ) • ± ~#/ ± / ± . gldh (u/l): . ± . e#/ . ± . #/ . ± . . in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous ~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [ ] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [ ] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no -chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of . - . parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non . . . . , parts per million (ppm) no, mean, n = ; ~ p < . (t-test) between smokers and non-smokers; p < . (t-test) between in-and expiration. about % of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than % {o. ppm before intubation vs. . ppm after intubation, ~, < . ), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [ ] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi , yoshihiro inoue , masao yoshida critical care and emergency center, and department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il- contribute to the occurrence of septic shock (circ shock : ; . the present study investigated the in vitro effects of lps, tnf-o~, il- and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il- , tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il- and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il- and tnf-c( enhanced lps-facilitated no production to a greater extent than il- or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. - uchimaru, morioka . japan. t. yolk , , i. ioannidis , w.j. kox and h. de groot objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: -morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin- ( mm), which releases both no. and o -., caused a time-dependent cytoreduction of % and % after and hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes --to form h and . in contrast, the addition of catalase (cat), which removes h to form h and , diminished the cytotoxic effect ot sin- to %, equivalent to high concentrations of snp ( mm), which solely releases no.. in addition, the amount of h formed by mm sin- equaled the amount ot enzymaticany produced h by mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration ( mm) alone were not toxic to endothelial cells, the combination caused an % reduction of viability, comparable to the effect observed with sin- ( mm) alone. moreover, toxicity mediated by high concentrations of snp ( mm and ram) was reduced by % under hypoxic conditions indicating synergism with no-and . conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with -. however, toxicity increases dramatically in the presence of h . this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no and no levels were measul*d sliectrophotometricallymonocyte cultures were done for itrs with or without lps+ifn-t stimulation mid no and no levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. )both no levels in the supematmlts of monocytes cultured with lps+ifn-t mid no , no levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. )based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [ u/ml]. after hours of incubation no-release was determined as nitrite (no ) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d , that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no -production (p<o, ). although we observed no toxic effect on cell viability, no -production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo nm] released % less no in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [ }jm] and bim [ioijm] reduced the no -formation by % and %, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d cells pretreated with d [ ~g/mt] released % less no than stimulated controls the inhibitory effect on no production decreased the later the inhibitore were added after stimulation; e.g. bim added and hours after stimulation reduced no -production only by % and %, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr , erlangen interleukin- (il- ) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il- ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the ' utr of the human il- ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable ( %) gc rich region. the translation of the type i il- r appears under a significant suppression and may limit the biological activities of il- by low level of expression. on the other hand, the type ii il- r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il- activity; inhibition of il- converting enzyme which cleaves pro-il-l[ , anti-il- ri, the il-i receptor antagonist (il- ra) and soluble (extracellular) il- ri and il- rii. il- ra is structurally related to il- and binds at °c to the il- ri with near equal affinity as that of bona fide il-i; however, il- ra does not transduce a signal. il- ra has been given to humans in pharmacologic levels (mean plasma levels of gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by % and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il- ra are in -fold molar excess to those of il-i [ . it is unclear whether these endogenous levels of il- ra are sufficient to block il- activity in disease. a single injection of ]l- or ifna in humans does not induce il- but rather high levels of il- ra ( ng/ml). prof.dr.l.a. aarden interleukin (il ) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il -anti-il complexes, suggesting that plasma il measurements represent a gross underestimation of il production in the patient. until now no natural antagonists of il have been described. the biological activities of il results from binding to a lowaffinity il -receptor (il r). the il /il r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp . studies on the structure-function relation of il revealed that disruption of the gpl -interaction site on il gives rise to a mutant il that, by virtue of its intact binding to the il r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin- (il- ), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il- and the ability of il- to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il- during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il- was found to be a potent angiogenic factor at concentrations ranging from - ng/ml. sequential fluorescein angiograms demonstrated that il- induced neovascularization by days, which regressed by weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il- . in addition, an il- antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il- has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th , thl and th cd ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il- are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il- also directly inhibits t-cell proli/eration induced by cross]inked anti-cd or anti-cd mabs (e.g. in the absence of professional apc), by specifically inhibiting il- gene transcription and il- protein production. il- also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il- , and tnf-a and the chemokines il- and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il- enhances the production of the il- receptor antagonist, a cytokine with anti-inflammatory activity. il-l produced by monocytes downregulates class ii mhc expression and il- production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il- is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il- also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il ) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd and cd memory t cells. ip- chemoattracts only preactivated (not resting) cd or cd t cells. hip-l~ preferentially chemoattracts gd t calla, while hip- more readily attracts the cd t call subset. the chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla- . these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln ', peaked by hr and was completed in hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin- (il- ) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of kd and kd. il- was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il- . phagocytic cells produce il- in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il- exert biological activity are t and nk cells. the major direct biological function of il- on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il- , produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il- to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il- , other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il- has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type (th-i) response and suppressing a th- response. the presence of il- during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il- , and inhibits the generation of th- cells producing il- . il- induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il- , however, il- needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il- -mediated inhibition of il- producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il- for thl cells and an inhibitory effect of ifn-y on th cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il- on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il- appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. , interleukin- and its role in the generation of t e cells, imm~ol. on peripheral blood monocyte/macrophages, il- behaves like il- in a variety of assays, and shows both similar and contrasting, activties with either il- or ifn-y. il- reduces inflammatory monokine and il- production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, , febs lett. , ) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi expression. il- produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il- . it inhibits niv-i production (montaner et el, , i. exp. mad. , ) , and interferes with hiv-induced cell fusion. the activities of il- and il- differ on t-ly•ocyte and large granular lymphocyte population. in particular il- does not exhibit the suppressive effects of il- on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il- and il-l share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. , embo j. . - ), explaining both thai common and divergent activities. the levels of il- ~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il- mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t cytotoxic lymphocytes express markedly higher levels of il- ~rna. in vivo, il- may thus be contributing, significantly to some of the activites previously ascribed to il- il- . recently, we cloned the human cdna encoding interleukin- (il- ). human il- is a non-glycosylated protein of aa with a molecular mass (mr) of kd and has biological activities on monocytes and b cells, il- , like il- , strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd (fc~rii). furthermore, il- , like il- and il~ , inhibited the production of pro-inflammatory cytokines il- , ]l- , tnfo~ and chemokines miplc~ and tl- by lps activated monocytes. both il- and il- enhanced the production of il-lra by monocytes. in contrast, il- lacked the t cell stimulating activities of il- . the responses of monocytes to il- and il- were not additive or synergistic, supporting the hypothesis that il- and il- receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il- , il~ and il- have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il- is unique in that it can modify inflammatory reactions without stimulating (as does il- ) or inhibiting (as does il- ) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of amino acids that is inactive until processed to a disulfide linked homodimeric molecule of t amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf- has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf- isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[ family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il- , pge and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il- and il- , have been characterized as critical in regulation of mo tnfa and il- , as well as in induction of t lymphocyte proliferation and ifn production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score > ) were analyzed for their il- levels, their in vitro proliferation to mitogen (pha) and their response after il- addition. since il- produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il- levels. increased patient il- production might also be resulting from the high levels of tnfa a known stimulator of il- . conversely, since il- augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il- deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il- found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il- production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il- were explored. increases in tgf[ may have partially contributed to the patients' depressed il- level, but elevated pge had no effect. addition of il- to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il- , pge ) and a dearth of other monokines (il- , il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells ( mmc) may result in the production of different cytokines including the interleukins (il) , , , and as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il- , l- , tnf-c as well as various mast cell growth factors (il- , il- , il- , il- , ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl bmmc/ml; ll.um ionomycin; - h). the actixdties of bmmc supernatants were assessed in specific biological (il- , il- il- , l- ) and/or elisa assays (il- , il- ). here we show that homogeneous populations of bmmc (> %alcian blue+/safranln-; in vitro age: weeks) generated in the presence of recombinant (r) rail- from normal balb/c mice produced modest amounts of l- and low or undetectable levels of il- , - , and - after induction with lp.m ionomycin only. however, a dramatic increase ( -to -fold) of these cytokine activities was noted, when in addition to ionomycin also human ( ) rll-la was provided during the induction period. this il- effect was dose dependent with a maximgm at - u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with ng/ml hrll- receptor antagonist abolished the action of u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll- or rmtnf-~. both mrll- and hrll- substantially enhanced ionomycin-induced l- production of bmmc in the absence or presence of il- . il- significantly enhanced il- and il- production while decreasing il- activities to abont - % of control levels, when il-i was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t antibody (ascites : ) abrogated the effects of mrll- . other mast cell-active cy~okines (] ,- , il- , l- , ngf, or kl) added to ionomycia-or l- /ionomycin-treated bmmc had no major effects on cytokine production. il- and il-i did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of " m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il- /ll- induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il- inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il- previously recognized as a product of th type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il- , and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of different httman cytokines, ilia, ill [ , illra, il , il , il , il , il , ils, ill , gm-csf, tnfa, ifn and tgf[ . , was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with % paraformaldehyde and permeabilized by . % saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[ _ , could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il- in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n= ) were incubated at °c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after , , , , and minutes plasma levels of tnf-alpha, il-i beta and il- were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from , pg/ml at min to pg/ml, pg/ml, pg/ml after , and min. il-ibeta ( , pg/ml, , pg/ml, , pg/ml, pg/ml and pg/ml after , , , and min) and il- ( , pg/ml, l,lpg/ml, , pg/ml, pg/ml and , pg/ml after , , , and min) plasma levels were increased min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il- to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr. , ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf- ) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf- ( . - . ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf- (> . ng/ml) potentiate production of the immunedepressive prostaglandin e . other investigators have shown that tgf-] can cause the appearance of cd (fc immunoglobulin receptor) on monocytes exposed to ng/ml of tgf-[~i for hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd is depressed in septic patients and therefore we hypothesized that tgf- could account for the down-regulation of cd observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[ for and hours at °c and examined ceils for cd and cd expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at °c alone. one displaying high density of cd had increased fluorescence over the homogeneous expression of cd in cells maintained at °c (baseline). the other population displayed decreased cd expression relative to the baseline cells. tgf-i~i ( - ng/ml) caused a shift of ceils from the high density into the low density cd population. this trend was observed within hours of incubation and was complete by hours. we observed a net decrease in cd expression f % for all subjects studied (p< . vs controls). phorbol myristate acetate ( ng/ml) also caused down-regulation of cd to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd after incubation with tgf- ( ng/ml) for hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd by tgf- deplete their surface expression of cd while generating cd . this down-regulation of cd by tgf- correlates with our clinical observations of lower cd expression on monocytes obtained from septic patients. for over years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw . and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw . cells, mif secretion was induced by as little as pg/ml of lps (e.coli l:b ), peaked at ng/ml, but was not detectable at lps concentrations > txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn . production of mif by lps-stimulated (l ng/ml) macrophages peaked at hr. expression ofmif mrna and tnf mrna by lps-stimulated raw . macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations ( pg/ml to p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately -fold after lps induction ( pg/ml). mif secretion also was induced by tnfoc ( ng/ml) and ifn? ( iu/ml), but not by il- and il- (up to ng/ml). lps and ifn had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif ( gg/ml) were found to secrete bioactive tnf~ (> pg/ml by l cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il- , il- , and tgf , have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il- , il- , il- , tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il- by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at °c for h in the absence or presence of lipopolysaccharide (lps) or tnfa. il- release was measured in the supernatants using a specific elisa. among tested cytokines, il- was the most efficient inhibitor of il- production by lps-activated pmn. il- was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il- production. northern analysis are under investigation to precise the level of the il- -and il- -induced inhibition of il- production by pmn. our data illustrate that il- and il- possess the capacity to down regulate the production of il- by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il- superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. female, wistar ( - gm) rats received either or pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with mg/kg ibuprofen lp prior to pg/kg bw cntf. rats treated with ,ug/kg bw cntf lost . _+ . gms bw as compared to freely-fed controls which gained . _+ . gms (p<o. by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (- . _+ . ). animals pair fed to the high dose cntf gained body weight ( . -+ . ), although weight gain was less than seen in freely-fed controls. rats treated with pg/kg bw had reduced body weight gain (+ . +_ . gin). food intake was also reduced by % in pg/kg cntf (from . +_ . gm/day to . _+ . gm/day; p<o. ) and was also unaffected by prior ibuprofen treatment ( . +- . gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight ( . + , gms vs. . _+ . gins), total protein ( . +- . gms/l vs . +- . gins/l) and albumin ( . + . gms/l vs. . +_ . gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il- and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box , gainesville, fl. . of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il- , have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il- are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp signal transd,uction protein, including il- and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il- and cntf also induced hepatic acute-phase proteins. thus il- and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il- -and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of to pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound ) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound ) or the phosphono-oxyethyl analogue pe- , structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd ÷-as welt as cd ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between / to / cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il- and ifnf, but not for il- or il- (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd ÷ as well as cd + t cells express cd , the high affinity il- receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il- receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t, cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)- when stimulated with calcium ionophore (eur. j. immunol. ( ), ). we now present evidence that human eosinophils contain preformed il- protein although they do not express mrna for il- as measured by rt-pcr. il- protein expression was determined using specific anti-human il- monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il- was re/eased into supernatant by % pure eosinophils after priming with gm-csf or il- and subsequent -min stimulation with paf, rantes, ionomycin or pma, however not with il- or il- , as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il- release in human eosinophils. the determined amounts of preformed il- protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il- in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il- concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il- concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il- in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by - % within days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. versus kin/day). they developed elevated plasma corticosterone levels ( _+ gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il- a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il- and tnf activity in plasma was not affected. after administration of .tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il- activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin (il- ). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il- ) and those of their specific antagonists interleukin- receptor antagonist (il- ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from patients with acute myocardial infarction at presentation in the coronary unit, every three hours for hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il- and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within hr whereas stnfr and l-lra peaked at - hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin- (il- ) is a multifunctionai cytokine known to be involved in important homeostatic processes. il- levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il- binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il- receptor (sll- r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil- r and il- are measured using elisa's. both elisa's are tested for interference of sil- r mid il- . the effect of sll- r in the il- bio-assay (b ) is tested. results: baseline levels are (mean + sd): . -+ . ng/ml in serum, . -+ . ng/ml in urine, . _+ . ng/ml in csf and . + . ng/ml in sf. there is no interference of sll- r in the il- elisa or bio-assay and no interference of l- in the sil- r elisa. conclusions: in all investigated biological fluids sll- r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin (il ) and to turnout necrosis factor ~x (tnfc . methods. bum patients (n= , -> % total body surface area) were studied weekly up to days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il ~-and tnfcz-binding t(cd ) lymphocytes was assessed by flow cytometry analysis. levels of il receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p< . ) increased up to weeks post-bum (means . + in non-surviving and . + . u/ml in surviving patients vs < u/ml in the control). within weeks of bum, the percentage oft ceils expressing receptors for tnfa and il [~ constitutively was elevated (by - fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range . - x j pg/ml vs < . x j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range - x - pg/ml vs - x j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl ~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of l lra may affect il -regulated biological responses including specific immune reactions. while studies suggest that il- is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il- contributes to depressed t-cell responses seen following hem; and ) how other agents, known to play a role in hem, effect il- release. to study this, c h/hen mice were bled to and maintained at a map of mmhg for h and then adequately resuscitated. mice were killed h post-hem to obtain splenic t-cells (nylon-wool purified). il- 's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il- to markedly improve the t-cell proliferative response [ . #g the marked increase in capacity of t-cells from hem mice to produce il- was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il- are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il- release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il- down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il- has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n= ) were randomized to groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received % etoh or ns daily for days by gavage. a % full thickness bum was induced hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed days post bum. splenic lymphocytes were cultured for days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma . ug/ml, ug/ml). splenocyte production of il- , interferon-gamma, il- , pge were measured, and lymphocyte proliferative response examined. results: il- production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of .- in a dose-dependent manner, indicating nitric oxide may modulate il- and interferon-gamma production in thermal injury. il- production is normal in etoh-burn animals. conclusion: il- and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il- production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin- (i - ), interleukin- (i - ), the interleukin- receptor antagonist (i - ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn , , , , , , and hours following operation. a portion of the results are shown (mean -+ sem). + -+ _+ one and two factor anova; *p< . , #p< . , §p< . , ¶p< . , for differences between groups i - was not detected at any time point. both ii-ira and i - increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for hours; the b-ira increase was a thousand fold greater than the rise in i - levels. i - levels increased up to hours after surgery. crp levels reflected maximum ii-ira and i - levels (r =. , p< . and r =. , p< . respectively). high ii- ra and i - levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i - ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin , ireland. change of il- and soluble il- receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il- plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il- receptor (sll- r) is considered to be agonistic to il- , unlike other soluble type receptors of cytokines. here we measured il- and sll- r levels in the serum and drain fluid from surgical field in order to investigate the changes of il- and sll- r after surgery and their origins. [materials and methods] serum and drain fluid samples from cases ( of esophagectomy and of gastrectomy ) were serially collected before and after surgery. il- and sll- r levels were measured by elisa. [results] ( ) serum il- : all cases reached the maximum level on pod-l, more precisely - hours after operation. ( ) il- in the drain : maximal il- levels in the drain were recognized - hours after operation, at almost the same time as serum il- . furthermore the il- values in the drain were much higher, about times, than those in serum. ( ) sll- r in the serum : all cases reached minimum levels - hours after operation and recovered to the preoperative levels a few days later (decrease ratio : . + . ~,, range : - ~'). ( ) sll- r in the drain : sll- r levels in the drain showed almost the same value and change as serum sll- r. [conclusions] ( ) il- is produced from the cells gathering around operative fields whereas sll- r is considered to be produced in the cells which do not gather around the operative fields. ( ) there may be a mechanism that down-regulates sll- r in the early stage of surgery. [objectives] il- plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il- concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il- . [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il- was measured by elisa and il- mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] ( ) serum il- levels reached the maximum concentration on the st postoperative day in all patients. ( ) the il- peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r= . , p< . , r= . , p< . , respectively). ( ) the peak concentration of serum il- in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p< . ), despite a similar degree of surgical trauma. ( ) peak l- concentration observed in a patient who underwent esophagectomy was about fold greater in the drain fluid of thorax than in the peripheral blood. ( ) il- mrna was demonstrated in leukocytes from thoracic and abdominal exudate at , and hours after surgery. in contrast, il- mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il- is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over years old show a different serum level cytokine pattern than pts under after a standard surgical procedure considered as a "medium strength trauma". patients and methods: pts( females males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a: pts under years(mean age: . +- )gr.b: pts over years(mean age: . _+ ). all pts underwent cholecystectomy and cholangiography. pts in gr.a and pts in gr. b underwent common duct exploration. spbintercctomy was performed in each group. on the day of surgery (pre) and on the st and th postoperative day(leo, po) : percentages of cd , cd , cd , cd and cd cells we measured by means of flow cytometry using moab. and levels of il- , il- , il- and tnf "in vivo" by elisa using moab. results: ere: cd % was . _+ in gr.a and . objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin (il- ) levels to determine a role of g-csf and il- in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il- levels were measured before and after surgery in patients with esophageal cancer (ec), and patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p< . ) and peripheral pmn chemiluminescence (p< . ) of patients with ec were significantly decreased compared to those of patients with gc at and hours after surgery. serum g-csf levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at and hours after surgery. serum il- levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at , and hours after surgery. significant inverse correlations (p< . l) between peripheral pmn count and serum g-csf and il- levels were seen at hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il- , may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin- (il- ) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system ( , , ). in our study we monitored il- and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il- and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) < and ct abnormatities or gcs < over hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over hours csf and serum. il- and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described ( ) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il- and il- antibody. astrocyte migration was tested in a chemstaxis chamber. results head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il- were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il- could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il- antibodies, purified il- exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il- was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o il- on astrocyte functions, il- promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il- stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il- represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of - mmhg for pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. animals received a bolus dose ( mg/kg) of tnf mab (celltech, berkshire, uk) at min after resuscitation (tn ). the control group (n = ) was treated similar to the tn group but received ringer's lactate (con). results: at min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph ( . + . ), hco -( . ___ . mm), and base excess (- . + . ram) values with pco ( . + . mmhg) and po ( . + . mmhg) in the tn with no difference to the con group. immediately after resuscitation ( min) plasma endotoxin levels were found to be increased in both groups ( . + . in tn vs . _ . pg/ml in con group) . prior to the treatment with tnf mab ( min) there was also no difference between plasma tnf levels of the two groups ( . + . in tn vs + . pg/ml in con group). treatment with the tnf mab at rain post-hs improved the hour survival rate to . % as compared to . % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy animals in the tn vs in the con group. no bleeding in the kidneys was found in the tn but in rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = ) was prevented in animals which died in the tn (n = ) group (( . +_ . vs . +_ . g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. -+ n=ll*$ -+ n= _+ n= * * p< . vs baseline :~p< . no anesthesia vs anesthesia thus ) tnf production increased - fold by - hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; ) anticd had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd does not involve tnf suppression; ) fentanyl anesthesia at hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from to yeas of age. the percentages of bum ranged from % - %. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of pgjml - pg/ml. peaks in the tnfa values were above pg/ml and were also associated with a peak in the stnfr levels. these levels began at < , pghnl within the in,st ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl there is a significant corresponding peak in il-trn (> ~ /ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin- beta l-ii ), intefleukin- (il- ) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the hours. patients' ages ranged from to years. iss varied from to and transfusion requirement from to units. five patients died after the study period. ] ,- was raised in / patients (max level , pg/ml) but was unrelated to condition or outcome. / showed a rise in il- b (max level pg/ml) which was negatively correlated to iss (i=- . , p< . ). tnfa was raised in / (max level pg/ml). peak tnfc~ was positively correlated with iss ( = . , p< . ) and haemorrhage (i= . but p< . ). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak ,- and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [ ] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [ ] . radioligand binding studies with ~ odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc ) for hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./( + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o o (mm h¢i): _k, (nm}: n (molecules/cell): - . ± . _+ - . ± . + - , ± . -+ - . + . -+ presented are calculated values on the idealized curve + % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [ ] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il- and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c, see, ° v bsa, ld ) and ( c, see, ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il- , - hr following injury and of try activity hr after scald, followed by elevated levels of il-i and il- at hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and postburn. il- increase peaked - hr after scalding and levels remained elevated - days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il- activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska , belgrade~yu. asadullah k ( ), woiciechowsky c ( ), liebenthai c ( ), doecke wd ( ), volk hd ( ), vogel s ( ), v. baehr r ( ); depts. of med. immunology ( ) and neurosurgery ( ) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l patients undergoing neurosurgery from the first preoperative day until at least day after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)- levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il- ( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il- and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit ( - ml/kg) hypotension phase (mean arterial pressure = map of - mmhg for - hours followed by adequate resuscitation). rats subjected to hs (map of - mmhg for rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin ( ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin ( ng/ml) for hrs at °c. results: at min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group ( _+ vs + pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats ( + post-hs vs + ng tnf/ml pre-hs) and baboons ( ± post-hs vs ± pg tnf/ml pre-hs). in contrast, the il- productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs ( ± pre-vs ±_ pg il- /ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., , new biol. : ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin- (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et- /et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et- on the release of histamine, serotonin, and leukotriene c (ltc ) by primary mouse bmmc (in vitro age: weeks) caltured with different recombinant mttrine cytokines (interleukin (il- ) and/or kit ligand (kl) in the presence or absence of il ) for two weeks prior to activation. et- ( x - to lxl - m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to % and % of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il- and il- . only when cultured in the additional presence of il- rather than in medium containing kl or kl plus il- alone, bmmc released histamine and serotoaln upon challenge with et- .furthermore, et- stimulated ltc biosynthesis up to -fold in bmmc cultured in the presence of il- . in contrast, the peptide was without major effect on ltc biosynthesis in bmmc culturd without il- . the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il- prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l- . et- ( " m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of - m. crude peritoneal cells (containing - % serosal mast cells) obtained from normal balb/c-mice released % histamine upon challenge with et- ( - m; rain). taken together, these resu/ts demonstrate that et- can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc production in mast cells. il- appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a (type ii pla ) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea , endotoxin, tnf-c~, il- and il- were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla and tnf-a in these patients were . ± . ng/ml and . ± . pg/ml, respectively, the two parameters being significantly correlated (r = . , p = . ). there were also a significant correlation between the level of type ii pla an il- (r = . , p = . ). there was no significant correlation between the level of type ii pla and il- . these in vivo findings suggest that tnf-a may be involved in the production of type ii pla . a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)- down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. , j. immunol. : ) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. , cancer res. : ) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. , embo j : ) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il- , tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il- the additional presence of rmull- for or days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack- ) nor kl-mediated proliferation or chemotaxis. in contrast, il- syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes . pre-incubation of bmmc with rhutgfl~ ( . , . , or . ng/ml) for h had no effect on kit receptor expression, although tgffil at concentrations of . - . ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il- , i,- , il- , and/or il- ). moreover, pre-incuhation ( h) of bmmc with tgf-gl (> pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il- -or kl-induced chemetaxis. when bmmc were preancuhated with rmukl ( ng/ml) for , . or days, a transient down-modulation of kit receptors with a maximum effect on day was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il- nor tgfi affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi cells from tnf-mediated cytolysis. material and methods: wehi subclone ceils ( x ), that are highly sensitive to the cytolytic activity of tnf, were grown on -well culture plates in rpm medium. after hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn ( - bp) were added ( . , and pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at nm. results: wehi ceils incubated with tnf ( - ng/ml) were completely lysed after hours ( % abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with . ng/ml tnf and jm odn showed still % of the absorbance observed in control ceils without tnf ( % abs). the protective effect of odn started at . pm, reached a maximum at ,um, and diminished at jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e , d- erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il- ), in the sepsis model of lipopolysaccharide (lps, mg/kg i.p.) induced hepatitis in galactosamine (gain, rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at hr and hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, mg/kg i.p.), rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il- levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, x mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, x unit/mouse i.v) r spectively. pretreatment with al and sod ( and hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il- into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee b'~ th~ recombinant mmlse tnf-o' ( . ~/rno~e j.p.) ~d oi~lps ~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il- release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients ( + years) in severe sepsis (sepsis score > according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma ( + years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. pl of whole blood were incubated either with / of a standard medium or with pl of a standard medium and pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours plasma concentrations of tnf-a, il-la, il- and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if- ,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients ( males, females, mean age +_ ) with severe sepsis (sepsis score > , elebute and stoner} were drawn on day , , , , and of disease. blood was immediately placed on ice and processed within hour. pl of whole blood were incubated with pl rpmi-medium supplemented with antibiotics and l-glutamlne or with pl of rpmi-medium and pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours samples were centrifuged and plasma was harvested and stored at - ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < , ) for circulating mean arterial tnf-a concentration ( pg/ml _+ sem} and central venous tnf-a ( pg/ml +_ sem). upon in vitro stimulation there was also a significant difference (p < , ) between released arterial tnf-~' { pg/ml _+ sem) and venous tnf-a ( pg/ml +_ semi. conclusions: these results are difficult to interprete but could reflect the influence of pao and sao on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il- ), tumor necrosis factor-a (tnf-c and interleukin- (il- ) the multifunctional cytokine intedeukin- (il- ) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il- -concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il- may be a potential substrate of these preteases. the serine preteases elastase (ec . . . ) and cathepsin g (ec . . . ) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin- (il- ) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il- leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin- . furthermore the loss of the biological activity of both, recombinant and natural human il- , was demonstrated by determination of the capacity of protease-treated il- to stimulate hybddoma growth ( td bioassay). these data suggest a possible downregulation of pathologically elevated il- levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il- , il- , _- -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il- , _- and il- were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to days, for a total of four tests performed (to, tl, t , t ). i .- was not detected in any samples. the serological profiles of the two cytokines .- and _- were similar; augmented levels were found at study entry and throughout the observation period, peaking at t and decreasing at t . however, in patients with sepsis, il- and _- concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il- and il- release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il- and il- production appears more significantly elevated, suggesting a role of il- and _- in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc and interleukin (il- ) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after hours and every hour. tnfct and il- levels were measured using the bioassays with cell lines wehi- ci and td , respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il- was found in all the patients' plasma samples and five patients' ( . %) ultrafiltrate samples. the il- blood level ranged from . to . u/ml (mean . , sd . ). the il- level in positive ultrafiltrate samples ranged from . to . u/ml (mean . , sd . ). conclusions: our preliminary results suggest that il- is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il- and il- and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd- mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first h with a peak h after clp at a significantly lower level than after lps. il- was measurable in serum only after h, significantly increased in spleen and liver after and h and in mesenteric lymphonodes from to h after clp compared with shammice. il- was significantly increased in serum throughout the first h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= ld ) to the sublethal (= / ld ) of three different lps (e.coli oiii:b and :b ; p.aeruginosa r ) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps :b induced as much tnf~ as the sublethal dose of lps :b ; furthermore, lps r , whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r ) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to h post lps administration moreover, the treatment with mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. ) circulating and organ levels of if are proportional to the dose and degree of toxicity of all the administered lps even if lps r was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if . to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if production. together with the total ablation of if , the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin- (il- ) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd- mice were randomized to mg/kg e.coli :b lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at , ', lh, . h, h, h, h, and h ( mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit pg/ml) and expressed as ng/g weight + sem (lowest detectable amount ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [ ~- p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present ( . + . ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced -fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was . + . ng/gwt at lh, reached maximal levels at . h ( . -+ . ng/gw-i) and returned to baseline at h. saline controls maintained their constitutive il-i~ levels. sb had fold more il- ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r = . ). separate mice were pretreated with saline i.p. orthe il- receptor antagonist (irap, mg/kg bolus i.p.) and were challenged rain later with . mg/kg lps i.p. or saline i.p. specific blockade of il- by irap decreased intestinal secretion at h and h post-lps challenge (p<_. . , student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content ( . % of total pituitary protein) and peak serum mif levels ( - ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps ( mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone ( % vs. %, p = . ). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps ( . mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from % to % (p = . ). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by . _+ . % in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin- (il- ) antibodies and il- receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il- in the serum does not correlate well with outcome. we hypothesized that this may be because il- acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il- levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n= in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. , j. inununol. : ) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after and hours of lps injection ( btg/ . ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels iron after injection amounting to only to % of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in % mortality in w/w v versus % mortality in +/+ mice . days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin- (il- ) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli :b , gg ip) into balb/c mice induces the rapid release of circulating il- ( ± u/ml at min). blocking endogenous il- using monocional antibody (jes - a , mg, h before lps) resulted in a massive increase in tnf production ( ± in lps+anti-il- treated mice vs ± ng/ml in lps alone, p< . , n= to mice per group) and an enhanced lps-induccd lethality ( % vs % in anti-il- +lps or lps alone respectively, p= . , n= mice per group). irrelevant igg rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il- during human septicemia. plasma samples were obtained from patients with gramnegative (gns, n= ) or gram-positive septicemia (gps, n= ) and from healthy volunteers. among these patients, suffered from septic shock at the time of sampling. il- levels were measured by elisa (detection limit: i pghrd). we found that patients ( %) had increased il- plasma levels (range to pg/nd). patients with gps had il- levels similar to the ones observed in gns (median: vs . pg/m, respectively). patients with septic shock had higher il- values (median: pg/ml) than septicemic patients without shock ( pg/ml, p= . ). no il- was detected in plasma from healthy volunteers. we conclude that il- is produced daring human septicemia. our experimental data suggest that il- might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, route de lennik, brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache i and sss). patients and melhods: septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i ( kda) were measured by enzyme immuneassays (norms values = + pg/ml and . _+ a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were + pg/ml and . + . ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p< . ). mean tnf concentrations for each patient were significantly greater in non survivors ( + vs _+ pg/ml p< . ); stnfr-i levels also were greater in this group, but the difference was not statistically significant ( . + . vs . _+ . ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = . p< . ). mean tnf levels were significantly correlated with apache ii (r = . p< . ) and sss (r = . p<o. ); stnfr-i levels were likewise correlated with both scores (r = . p< . and r = . p< . respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il- ). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of pg/ml and ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in and out of the patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il- . multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il- . peak plasma levels of both lif and il- correlated inversely with patients survival duration. cox's analysis for plasma levels of lif > pg/ml yelded a hazard ratio of [exp ( . )= . ]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human kda tnf receptor (stnfri) and the kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: points). serial blood samples were obtained within h after diagnosis of sirs, every hrs for the first hrs and every hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o ) compared to healthy controls (normal value: tnfrh , ng/ml; tnfrii: , ng/ml). the serum concentration of tnfri ( , ng/ml) as well as tnfrii ( , ng/ml) were significantly higher in nonsurvivors (n= ) compared to survivors ( , and , ng/ml; n= ) at the onset of sirs and during the course of the inflammation response (p< , ). a positive correlation exists between both receptors (r- , ; p < , ). increasing levels and maximal values of stnfri ( ng/ml) and i ( ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in out of patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. - is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first hours after administration of il- . in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il- and il- using immuno assays. in these plasma samples we also measured phospholipase a activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il- therapy the cytokines tnf, il- , and il- are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il- . objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin- (il- ) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il- by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of healthy volunteers and cultured at a concentration of . • ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after hours. using elisa, tnf-c~, il-ib and il- concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il- ] and il- by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il- in ng/ml as mean _+ standard error. ca/ml . . ( ) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity ( , x - m) as the fab-fragment ( , x - m) but bind the antigen with a eightfold lower avidity compared to the parental mab ( , x - ). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl- and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l , the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a -fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies ( ) reveales that the glucocorticoid antagonist ru , nufepristone, sensitizes both endotoxin responder and nonresponder, c h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and hrs following lps injection, ru significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru . however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru treatment. these studies suggest that ru aggravates lps lethality via factors other than tnf in endotoxin non-responder, c h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi response in - % of the pbmc after - hours of cell stimulation. in contrast, il and tnfc~ containing cells was only detected in - % of the pbmc. the induction ofil , il , il , and ill , was weak or completely absent. thus, the response indicates activation of th cells. however, illc~, illl , illra and il , were consistently found and gradually produced, peaking at hems in approximately - % of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of rag , , -trinitrobenzenesulphunic acid in . ml % ethanol (n= ). control animals received an isovohlmetric instillation of normal saline (n= ). faecal pellets were collected before induction of colitis (day ) and throughout the experiment. after days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day -#p= alld the total ftnf measured during the study correlatcs with the cms oll day -p= . there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice ( - g) were primed intraperitoneauy with either lipopolysaccharide (lps) ug]anlmal or saline. five days later, the animals were subjected to hypoxia ( % ) for , followed by of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at , , and and assayed for tumor necrosis factor (tnf), prostaglandin e (pge ) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia ( %o disease , berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl . we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il- receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps ( ng/ml) supplemented with different concentrations of mps ( , pg/ml, mg/ml). after hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il- ra, and tnfbp-i (tnfsrp ). il-la, il- ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to . + . %( mg/ml) by mps in a dose dependent fashion, whereas about % decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased times more in the supernatant of lps-stimulated pbmc with gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional -adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that -ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected times with ! -ar agohist isoproterenol ( . mg/kg i.m. and . mg/kg s.c.) , and hours prior to a lethal dose of lps injection ( mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. ) . these data suggest that -ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at °c in rotating polystyrene tubes. samples were taken before addition of x ng/l e. col± endotoxin and after hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin- (il- ) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of mg/ml or mg/ml or with the monoclonal lipid a igm antibody ha- a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il- values were found.in the plasma. cytokine values at hours: tnf-ec pg/ml (mean) and l- pg/ml (mean). preincubalion with mg/ml standard immune globulins reduced il- values by % (mean) while there were no effects on tnf-c~ values at hours. a slight reduction in il- values was also found with the lower concentration, mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il- nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha- a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced l- release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha- a had no effect on the il- or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto % of the estimated blood volume was withdrawn over - rain, to reduce bp to - mmttg and co by / mad withheld for rain. shed blood ( %) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of %. zymoeen activated plasma ( , gg/ml) was given rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r , rat igg , mgkg i.e. n= ) or emine (nffi ), rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p< . ) higher in saline compared to r -treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p< . ) higher in ealine ve r animals at and h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p< . ) attenuated in r treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin- (il- ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il- in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, sprague-dawley rats were studied. anesthetized rats were divided in groups, implanted with alzet r minipumps and randomized to receive the following treatment for days:i: gg/kgbw/min of il-lcc; ii: ~tg/kgbw/min of il- receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a % bsa, full thickness scald burn and the wounds then infected with + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first days and improved food intake between days and . we conclude that blockade of il- after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il- . ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il- producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx ( mg/day) in septic patients resulted in tnf and il- productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( ml/kg/day ). the prevention of lpsinduced in vitro tnf and il- production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam- in sera of septic patients was significantly higher ( - ng/ml) than in normal individuals ( - ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy rgyi medical university, institute of microbiology, szeged, hungary h- ddm t. . e~i~im~/tii, septic sh ~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit ' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il- concentrations. methodology female wistar rats (wt - g) (n- per group) were prefed with % tanrine, . % glyciuc ( in tap water) or tap water (tw) for days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received . % saline subcutaneously ( mls/ g) post-procedure. animals were venosected by direct cardiac puncture at or hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at - °c was completed within hours. tnf and il- were measured using wehi and b bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il- estimation revealed a statistically significant reduction at hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin (il- ), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after days systemic blood was harvested to determine the circulating levels of il and tnf. a second group was treated with thy for days and then received a gavage wide lxl escherichia coli and a % burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had mice/group. in separate groups (n= /group), receiving the same treatments, mortality was observed for days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av difference. it was mclean in a study reported in that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are , neutrophils per cubic millimeter of blood, a kg man would have ~ . xi u circulating neutrophils. when activated ixl ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate - . xi nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of - . ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il- , il- , and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m populations; it decreases splenic m but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon- offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin- , which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il- and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il- and il- +m-csf treatments increased the production of tnf in burn day macrophages and progenitor cells compared to normal cells. il- +m-csf+il- increased the production of il- by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il- +m-csf increased the mrna for tnf in the -day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il- /~ actin mrna indicated that il- , il- +m-csf, and m-csf increased the il- ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~ servojf of appr m ~ separating i~ eucayotic ceils of the hosl ttom l ~ bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (. u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= %' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h -receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h -receptors by h -agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,- decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il- , and , and shared mscrophags funclion with loss nr f and c b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we g~ven in doses of ~ . to ~ , u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~ animals, interferon ~amma was a so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to ,s uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< , ). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c , interleukin- (il-ii ), interleukin- (il- ), and interleukin- (il- ) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin- (il- ), interleukin- (il= ), transforming growth factor- (tgf-j ), and the newly sequenced interleukin- (il-i ). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u . in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il- b and tnf-a. the il- receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p ) and type ii (p ). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il- , il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il- and l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, ) early decreases in total t and helper t cells in the peripheral bloo~ } lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and ) severe and prolongei deficiency of interleukin- (il- ) prcductlon by peripheral blood mononuclear cells (pbmo). this il- deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il- deficiency end shown that the defect lies post-transcrlptionally since mrna for il- is also depressed following thermal injury. we have also investigated the potential of a defect in ii- receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il- r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd +) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: ) occurrence of activation-induced cell death (aicd), ) activation-related phenotypic and functional_ diversity in the pool of peripheral cd + t cells. initially, (up to days post-bum), peripheral blood lympliocytes from severely injured (> % total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l (cd ) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd +/cd ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il- secretion and downregulating excessive il- and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either . g/kg of e in a % solution by garage (ge), or . g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a % body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p and p were both - fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p and p during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. decker, d., sch ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages -ii according to me sherry), were - years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn h before surgery, immediately before incision (after anaesthesia), h and h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in laparoscopic and open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were . the interleuldn- receptor and im soluble form (cd /scd ); . the activation antigen fd- and its soluble form (cd /scd ), a member of the nerve-growth-factor receptor family; . the cd ro epitope which characterizes t memory ceils; . the trausferrin receptor cd ; . the soluble adhesion molecule icam- ; and . the cytokines interieukin- and interleuldn- . on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to h before surgery. h after incision the tra index in open cholecystectomy showed a distinct - fold increase, whereas in laparoseopic surgery a mere - fold increase was noted. h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was . % (range %- %) of total body surface and mean age was years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd and most strongly positive for cd . this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd and cd positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd +) was unchanged and nk (cd +) cells were decreased by ahnos[ % compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd were unchanged. cd (il receptor) and cd (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days ( ) t, , , and post injury. we analyzed the cumulative data for interleukin- beta (il-i[ ), il- , il- as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d post-trauma and recovery thereafter. *p < . , ** p < . vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il- and ,- , suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r= . ). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss> , n= ). i - , - , g-csf, fpa, and c a -levels differed significantly (p< . ) between the iss-groups (>-< iss ) at the time of admission, whereas on day tnfa, c a, - , and ealpi showed significant differences. beyond the first week, major differences were restricted to pge and c a. the formation of two groups with respect to later multiple organ failure (mof < ; mof > n= ) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd (monocytes), cd /cd (i - r + t-cells), and cd /cd (th calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving % or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma ( % tbsa). following a % tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a % tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of % tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with % and % tbsa thermal injuries, respectively. a % tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a % tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm and the office of navy research n - -j- . division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut . post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. cd- mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, cd- mice were randomised into control (c), l or s groups and peritoneal cells studied at hours for bacterial phagocytosis and killi:~g, superoxide ( -) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd- b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac- was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac- expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in ,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. - mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a ,c chamber for recording by real-time video confocal microscopy. the cells were subjected to msec, v/era, a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was , .c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with mm mg%. experiments were repeated with mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. patients ( males females,mean age: . _+ ) with gallstone disease and with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the st and th postoperative days (pre, lpo, po) percentages of cd , cd , cd , cds, cdi were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il- , il- , il- , il- , tnf) were measured in pts. of each group by means of elisa using moab. _r. esults:variations of il- and il- were not s.s.. il- increased but differences between groups were not statistically significant (s.s). il-i decreased on po and increased on po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p< . ).tnf decreased in the l.g. and increased in the th.g. on the po, the difference was s.s(p< . ); on po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on po, except for %cd cell that increased on lpo and decreased on po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< . i), but differences between them were not s.s. in contrast, igg decreased on po (p< . ) and increased on po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, % nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia, and hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound or hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd ) and helper-t-cells (cd ) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd ) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd ) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd +hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd +cd +leu -pos) was increased just after anaesthesia being decreased at g and hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples hours after the operation with recovery at hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at hours (p<o. ) and hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in trauma patients on hospital admission and x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age -+ years ( m, f), mean injury severity score (iss) + , sepsis ( patients), sepsis syndrome ( patients), ards ( patients), renal failure ( patients). ige increased in patients ( %). the mean increase was ng/ml (range to ng/ml; % ci was to ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p= . ) and renal failure (p< . ). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p> . ). plasma ige increase was not related to severity of injury by iss score (p = . ). the mean day to highest ige was . -+ . . the day sepsis was first observed preceded the day of highest ige by . + . days. there was a significant association between the day of sepsis onset and the day of highest ige (p= . ). eight of nine patients with sepsis syndrome had > % increase in plasma ige from admission. one patient's ige levels were normal ( - ng/ml) for days and then increased to ng/ml over the next days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il- , which concurrently regulate production of ige and il- receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during hrs. adult male wistar rats ( +_ gin) were submitted to hs (sbp tort) and after t hr (shock i hr) and hrs (shock hrs) hsl (nac . %, . ml/kg) treatment, e. coli (i ) was injected into the portal vein ~tci (n_> ). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p< . , ~" p< . and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung uptake was increased and liver and spleen uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after hr of hs hsl reatment, diminishing by hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after hr hs hsl treatment, returning to normal values by hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of rabbits, labelled with indium-tropolene and reinjected intravenously into the rabbits, i rabbits received an infusion of escherichia coli endotoxin ~g/kg, while i rabbits were subjected to a major sham operation and i rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and , and hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd , cd , cdlla/b, cdis, cd , cd , mhcii and cd /cd ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd /cd ratio while the cd positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to % of initial values following endotoxaemia and to % following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd cells compared to cd positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin- (il- ) and interleukin- reeeptor(il- r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at , i, , , days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il- stimulated by concanavalin a(con a): the sera from the rabbits days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, kd,milipore; centricon- , kd,amicon), that are less than kd, between i and kd, and more than kd. each group of the substances also was tested for the expression of il- and il- r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il- by the con a-stimulated splenocyte in mice; ) the sera from the rabbits days after injury had more profound suppression than other injured sera; ) there was a marked band at about kd in sera from the rabbits days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; ) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il- and il- r than other groups substances, of which molecular weights are more than kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; ) there is kind of substance, of which molecular weight is about kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; } the substance can depress the expression of mrna of both il- and il- r. research institute of surgery daping, chongqing, p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m( results in decreased antigen presentation, induces tgf- and pge while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il- . consequently, here we investigated rnonokine production in trauma patients (n= ) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t ) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days - post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the - days post injury period became higher in alcoholic trauma patients. furthermore, over days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml - days post-injury days post injury stimulus ale. pt. pt . . . . immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol ( - mm) exposure resulted in a significant down-regulation of tnfc~ (p< . ) and il- (p< . ) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd + receptor, the proinflammatory mediator il- , the macrophage activating factor ifn- ,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days , , , and after mechanical trauma of and after bum trauma of patients (mean age ~ years; mean iss ± pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il- and ifn- in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly ( + u/ml) compared to controls ( + u/ml) as well as mechanical trauma ( + u/ml). il- showed a significant suppression following mechanical trauma ( + u/ml) vs control ( + u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than % of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l- and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il- agonist) in eutrophic rats subjected to either % tbsa cutaneous scald injury (bi), muscle scald injury of equivalent % tbsa (mbi), sham muscle bum (resection of skin only, up to % tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation ( mukg i.p.). separate rats were infused with mg/kg e.coli :b lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount . ng/gwt). il-lo~ was constitutively present only in the skin ( + . ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at . h (in the liver, bi = . _+ . ng/gwt, sbi = . + . ng/gwt, p _< . ; in the lung, bi = . + . ng/gwt, sbi = . + . ng/gwt, p -< . ). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint already ( min real-time). these levels increased in parallel until min and became eventually different by log at - . h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il- ~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il- c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma . j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of mmi-ig for rain. the animals were then resuscitated with either times (x) the shed blood vohune as lactated ringer's solution (lrs) or x lrs + lx % lies. sham mice were neither hemorrhaged nor resuscitated. at or hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il- ( alternatively pmqb showed no differences in il- release between all groups at and h, while sm~ from the lrs + hen group showed a depression at h. tnf production by pm~ was depressed in all groups at h and remained so in the lrs + hes group at h. sm~b showed decreased tnf release values in both hem groups at and h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il- ) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in patients with multiple trauma (median injury severity score = , ). blood samples.were collected shortly after injury and after , , , , s and l days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p< , s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury ( - h) at , eki/ml (median), decreased thereafter to , eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day revealing the lfighest level to la followed by pa (= % of la-sac), ec (= % of la-sac) and vc (= % of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day ). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day and . however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r> , ; p< , ) most often between antibodies of igm-elass ( %) followed by igaclass ( %) and lgg class ( %]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin- (ii- ) and c-reactive protein (crp) were measured in patients. i female and male, ageing from to with a median age of . blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, and hours after the operation and then every morning until the th postoperative day. blood was drawn into heparinized tubes (i iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. - levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p . ) in endotoxin plasma occurred during surgery, culminating in a peak (median value of . eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the th day after surgery, whereas those of interleukin- rose at the end of the operation and were at their highest hours later (median value of . pg/ml). crp levels were also high postoperatively with a median value of mg/l, and were markedly raised on day ( mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and - during the operation was establisthed (p . ), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of - , and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or - plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map ± mm hg) for minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at and hours following hs for quantitation of slga by ria. animals were sacrificed at hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled and days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day in the control . + . , in group . + . , in group ii . + . , in group iii . + . , in group iv . _+ . , in group v . _+ . x ( ). the evident increase in bmc yield in all groups continued until day . increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from through , and who had serum cholesterol assayed during the first hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< . level. results: trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first hrs. patients had cholesterol levels less than mg/dl; of these ( . %) died, ( . %) developed ards, ( . %) developed acute renal failure, and ( . %) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was times greater and risk of multi-organ failure times greater in this group than in those with a normal serum cholesterol (>if mg/dl; patients; p< . ). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n= ) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n= ) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi ) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f ~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p< . ) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa which was more pronounced in c (p< . ) and most in b (p< . ). similar results were found for pge~ and pgf =. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p< . vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r= . , p<o.o ) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt . + . tt , +_ , n.s. pla-ca(s) mt . +_ . tt . _+ . n.s. goris mt . - . tt . _+ . n.s. iss mt - tt - p< . we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim nez f j, l pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha- antitrypsin (aat), alpha- macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of patients, males and females (mean age: years), with compound fractures. % of them were located in tibia and fibula. intercurrent infection developed in patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days , , , , , and . a control group of healthy volunteers was also evaluated. aat and cp showed increased values from day which kept elevated untill the end of the study. amg elevated from day . when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il- , il- , tnf, pge and tgf-b have all been implicated in these immune system changes. in vitro, pge causes many of the immune system changes following hs, yet the time course of pge release has not been well deliniated. the aim of this study was to determine the time course of pge release by peritoneal and splenic macrophages following hs. c h/hen mice were bled to a mean bp of + mm hg for minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at , , , , and hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for hours. the supernatants were collected and frozen until assayed. pge was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge release by peritoneal macrophages was significantly elevated at , and hours over control. it was maximal at hours, but by hours it had started to decline. splenic macrophage pge release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge is significantly elevated at hours following hs, and reaches a peak at hours. pge release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in patients ( male, female, mean age . ), admitted to our icu following serious trauma (mean iss= ) and in control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was . (range - ). patients didn't develop organ failure(nof-group), patients survived, developing mof (s,mof-group), patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days - . n and crp levels showed a parallel release peaks on the days - and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values - h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats ( g) divided in groups (observation time hrs): a: mild hemorrhage by sequential blood sampling ( . ml each)( , , , , , , , , hrs). b: a with hemoperitoneum (hp)( ml/ g), c: hemorrhagic shock: blood withdrawal of ml/ g over min and resuscitation with ringer's lactate ( x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); fe labeling of erythrocytas (application of ci fe i.a. into a donor rat, days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at and hrs, respectively ( + vs. + ; +_ vs. +_ ). hp reduced this increase ( +_ vs. + ; + vs. +- , p<. ). i-ip significantly increased hemoglobin between to hrs in mild ( . +_. vs. . +. ) and severe hemorrhage ( . +. vs. . +. ). in mild and severe hemorrhage comparable maximal resorption ( fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at hrs (mild: +_ vs. severe: +- counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: / ; hp: / ). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at hrs a significant higher transferrin level than with hp alone ( . +_. vs. . + . ). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during days after cct. antigenic challenge was made on day i, , or . number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day and reached a maximum when antigen was presented on day after trauma (pfc number . times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, & , prostaglandin e (pge ) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (> %.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output > cc's per hour had et levels drawn on admission, at i, , , and hrs. et levels were measured by radioimmunoassay. mean levels were elevated at ± pg/ml at all time points versus levels in healthy controls of ± . in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin these processes since it eradicates the influence of infectious factc~rs. dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il- , saa and neopterin, the percentage of cd + cells, the in-vitro il- synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of patients with cholecystolithiasis undergoing either laparascopic ( ) or open (i ) cholecystectomy on consecutive perioperative days - , , and . there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients ( versus minutes, versus days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i + lng/ml, d + ng/ml) versus lap group (d- + ng/ml, d + ng/ml), while in serum il- levels we saw a less steep increment in the lsc group ( -fold from d- to d ) compared to the lap group ( -fold from d- to d ). the analysis of cd + receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d - : . + . cpm, d : . + . cpm) compared to the lsc group (d -h . + . cpm, d h . + . cpm). in both groups il- synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors ( - and kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il- mrna and il- production was detected. the nucleotide sequences of the human il- gene regulatory region bounding by the splenic nuclear proteins were determined between + - b.p. the il- trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il- production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il- gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania non-pregnant patients from rural areas were followed. of preoperative patients % had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied patients of which % had a parasitaemia on admission and % had postoperative malaria. half of the surgical patients came from rural areas, whilst only % of those with malaria lived in the city (with much less exposure and immunity). % underwent major surgery and % minor. bloodtransfusions ( % with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about % of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that % was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co derived from [i- c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol . ~g/ml, glucagon #g/m , epinephrine ~g/ml, insulin t~u/ml; traumaeortisol . ~g/ml, glucagon /*g/ml, epinephrine ~g/ml, insulin ~ij/ml. analysis of twenty subjects showed a reduction of ° ~mp shunt activity by lymphoeytes and a ] % reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < . . lymphocyte glucose uptake was also reduced by trauma hormones p~ . . it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence f.neutr phils. there~bre, neutr pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln- (i ..- ) and mo~tocyte chemotacde pop, de (x¢cp- ) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from ipf and sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii. arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i ( . ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf ( . ng!mg) in comparises to . normal individuals ( . ng/mg) and to patients w~th obreic bronentis (cb) (~, rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp- e.qu/va]ent) was strongly increased in sareoidosis ( . ngjmg) as well as ~n f pag,nts ( . ng/mg). norra.al indlvidu~s and cb patiants hzd a . or -fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il- ievei~ ( . and . rig/rag, respe~veiy; nomzls: . rig/rag; cb: . ng/mg) mad nvatropmi ohemotax~ ( . ~'~d . nnmg, res!z~ztiveiy; aormals: . ng,'mg; cb: l ngmg). these data suggest that increased ievels of born mcp. ~d il- may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ ( fold vs. lps or paf alone), ll-lg ( fold), kc ( fold) and il- . taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day , hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays - and further observation and measurements at days - . from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight kg) neutrophil counts ( e pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood ( e cpm/ , pmn), and of blood-and bal-isolated pmn ( e cpm/ , pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca + second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in polytranmatized patients (mean injury severity score = ) arterial and venous blood samples during days. daily evaluation of horowitz-quotiant (po /fio ), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day and (day : +. ; day : . +. ). plasma lactate was significantly increased at day l ( + ) and day ( . + ). hurowitz-quotient (day : + ) was low at day ( + ) and day to ( + )(p<. ). at day a substantial rise in venous pmnl-superoxide production (day : . +_. , day : . +. , day : . +_. ), oecured with significant increase in plasma lipid peroxidation (day : . + . ; day : . + . ). pivin~-myeloperoxidase activity was high at day ( . +--. ) and then continuously declined (day : . +. ). plasma antiexidant activity (glutathione pemxidase) was reduced by % at day (day : . +. ; day : . +_. ; day : . +. ). whereas basal ca + concentration remained unchanged (day : +_ , day : +_ ), fmlp-stimulated cytosolic ca + mobilization increased at day (day : + , day : , day : + ). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca + mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, homburg/saar donnelly sc, haslett c, dransfield i, robertson ce, grant is, carter c, ross ja, tedder tf. dept's of respiratory medicine, accident & emergency, intensive care, surgery, university of edinburgh, scotland and dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n= ), perforated bowel (n= ), and multiple trauma (n= )), of whom progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o. ) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity ( _+ pmol/i vs _+ ; p - . ), but not the release of neutrophil related mediators ( . _+ . ng/i vs . _+ . ; p= . ). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb ( . + . % vs . _+ . ; p = . ). although not significant, nac prevented also the increase in elastase levels in lavage fluid ( . + . pmol/i vs . + . ; p = . ). it is concluded that nac is able to protect against the inactivation of a -proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion ( rain) and repeffusion ( min). sprague dawley rats were randomized into: control (n=i ); ischaemia repeffusion (ir) (n= ); and ir treated with gtn ( ug/kg/min) during repeffusion (n= ). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n= per group). control _+t "* _+ _+ ** bal pmn/mm l+- " + # _~ *p< . , #p< . v control/gtn;**p< . v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from . + . to . _+ . mcf after minutes repeffusion (p<. ). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin , ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, #g in . ml saline). three groups (n= each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh ( ~tg in . ml saline) at , , and hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f , = . , p<. ), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<. ). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<. ), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia ( mg/kg, i.p.) in female balb/c mice ( - g) that were then randomly assigned ( - mice per group) to receive at time and hr later: control saline thjectioas (i.p.), injections of ~-msh ( ~tg), gentamicin sulfate (i mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in patients ( female, + years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and min after declamping. arterial pla concentrations were higher than venous ( + vs. + ). while cytosolic basal pmnl ca + concenlxation was not altered at the end of ischemia (v: . + ; a: _+ ) and during reperfusion (v: + ; a: + ), fmlp-stimulated cytosolic ca + mobilization showed a significant arterial increase as compared to venous ( _+ vs. + , p < . ). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by ) pulmonary membrane damage possibly due to pmnl-degranulation, ) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and ) a significant arterial increase of pmnl cytosolic ca + mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca + messenger system by inflammatory mediators (e,g. ii- , tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p cell) is not only an important target, but as recently shown it is also capable of producing h . the aim of this study was to investigate the influence of endotoxin on h release of p cells. we obtained p cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h release of fresh isolated cells and on cells cultured for days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for hours with p.g/ml lps and tested the conditioned medium (mcm) on cultured p cells. p cells h ex vivo were _> % pure, _> % vital and had an basal h release of . _+ . nmol h per hour and million cells. adding p.g/ml lps to the incubation medium the h release decreases slightly but significantly to . _+ . nmol. adding . p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h release increased -fold to . _+ nmol. pma induced h release decreased to . + . nmol after addition of p.g/ml lps. after culture days the p cells were _> % pure and showed a pma inducible h release of . _+ . nmol addition of p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h release up to . _+ . nmol. addition of mcm to cultured p cells increases pma-stimulated h release to . +_ . nmol. the release decreased to . _+ . nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > % in all experiments. the results show that lps has an direct effect on p cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h release of p cells could be important for generating internal oxidative stress in p cells before external oxygen radicals exceed. the produced h did not necessarily damage p ceils, but it can effect surfactant metabolism, especially when extracellular h release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled patients with severe lung injury after sepsis ( ) or polytrauma ( ) and obtained multiple blood samples ( days) for endotoxin, tumor necrosis factor e (tnfa), interleukin (il- ) and interleukin (il- ) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l and il- were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, . ng/ml) for hours at c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao /fiao quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < ( . ± . eu/ml, mean values ± sem) versus the patients with a sli > ( . ± . eu/ml, p<o. ). the basal plasma concentration of ll- also correlates with the sli index (p<o.o ). the delta tnfe and delta il- values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n: ), the deceased patients (n= ) showed higher endotoxin level, a reduced tnfa and il- releasing capacity of the blood and higher basal il- levels. we can conclude that endotoxemia, high il- plasma level and a decreased capacity of the blood to release tnfa an il- under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il- in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado ) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($ or $ ) of the right and left lung through a bronchoscope on days l, , and days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado unexceptionally deteriorated postoperatively and the mean aado in patients reached the maximum (mean ± sem; _+ mmhg) on the rd postoperative day. the mean concentrations of il- , ne and neutrophil count in balf also increased postoperatively and reached their maximum level ( + pg/ml, -+ gg/l, and ± x cells/ml, respectively) on the rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il- and ne levels in balf (r= . , p= . ). also, significant correlations between aado and the neutropbil count, and the concentration of ne or l- were observed. in contrast to il- , neither il- , il- nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il- increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes ( mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats h prior to pma exposure ( pg/kg, intratracheally) and treated rats were killed h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp- ,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n- ) weighing .d- . kg oaselinepao> kpa and mean pulmonary arterial pressure (mpap)<i. kpa were included in this study. anaesthesia was induced with thiopental - mg/kg bw and "blind" jntubation was performed. a f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose mg iv or metylprednisolone mg iv min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap( . -+o.~ kpe ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp- or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients ( sepsis, trauma) with an fie of . + .( , a peep of . ± . , and a murray score of . ± . were included in this study. healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) . ~: . * . ± . *= < . , **=p< . condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io ), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of serum samples were collected from patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n= ) and non-septic (n= ) patients. patients with ards (n= , . + . ng/ml) and with respiratory disease other than ards (n= , . +- . ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n= , . _+ . ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using -hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase ( .i- . %) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej a - ~ia~ystok, poland, tel/fax + . in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) - ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. : , ) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n- -and n- -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf ( um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of -series lts and -hete (total sum of - ipoxygenade products rd. and rd. pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of - ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. pmofml). eicosapentaeooic acid ( um) elicited exclusive generation of -series lts and -hepe (total sum rd. pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived - ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. pmol/ml). we conclude that the paf-evoked - ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n- -versus n- enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr- , incubated with either pmn or mnc for hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin (cx ) which forms channels between cells capable of pas.~ing any molecule smaller than d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > adjacent hepatocytes within seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx is markedly decreased while the secondary gap junction protein cx is either unchanged or even increased. while cx readily effects electrical coupling, molecules > d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $ although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., and h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci , peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for , and hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after minutes of hepatic ischemia and hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to min of hepatic ischemia followed by reperfusion (rp) and injection of . mg/kg salmonella enteritidis endotoxin (et) at min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from + u/l (i/rp only) to + u/l at h rp. inhibition of kupffer cells (kc) with gadolinium chloride ( mg/kg) attenuated liver injury in this model by %, however, monoclonal antibodies (cl , wt ) directed against adhesion molecules ( integrins, cd ) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time ( + pmns/ hpf; baseline: + ). isolation of kc and neutrophils from the postischemic liver indicated a -fold increase of the spontaneous superoxide formation only in the kc fractions [ . + . nmol o -/h/ %elts (kc ); . _+ . (kca) ] at h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments ( h), the antibody wt ( mg/kg) attenuated liver injury by % at h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es- and gm- ) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type (scri), mg/kg iv after vascular occlusion (n= ) or by depleting the complement system using cobra venom factor (cvf), . mg im, and hours before ischaemia (n= ). non-ischaemic rats (n= ) and ischaemic non-treated rats (n= ) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c and c were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p< . ) and haemoglobin saturation (p< . ). c and c were present in the endothelium of the ischaemic control group. no deposits of c or c were found in the cvf group. few c and no c were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, control and cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to + mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by %.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [ mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < . ) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < . ); fhm ( < . ); and liver blood flow (p < . ) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < . ) in cirrhotic rots. tnf levels were higher (p < . ) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin (cx ) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx to % of sham controls. immunostaining of liver sections using anti-cx revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles hrs after animals were treated with lps, suggesting the internalization of cx without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at hr post injection . % ofpixels exceeded threshold, compared to . % in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca ÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca ÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats ( g, n>_ /group; pentobarbital mg/kg) with hemorrhage for rain at mm hg. reperfusion by ringer's lactate ( x maximal bleed out/ min) and % of citrated shed blood. tnfcz-moab (tn , ceutech, mg/kg in . % nac ) infused during flrst min of reperfusion. at baseline, end of ischemia and min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " hepatocyte incubation ( mg w.w./ml) with caci ( . + + + mbq/ml) for rain (ca influx [slope, /mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca + (ca +ex) [nmol ca +/mg protein]; ca + membrane flux [nmol ca +/mg protein'min]). during incubation, aliquots ( ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca +ex and membrane ca + flux were significantly increased at both, the end of ischemia ( . +. ; . +. ) and reperfusion ( . +. ; . +. ), as compared to sham-operated animals ( . +_. ; . +. )( <. ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca +ex ( +. ) and membrane ca + flux ( . +. )(p<. ). fast ca + influx was significantly increased by epinephrine in hepatecytes from sham-operated rats ( . +. vs. epi: . +. , p< . ). this hormone effect was not observed in isehemia ( . +. , epi: . !-_. ) or reperfusion (untreated: . +. , epi: . +. ; tnft~-moab: . _+. , epi: . +. ). conclusion. the present study clearly demonstrated hepato-cellular ca + overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca + fluxes and hormone ca + mobilization suggests disturbances of membrane ca + transport mechanisms, e.g. through ca +-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca + buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing - kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. . fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive % of tbs a third degree burn. eighteen hours after burn. gg/kg e. coli lps was intravenously administered over rain. ali animals were studied for additional hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc ) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc -indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc . inositol(l, , ) triphosphate (ip ) has been proposed as a second messenger for calcium mobilization. the addition of ip at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip receptor binding was performed with tridium label ip . the results shewed that the ip binding was significantly depressed by - % (p< . ) during late sepsis ( hrs after clp), but not in early sepsis ( hrs after clp). the ip binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p< . ), but not on smooth subfraction. since ip binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h ) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h# a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was consecutive patients with liver cirrhosis admitted to the department of surgery over a year period from january to december were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c , c ) were measured and functional assay for complement (ch ) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c , c and the functional chs complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch ) showed marked impairment in child's c patients (p< . ) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p= . ) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o per . ml medium fi +dmem ( : ) with % fetal calf serum for days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by - % (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than . times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., ) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., (kudryavtseva et al., , the ability of metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in consumption supported by nad+-iinked substrates, but showed almost no change in consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state oxidation of site i substrates and with the increase in state oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca + release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca + cycling was prevented, either by chelating ca + with egta or by inhibiting ca + re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca ÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally mmol/kg and mmollkg, respectively, and lps ( mg/kg) given h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase h after lps injection. the degree of hydrogen peroxide (h ) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [ ]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: .evaluation nfthe course of the ees due to the biochemic changes .determination of the influence of the ees on the activity of lysosomal enzymes in liver cell .deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: .activity of acid phosphatase in the venous blood serum. - .total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after hours of the experiment). conclusions . essential increase of activity of lysosomal enzymes was observed in the ees. . increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia ( min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. : g , ) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. : , ) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. : , ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats ( - g) were subjected to rain of hemorrhage (mean arterial pressure mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from . + . txmol/g to . _+ . ( min shock) indicating severe ischemia, and recovered to . -- . at min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by % compared to baseline levels ( . !-_ . i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was . + . nmol o -/min/ ecells (kc ) and . + . (kc ) in controls and did not change significantly after shock and rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after min of hemorrhagic shock and up to rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after weeks animals were sacrificed and livers were "perfusion fixed" with % glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as -fold of basal levels at the end of min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: ( ) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda ( ) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. ( ) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan , r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing - m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps( .gg. ) . rag/kg intraperitoneally. at the end ,:,f l,gand hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at - hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a c in wlstar rats was developed by tigatlng the common bite duct and injecting d. mt solution of e. goli % b, (g× ' efm/mt) into the bite duct. the mortality rate was ~ at h after aoc. at , , , h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at h after aoc. at h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p< . ), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at h after a c (p< . og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho ensis of hepatic dysfunction and m f following h . " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il- , il- , l- , i , tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il- and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: ( ) the same immurzoassay (elisa) yielded comparable results in different laboratories; ( ) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; ( ) assays for soluble receptors yielded less discrepant results; ( ) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately % for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p and tnf-r p . these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin- and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta- -microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, ) increasing neopterin levels predict rejection episodes in allograft recipients ) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv- infection ) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease ) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv- infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ -proteinase inhibitor (a pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage , m nchen, germany procalaitonin (proct) a aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as hours after the andoto:edn release and readied maximal level with a to b-fold increase h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii- , that reached peak levels at h and h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about daltons is unable to cross the kidney and the c_n barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau . ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i to fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp ) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin and interleukin (ill, il ). class acute phase genes are mainly controlled by ill and by combinations of ill plus il ; class genes mainly by ii, . the human c-reactive protein(crp), serum amyloid a(saa) and complement c genes will be discussed as examples of class genes, and rat c~ macroglobulin as a prototypical class gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class genes use the transcription factor nf-il or c/ebp as the key factor to mediate cytokine responsiveness; class genes use a different, so far unidentified factor to achieve responsiveness to il . ongoing efforts to purify this factor, called the il -response factor (il -rf), from rat liver nuclear protein extracts will be described. the il -rf apparently mediates the effects of il in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il as nf-il . the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex ~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf sioi to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to % and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than % when sepsis is present or when at least organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of to ml/ g/min are reflected in isoelectric eeg, and values about t ml/ g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about ml/ g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm , berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e synthesizing m~ and a simultaneous decrease of functionally competent cd + and cd + lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin- (il- ) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il- , most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper- (thl) secreting principally il- and ?-ifn and t-helper- (th ) acting principally in inducing antibody formation with a secretion of il- , il- , il- and tnf-[ has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u , tnf-c~, il- , il- ) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: % of all pmn's in this compartment show downregulated or non existent ~-integrins within hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr + receptors (cdll/cd complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+ ) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in- (il- ) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge . administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il- production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il- after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen - hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc gradients. supranorma[ delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. . myocardial depression with a drop in delivery (do~) . redistribution of blood flow between the different organ systems . inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ supply may therefore be inadequately matched with cellular needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april to august patients, aged from months to years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in out of these patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between - s and an activated clotting time between - s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four days. in the conventionally treated group % survived and in the additionally with ecmo treated group % survived, representing a % survival rate in patients whose risk of death is considered more than %. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med ) . in contrast, mean skeletal muscle po was found to be abnormally high in patients with sepsis ( _+ , mmhg, n= ) compared to patients with limited infection ( , + , mmhg, p< . , n= ) , to patients with cardiogenic shock ( , + , mmhg, p< . , n=ls) and to healthy subjects ( , _+ , mmhg, p< . , n= ). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po was higher ( , _+ , mmhg) on days with severe sepsis than on days with intermediate state ( , _+ mmhg) and than on days without sepsis ( , _+ , i mmhg) . thus, a decrease of abnormally high skeletal muscle po was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po with the decrease of apache ii score or elebute score as well as interleukin- serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il- and c a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on consecutive pat. after elective open-heart surgery, we had determined ele daily for at least pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n = patient-days, both ele and sirs showed significant (p< . ) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: . vs. . ; icu treatment days: , vs. . ; days on mechanical ventilation: , vs. . ; days with vasopressor requirement: . vs. . ; number of microbiological findings: . vs. . ), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of . for ele (ele >_ on >_ days, accuracy: %) compared to . for sirs (sirs present on > days, accuracy: %). accordingly, ele roc curve areas for both overall ( . ) as well as sepsis-related prognostic evaluation ( . ) were significantly (p< , ) larger compared to sirs ( . and . , resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate ( % and %, compared to % and %, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. , d- munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score , while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation . four of the nine patients died. apache iii scores at h were lower in survivors (s) than in non-survivors (ns), ( -+ vs -+ p< . ), while apache iii scores at admission were not significant different between s and ns ( -+ vs -+ ). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an fold increase of tnf-a bioactivity with[r~ hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il- [ and il- immunoactivity. tnf was measured by wehi bioactivity, il- [~ and il- immunoactivity were determined by elisa. the sensitivity was pg/ml for tnf, pg/ml for il-ll and pg/ml for il- , respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day , p< . , the pattern of cytokine production by pbmc upon lps stimulation over the first h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin- (il- ), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, polytraumatized patients were prospectively studied (mean age y, % male, iss ). serum and edta-plasma samples were taken in h intervalls until the patient left the icu. il- , tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, ) with at least of clinical signs: ( ) tachycar-dia> /min, ( ) temperature > , °c, ( ) blood pressure < mmhg, ( ) mechanical ventilation, ( ) leukocytosis > . /ml, ( ) thrombocytopenia < . /ml and ( ) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day after trauma. results: of patients developed a systemic sepsis ( . %), and died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il- ( vs. ng/l; p= . ), crp ( vs. mg/l; p= . ) and tnf ( vs. ng/l; p= . ) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il- , tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n= ) was determined by the injury severity score (iss). lidocaine is given at a dose of mg/kgbw over rain. i.v. and is metabolized in the liver by a cytochrome p- mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the ~t and the ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin- (il- ), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~ , stnfr ) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il- , tnfot, stnfr , stnfr ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il- was detected in an biological assay using the/l- dependent -cell line / . detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (> mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with ( ± mg/l) or without ( a: ) multiple organ failure (mof) nor in survivors ( ± ) or non-survivors ( :t: ). in contrast, l- was elevated in patients wilh mof (mean pg/ml, range - pg/ml). il- levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il- and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r= . ). patients could be divided into two groups by values for stnfr~ and stnfr : the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il- correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg , kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in consecutive patients with multiple injuries (iss . ) the inflammatory response to trauma was prospectively studied. the patients were divided into groups according to their iss points. additionally, the alterations after secondary operations (> hr) were determined (msteosynthesis of the femur (n= ), pelvic girdle (n=ll) and spine (n= ), facial reconstruction (n= ), smaller osteosynthesis (n= ) and others (n= )). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every h, beginning on admission of the patient to the emergency room for a period of hr, and in the operative patients on the morning of the operation, at the end of the procedure and every hr during the first two days. results: lactate, neutrophil elastase, heart rate, po /fio -ratio, and other parameters discriminated significantly between the injury severity groups during the first hr (kruskal-wallis-test, p<. ). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<. ) between the types of operations for lactate, heart rate, po /fio -ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss to ) or moderate (iss to ) accidental trauma for neutrophil elastuse, heart rate, po /fio -ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~ years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a -year period, patients were consecutively admitted into the icu; and during a -year period, patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o ), sepsis (p<o.o ), and mortality rate ( % vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o ). of the total group of , patients, there were ( %) patients years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o ). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients ( % vs. % in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of . ( % confidence limits, . - . ), age did not increased [odds ratio . (i. i.i )], while admission to the sicu reduced risk of death by a factor of . ( . - . ). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan , ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il- , l- ,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n= ) and non-survival group (n= ).tn these patients,ll- , l- ,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were points in survival septic group, l l points in non-survival septic group and points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p< . . results: il- levels in non-survival septic group were higher than in both survival septic and los group.ll- levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/ ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha- -maeroglobulin, c estemse inhibitor, antithrombin ill and alpha- -antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in patients and continued postoperatively in patients, % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication - days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by - days. dept. of surgery m'alm general hospital, s- i malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of patients admitted with burns covering more than per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: out of burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were . -- . eu/ml, significantly higher than that of patients without mof ( . - . eu/ml) on , , and days postburn (p< . -- . ). significantly more incidence of positive endotoxin test (>_ . eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p< . ). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp- , and gp ) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs expression) was significantly elevated (p< . ) and correlated well with severity of disease (f . ). no significant change in surface expression ofthrombospondin, gmp- or gp was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (! < . ) that correlated well with severity of mof (gmp- , r= . ; thrombospondin, r= . ).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a -year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period - i patients with abdominal sepsis were treated at the icu at our university hospital. patients were women and men with a mean age of ( - ) years. in cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for (- ) days out of which (- ) in the intensive care unit. out of patients ( %) died in median after ( - ) days. the primary cause of mortality was multiple organ failure ( / ; %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin ( %) and in % cultures grew multiple bacteria. patients bad organ failure and multiple organ failure. / patients ( %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about / of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january to september patients, mean age + years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for . _+ . days. diagnosis of sepsis was confirmed in cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, -patients with sepsis, the control group consisted of patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c ,c ), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c (mg/dl) c (mg/dl) k +- + _+ + +- -+ " -+ * _+ " -+ ' _+ " +_ '* -+ ** -+ "* -+ "* _+ " in a prospective study we investigated serum of severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n = ) was investigated as. well as serum of patients undergoing elective herniotomy (n= ) hours before (op i) and hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain :k :h . / suspension with a final concentration of - cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co . _+ . opi . _+ . opii . _+ . * tri . _+ . "* trii . + . ** (*:p< . ; **:p<o. ) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied patients with sls (group i: age . ~ . years); they were compared to control patients (group : age ± . years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone ( mg/ hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in pts. preoperatively pts in group revealed a significantly lower cardiac index ( . ± i/min/m ) compared to group ( . ± . i/min/m ; p < . ) higher left atrial pressure (group i: . ~ . mmhg; group : . ~ . mmhg; p < . ) and lower ejection fraction (group i: . z . %; group . ± . %; p < . ). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: , ± , /ui; group : , ~ , /ui; p < . ) and core temperature hours pop was higher (group i: . . °c; group : . ~ . °c; p < . ). serum lactate was already elevated on arrival on the icu pop (group i: , z . mmol/l, group : . ~ . mmol/l) but dropped within hours (p < . ). icu stay (group i: . ! . ; group : . ± . days; p < . ) and mortality over months (group i: . %; group : . %; p < . ) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. patients who were admitted to the intensive care units between january and july ' with polytrauma, sepsis or those requiring ventilatory support for more than hours were selected for the study. the patients were randomized into groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored hourly and the patients underwent an upper gi endoscopy hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the patients selected for the study, had undergone cardiac surgery, had polytrauma and had sepsis. the change in average pre and post drug gastric ph was + . in the omeprazole group, + . in the ranitidine group and -). in the placebo group (p < . ). endoscopic evidence of stress ulcers was seen in % of patients not on prophylaxis, . % of those on omeprazole and . % of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, % of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < . ). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin- (il- ) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn- receptors (sil- r). the aim of this study was to evaluate: the significance oftnf c~, il- and sll- r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il- and sll- r. we examined patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, ,- and sil- r were measured in septic patients and in adult healthy controls. tnf ct, il- and sil- r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il- and sil- r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll- r serum levels were significantly (p< . , student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ] ,- levels. we conclude that only the increased serum sil- r levels were predictive of bacterial sepsis within hours after the first septic signs. tnf c~ and il- levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed children with light and children with severe course(l- class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch , level of c -c , factors b told d, c a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group ( %- st and ioo%- nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c , c components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~ ) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr der j, braun j, rennekampff ha, schr der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in trauma patients with an injury severity score (iss) > (mean iss = ; mean age years) lymphocyte and neutrophil phenotypes cd (t-cells), cd (t-helper cells), cd (t-suppressor cells), ratio cd /cd , cd b (receptor for cr ) and cd (fcriii) were measured on day , , , , and post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd ) and il -receptors on t-helper cells (cd /cd were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd were significantly lower an day , , and in patients who developed mods (p< , ) compared to patients without mods and a healthy control (p<o,o ). the expression of il receptors on cd was significantly different only on day . no differences could be measured in lymphocyte phenotypes cd , , and cd /cd -ratio as well as in expression of cd b and cd on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > /zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the consecutively admitted patients to a surgical and a medical intensive care unit during -ye r period, ( %) had arf. arf mortality was %. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o, ). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p< . ), the number of osf before was higher compared to after arf in both groups of patients (p< . ). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p< . ). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p< . ). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama ; : z - ~ ) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of , recent admissions to hospitals in the united states and western europe, we identified , patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors ( % of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original , patients with a receiver d_perator characteristics (roc) area of . and in two independent databases, the first independent database contained hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc= . ); in the second, the placebo patients withtn the recently completed phase ill e~aluatlan of il- ra (rocffi . ). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low % to a very hlglt %. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il ( ) -to use a model for risk of death either from a score (apache ii, lii, ( ) saps ii) or directly (mpm ii system ( )) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system ( )) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii ) before any therapy. -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: ( ) toxicity models with injection of bacterial endotoxin or exotoxins; ( ) live or killed bacterial challenge models; ( ) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: ( ) hemodynamic parameters; ( ) modulation of host-derived inflammatory mediators; ( ) resolution of end organ injury; or ( ) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in %- % of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field ( ). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa pi, tat), cytokines (il- , il- ), and soluble intercellular adh sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il- , il- and tnfe) as well as breakdown products of complement proteins (e.g. c a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, ) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd mab wig was able to down-regulate production of il- , il- , ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin- receptor antagonist (il-lra) but not of il- ; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il- ), some only by solid-phase detection systems (anti-ll- , anti-ifn% anti-tnfc . infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il- in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il- , and il- . self limitiation of this process might be due to demonstrable instant release of il- ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il- , il- , tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. ( ) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi ,hokkaido,japan $ immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h mon departmen~ of surgery, san frandsco general hospital, urdversr , of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi is down-regu/ated, along wl~ cd and cd . thus responsiveness to lipopolysacchmdde ( ) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e . in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge . ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s! otlse. by examining m n cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il- was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than pg/ml (normal: less than pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il- level and the number of organs showing failure (r= . ). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p< . ). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i- - , kumamoto , japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock ( ). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of mg/ rain, followed by a continuous infusion ( mggh). with hemodynamic improvement (dopamine < gg/kg/min) the infusion rate nfhc was tapered over a period of days. phospbolipase a (pla ), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after days dopamine requirements decreased to less than gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day and ) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla , and finally by the relapse of septic shock in four patients on days , , and o. a second infusion of hc again resulted in shock reversal and in a decrease in pla , crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il- , il- , and il- is attenuated by- rag hc prior to endotoxin challenge in humans ( , ) . cytokines like tnf, il- , il- , and il- are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: patients with an iss nf more than points who could be treated with at iii administration within rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of % by using the following calculation: at iii to administer = ( % -at iii measured) x body weight. at iii was given every six hours for a period nf hours and on the th day. patients were followed up throughout their stay in the icu but at least days. in this abstract the data of the first patients are included. results: the patients' median injury severity score was points with a median age of years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received , units of the inhibitor on average during the first days. with our regimen, the at iii levels could be kept between % and % of normal, whereas control patients an at hi concentration of % to % was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to % of normal. white blood count, pmn--elastase, interleukin and and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii group ( . vs. . days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of %, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. patients with severe multiple trauma ( treatment [at-ill] -- controls [c]) were investigated. study cdteda were age > and < years, injury severity (iss) > points and glasgow-coma-scale > points; randomization and treatment has to be started within hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp- , a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp- in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. h later bk was injected ia and the pain score ranked from (no responses) to (vocalization). cp- at . umoles/kg completely inhibited the pain responses for a period of . - h. cp- at . umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a - h period by an intraplantar injection of . % carrageenan. the abdominal constriction response o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp- . when ul of . % formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp- significantly inhibited both the first ( - min) and second ( - min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at °c for sec the increase in paw volume was significantly reduced by pretreatment with cp- , . umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (- °c for sec) to the dural surface following a craniectomy. cp- at . umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp- , may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi , a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats ( - g.) received a % liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either . mg/kg/hr rbpi (phx-bpi, n= ; sh-bpi, n= ) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n= ; sh-con, n- ). various parameters were measured h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi . blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il- levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at - rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of - mmhg for rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. rbplg. was administered at a total dose of mg/kg i.v. ( . mg/kg at the -eginning followed by two doses of . mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi . results: imrffe?diately after resuscitation ( min) the detected plasma endotoxin levels in the control group (mean = , range = - pg/ml) were almost neutralized by rbpi treatment (mean = , range = - pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points and min of experiment (means: and in bpi vs , pg/ml in the control group). the -hour survival rate was improved from / ( . %) in the control to / ( %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. rabbits weighed + grams divided into two groups. group i included tabbits were treated with allopurinol mg/kg for seven days before induction of haemorrhage. group ii as a control included rabbits. all rabbits were subjected to % arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was out of while in group ii it was two out of . our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, pigs were first anesthetized, then injured with joules of energy to the posterior thigh, then hemorrhaged - % of their blood volume. after i hr of shock, all the shed blood plus x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine ( . mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after hrs, a septic challenge was administered ( bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for min. baseline values and units were cardiac index _+ ml/min/kg (ci), arterial po + mmhg(pao ), base excess . -+ meq (be), physiologic dead space fraction +_ % (pds), and tnf . + . units/ml. baseline gastric ph was . -+ . and . _+ . in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao * be* gastric* pds* peak* rain rain rain ph min tnf ranitidine _+ _+ - . ± . bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin- (ii- ). this study was designed to clarify the role of lps and - in the metabolic response to bum injury. twenty-five burn patients ( -+ %; + % ft bsa burn; _+ years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i - were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b ( , u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic ( . +_ . pg/ml; normal < pg/ml). i - did demonstrate a significant correlation with cole temperature (tr~ = . + . ogi - , p= . ) and with nitrogen excretion (nou t = - . - . ogi - + . tr, p= . ). administration of polymixin b had no effect on metabolic rate, temperature or i - levels but did reduce nitrogen excretion resulting in more positive nitrogen balance ( .t grn/day vs. - . gm/day, p= . ). although bum injury does not produce an obligatory endotoxemia, i - does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within rain and shock state was hold for h at a map of mm hg (cardiac output of %). following adequate resuscitation with % of shed blood and twice of this volume as ringer's solntion, animals recovered to map > mm hg and co > %. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at , , or hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [ mg/kg, celltech, uk) , pentoxffylline (ptx, mg/kg, hoechst, d), and a paf antagonist (web , boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n= - group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence - of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from , to about pglml, but did not abolish it. the inhibition of the lps( . ug/ml)-induced tnf production occured already in presence of , pg peptide/ml and peaked at the concentration of , ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp- and tp- ), thymocartin did not enhanced the pwm-induced pge production in pbmc cultures up to a concentration of ng/ml, inclusive. the selection of thymocartin (tp- ) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about % registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: patients (pts) formed control group a. group b pts received x mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d ) intravenously to block prostaglandin e induced suppression of cmi response as well as mg thymopentin (tp- ) subcutaneously preop., on d and d to augment cmi reactivity. in vitro investigations preoperatively, on dl and d included measurements of the percentage of cd + t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)- as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il- as one cytokine primarily produced by th -cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d were used as in vivo tests for thl-and th -induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd + th cells, il- /il- ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p< . vs.bl). in group b pts no change in cd + th cells, il- /il- ratio, or dth skin response was observed (p< . vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: .these results clearly indicate a significant suppression of th induced cmi response, while th induced response remains normal following ohs. .these alterations may gain clinical significance whenever a postoperative infection requires a th response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing - g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il- . they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from to . results.it was found that in % of infants with - classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then times, ratio th/ts was reduced to . or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c a subcomponent were increased but the level of c , c ,, c and c components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i- mcg/kg per day and mg/kg per day accordingly during a week in children with - classes el heaviness. aiter - days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of ( - )-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c h/hen or c h/hej mice, to wkolds of both sexes were used through experiments. highly purified ( - )-{ -d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b- . induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c h/hen and c h/hej mice, whereas lps only induced such activities in c h/hen mice. most effective dose of bgc was ug/ml. ) bgc-dependent proliferative response of c h/hen splenic cells was not detected under the culture condition used. ) induction of tnf~ prodction was apparently observed in bgc-stimulated c h/hej pd cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~ activation, intracellular signaling pathways of p~ by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il- in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il- . under anesthesia, -weekold male c h-hej mice were subjected to a % scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of organisms of p. aeruginosa hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells ( x ) intraperitonelly after septic challenge. the mice were scored for survival daily. on day after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il- and il- in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il (n= ) were raised to . _+ . and _+ pg/ml, while the levels in lak treated mice were reduced to . _+ . and _+ pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included operated patients, of which received mg indomethacin before and , and days after surgery (group a) and patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd ), suppressor (cd ), nk-cells and interleukin- and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day and , and days after operation. in group a we detected a significant (p< . ) increase in cd /cd ratio on day while no differences were ~oted in nk-cells or cytokine production in both groups (table ) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. female cd- mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p< . vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p< . ). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p< . ). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin , ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h -antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. -aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi )(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p< . ) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi and plasma cholesterol (p< . ) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, -aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, x u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. -hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the -aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in neurosurgical centers and included patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and groups of tirilazad in escalating dosage ( . - mg/kg/day). the clinical outcome was compared for these groups and cerebral a~giography was performed in % of patients on day - follovang sah, to study the effect f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z patients sufferin s from severe ap (c-reactlve protein > me/l) we determined on day and day after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i patients were randomly treated with ug/day of se for days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only / of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains % hes and % chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats ( - g, n> ; pentobarbital anesthesia mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate ( ) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and ( ) whether dfo-hes could prevent this tissue damage. methods: in rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to groups: one group was treated with ml dfo-hes ( mg/kg iv), the other rats received solely ml of the carrier starch solution. , , , and min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> . eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p< . ) in the lies group already min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p< . ). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p< . ) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca + levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose ( mg%) and bovine albumine ( gm%) for min at °c.it composed of groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo& mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were % in group ab and % in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a , leukotriene b and leukotriene c are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after , , , and minutes plasma levels of thromboxane b (txb ), leukotriene b (ltb ) and leukotriene c (ltc ) were determined via elisa technique. another volunteer had taken mg aspirin one day before taking the blood sample (no ). results: txb plasma levels increased from pg/ml at min to pg/ml, pg/ml, pg/ml and pg/ml at , , and min (p< , ) . ltb and ltc plasma levels showed an increase during the first few minutes (ltb : min: llpg/ml, min: pg/ml; ltc : min: pg/ml, min: pg/ml (p< , )) followed by a decrease to normal values at min. in the sample no the cyclooxigenase-pathway was completely inhibited, the txb plasma-levels did not alter at all, whereas ltb and ltc -plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il- secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il- productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early th century, however the protein was not isolaled until . the human gene was identified and cloned in , which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a - day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet ; : - ) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p< . ) in patients receiving allogeneic blood (n= ) or operated without blood transfusions (n= ). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n= ). in contrast, specific igg-levels increased sigmficantly (p< . ) in patients receiving allogeneie blood (from . + . to . _+ . ie/ml) whereas in patients receiving autologous blood a smaller increase (from . + . to . + . ; p= . ) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l . + . to . + . u/ml) compared to those patients with autologous blood ( . + . to . + . ). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin- (il-i), interleukin- (il- ) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during - hours after surgery in patients undergoing arthroplasty ( hips and knee). kf, il- , it- , thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il- concentrations ~ere analysed at the beginnlqg and end of the - hour blood retrieval period. in a separate study ( hip arthroplasties) f~f, il-i and il- ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il- coocemtratiems rose from a median value of to pg/ml, p<o. , during the blood ret.rieval period. in c was detectable in patients, range: - pg/ml. il-i was not detectable in the patients. in retrieved blood tag was > mg/ml in all samples (ref:< . mg/ml) and at was . - . units/ml (ref:o. - . ) . the il- concentrations in retrieved blood was > pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n= ), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: - pg/ml) and il-i (range: - pg/ml) ~re present in all samples but ii- (range:o- pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients ( . %) were perioperatively transfused. thirty-six ( . %) patients were given autologous blood , while ( . %) received homologous blood. no patients received both autologous and homologous blood. twenty eight ( . %) patients developed postoperative infections. non transfused patients had a . % infection rate , those receiving autologous blood had a . % infection rate, whi]e in the homologous blood group the infection rate was . % (p < . ). univariate analysis showed that infections were significantly related to operative blood loss (p< . ), length of operation (p< . ) blood transfusion (p< . ) and attending surgeon (p< . ) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, month old rats were transfused intravenously with syngeneic heparinized venous blood ( x ml, every other day), and , and days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a : : . : . mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of . microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over - hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of - -weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il- . we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium- m ( mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a % topload of leh ( mg of phospholipid, . g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, % at minutes and % by hours. the spleen accumulated activity at a slower rate, % at minutes and % at hours. at hours, autopsy biodistribution studies revealed that approximately . % of the dose is in the blood pool, . % in liver, . % in spleen, . % in lungs, . % in muscle and . % in urine, with trace levels in kidney, brain and heart (< °/o). in a hypovolemic model, rats were % or % exchange transfused with mtc-leh. in the % exchange model, mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at hours. with the % exchange, % of the leh was in circulation at hours. the interaction of leh with platelets labeled with indium- was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha ( ) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens ( ) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine ( ) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength ( ) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t - years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. o]~ga- pufa there continues to much interest in the application of the mega- pufa in clinical nutrition. the basic principle has been that the mega- pufa will displace arachidunic acid and result in a decrease in eic san id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega- incorporation with continuou~ enteral feeding both in control and endotoxic animals within days. this includes the liver, spleen, circulating and alveolar marc phages and the lung. this incorporation resuls in significant changes in the eicosan id production including pgf and ket -pgflalpha. there is improvement in the cardio-vascular reep nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of mega- pufa. clinical studies using short term entsral nutrition with mega- either alone or with other enteral supplements in a number of clinical settings have shown significant mesa- incorporation and decreased eicosan id production. these positive results must be discussed with the additional evidence that long term omega- supplementation decrease eic san id production but als induce a state of immune suppression that is capable of increasing transplant sunvival. these ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf- mice aged - wks received % tbsa fullthickness dorsal burns & were resuscitated with cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast hrs, chow hrs). at hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p< . , ** p< . compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of adult icu patients who required enteral feeding for > days. patients entered the study within hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least ml/hr by hr after event. results. both groups tolerated administration of formula well. for patients fed > days, the median los was % shorter (p=o.ol) for the--supplemented group ( days) compared to the conventional group ( days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o ). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia ( vs. days) and skin/soft tissue infection ( vs. days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into groups ( n= each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within hours following surgery ( ml/hour). it was progressively increased reaching a full regimen on day . on hospital admission and on post-operative day and , the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day , , . the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day . comparing post-operative day versus day a significant increase of prealbumin (p< . ) and rbp (p< . ) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p< . ) and a significant recovery in delayed hypersensitivity response (p< . ) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent ( % in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = % vs vivonex ten = %), mean mof score (immun-aid = l.b vs vivonex ten = . ), or mortality (immun-aid % vs vivonex ten = %) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> ), patients receiving immun-aid appeared to have fewer septic complications ( % vs %) and their mean mof was significantly lower ( . _+ . vs . + . , p = . , student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from patients in septic shock and heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for minutes. pmn (p{o, ) and monocytes (p<o, ) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the %-bcaa vs %-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o for all). clearances of bcaa increased with increasing bcaa dose (p<o.o ); arg and gln clearances were directly related to the bcaa clearances (rz=o. , p<o.o i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation ( gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with x t.spiralis larvae. thirty to fifty days later rats received gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o mucosal mast cells and their subsequent degranulation. mast cell-derived -ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group ( % vs %). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m- polyunsaturated fatty acids (pufa) from safflower oil (so) or (o- pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at % in the diets was adjusted to provide % each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for days, lipopolysaccharide (lps, mg/ g) was injected through the tail vein. the percent of animals surviving in the mo group was % ( / ), and did not differ significantly compared to % ( / ) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so ( . + . ng/ml) and those fed mo ( . + . ) did not differ significantly. the levels of prostaglandin(pg)e , -keto-pgflc~ and tumor necrosis factor-ct (tnf-c were determined min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and h after the lps administration (lps+). the data (mean_+sd; n= ) are as follows. group these data indicate that a marked reduction in the plasma levels of poe and -keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a . % reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf- outhred female mice ( - g) were fed purina # chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from to days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a- desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for weeks. the incorporation of dihomo-y-linolealc acid (dgla: : o)- ) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between - % compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps ( p.g/kg body wt), the plasma levels of both pge , and txb , were significantly decreased (p< . ) by nearly % for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a- desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a (pla ) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was % ( / ) which was markedly higher (p< . ) compared to only % ( / ) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly % reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c bl/ mice that have been stressed by immobilization. the proteins have been labelled with ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of ss-methionine reveal about protein spots, from which a small fraction -about are altered in both qualitative and quantitative manner. the major changes are in the range of to . d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery days and days preoperatively and on the ist and th postoperative days (study-days i, , , and ) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived days preoperatively an enteral nutrition enriched with omega- fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a ( . pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb (mean values . ng/l x i~ cells) compared to the placebo group ( . ng) at study-day [p, . ). in contrast, the capacity of neutrophils to produce ltb was not significantly different in both patients populations at study-days and . the omega-oxidation of ltb to its biologically less active metabolites oh-ltb and cooh-ltb was not significantly different in both groups. however, neutrophils from group f patients generate more ltc at study-day (p( / ). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega- fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega- fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega- fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution ( kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn ( , g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide ( , g n derived from ala-gin = % gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o, ). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p< , ). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p< , ). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o ~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai y erodes both fimetianal and stm~ tissue. wiu _b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~ po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh iu daily three days before the experiment (gh- ), another group treated with gh ie at the start of the surgical procedure (gh- ) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u- c-glutamine. substrate fluxes were measured one (lh) and five ( b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh- : . + . mg/kg, gh-i: . +_. . mg/kg, con: . +- . mg/kg, p= . ). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh- : . + . gmol/min at lh and - . + . in.tool/rain at h, gh-i: - . + . i.tmol/min at lh and - . + . pmol/min at h, con: - . +_ . g mol/min at lh and - . + . /amol/min at h, p= . , p= . ), whereas the absolute uptake of glutamine in hindleg was unchanged (p= . ). glucose uptake in the hindleg was decreased (p= . ). net glutamine uptake in liver was attenuated (p= . at h), whereas net glutamate release from liver was increased (p= . ). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of , ; , or , i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from . calories per kg body weight to . calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus . to plus . g n / days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either u of rhgh bid (sc) or no alternative medication. the study period lasted days. from daily hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days and to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf and igf , igfbp were measured daily during the study period, on day a hour profile of gh was.performed. samples were collected each minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf differed significantly between the groups for the whole study period, but not igf and igfbp- . the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n= , age range - yr, bmi . + . kg/m ) or for days additional gh ( x i.u.s.c.) (gh group, n= , - yr, . + . kg/m ). metabolic studies (calorimetry and lsc-leucine infusion) were performed (test a) and days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the -week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was + ; + (co) and + ; ± (gh) pmol/kg ffm/h (n.s.). protein breakdown was ± ; + (co) and + ; + (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was ± ; + (co) and ± ; + (gh) pmol/kg ffm/h (p< . for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days - : gh + vs co ± g; p< . ). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf- levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf- . this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: patients (both sexes, age: - years, bmi - kg/m ) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received ug rhlgf- /kg body weight/twice daily or placebo during treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition ( g chng, o. g aa/kg) troughout the whole study period. cumulated nitrogen balance, -methyl-histidin exretion in urine as well as serum-igf- levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to groups (n= /gronp): gh-high ( + mg/kg/day), gh-iow ( a mg/kg/day) or control (saline). following subcutaneous treatment (every hours for days) with gh or saline, mice were challenged i.p. with e.coli (lxl /body). survival rate was recorded every hours. in the next experiment, gh-high and control animals were sacrificed hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for hours with lipopolymccharide ( p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf- ) and the insulin like growth factor binding protein (igfbp- ) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf- and igfbp- . materials and methods: patients sheduled for esophagus resection were investigated once they had given written informed consent. patients showed acute alcohol abuse and were treated with clonidine postoperatively• patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf- and igfbp- were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf- and igfbp- levels showed no differences between the groups. discussion: igf- and igfbp- plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n= , mg/kg/day), low dose igf-i (n= , . mg/kg/day) or saline ( = ) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for hours with lipopolysaccharide ( ).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l bioassay. results igf-i improved the survival rate at a dose of mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than /~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated patients ( scn, cyclic neutropenia) with r-methug-csf (filgrastim). thirty of patients with scn and all cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than /~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between . and ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations hours after administration. these levels were -fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately - -fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day of gestation (parturition in rodents occurs on approximately day ) survived a lethal challenge of group b- hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res : , ) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood : , cairo, pediatr res : , ) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. infants < hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm ( - wk), preterm ( - wk), and term (> wk) and randomized to receive either a placebo or , , and gg/kg/qd or and p.g/kg/bid of rhg-csf infused by pump over hour for consecutive days. cbcs were obtained at , , , , and hrs. tibial bone marrow aspirations were performed hrs after study entry and differential counts and cfu-gm pools were determined. c bi expression was determined at and hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at , and hrs following administration of both and ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred hrs after and ~tg/kg/d ( - %) (p< . ) and ( % -+ %) (p< . ), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool ( _+ % vs. + %) (p< . ) (placebo vs. ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo ( . _+ . vs. -+ ) (colonies/l(p cells/plate). c bi expression was significantly increased hrs after bg/kg/d of rhg-csf ( + % vs. +- %) (p< . ). peak serum g-csf levels occurred at hrs and were dosedependent. the half-life of rhg-cse was . + . hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. of patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research. : , ) . methods: awake ylo beagles had serial cardiopulmonary and laboratory studies before and for up to days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until days after clot placement, animals received high (n= ) or low dose (n= ) g-csf or protein control (n= ) subcutaneously. results: survival in high dose g-csf animals ( / ) was significantly improved compared to low dose ( ) and controls ( ) (p< . wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day after clot, p< . ) and mean arterial pressure (day , p< , ) compared to low dose and controls. high dose rcg-csf increased (p< . ) peripheral neutrophil numbers both before and after clot implantation ( hours to days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p< . ) rise (day ) and fall (day ) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin ( to h, p< . ) and tumor necrosis factor (tnf, h, p< . ) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and or hours later. lps-inducible tnf, il- , stnf-r p and il-lra were assessed in the supernatant of whole blood incubations stimulated with ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about % hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p which was indetectable in blood incubations from untreated donors increased dramatically hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as /.tg/kg iv every h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either or days before or hours after cp. results. mortality was reduced from % to about % with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin and lactate were attenuated the first hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin and lactate are suppressed the first hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels h after lps administration. gm-csf pretreatment ( ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin- (il- ) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il- -tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in hs. continuous infusion for days. safetyandefyieacy will be assessed till to day . the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > (score corresponds to expected mortality rate of %).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than , were included in the study. filgrastim was given by daily continuous intravenous infusion for days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > apache i score points on day after onset of treatment. results: none of the patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to . + . /~tl (mean + sem) and reached a level of . +_ . /tal at day after onset offilgrastim therapy. the apache ii score initally was + . (mean + sem) and as an indicator of filgrastim response a decrease of points ~dthin days oceured in out ot patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= ) or colorectal (n= ) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il- , which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of pg/ml (preoperative < pg/ml) on day and day to after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < , ) correlated with infectious complications in patients whh gastric cancer (n= / ). secondly patients could be arranged into two groups according to an anergic (n= ) or normergi¢ (n = ) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= / ) or colorectal (n= / ) cancers. interestingly we found a significant correlation (p < , ) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= ) within the pre-and postoperative period resulted in reduced mean values (about %) for ifn-ff release (preoperative means llo pg/nfl), if compared to patients with normergic dth (n= , preoperative means pg/ml). similar, but less significant results were obtained for il- secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il- ) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s orl o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe -gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)( m$/k bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after weeks was significantly decreased by administratlon of rh~-csf(p< , ).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached . %. nextly,treatn%ent wlth rhg-csf(s ~ $/k body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p< . ). iv~oreover,functions of neutrophlis which were phagocytic activity and h p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~( ~ /ks b ody wt) (p< .( ). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < /mm ) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < /ram ) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of to mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within to days. in most of them, spectacular correction was observed within hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed ( cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, , rue de s~vres, hopital necker, paris, france. changes in serum g-csf and il- after surgical intervention hitoshi toda , atsuo murata , hidewaki nakagawa , takesada mori , nariaki matsuura osaka university medical school, osaka, wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin (il- ) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il- and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l- was less than pg/ml and g-csf less than pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il- levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il- (x) was y= . x+ . (r= . , n= , p< . ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il- was pg/ml and g-csf pg/ml respectively. the estimated value of g-csf was pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than were treated with filgrastim ( . - l.tg/kg/day) for prophylaxis of sepsis on days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced ( - reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of % +- % ( % - %) compared to pretreatment values of %. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p< . ). before initiation of csf treatment urinary neopterin was similar in both groups of patients ( +/- and +/- lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day the mean value was about % above the basal level (p< . ). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than fold induction was noted and the difference between g-csf and gm-csf was highly significant (p< . ). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp- human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r a and pot ca . john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, . at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i. ], dmpg[ . ], cholesterol [ . ] , and monophosphoryl lipid a [ - gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected - days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing , or gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds for control (liposomes with no lipid a) mice was x bacteria; ld for mice vaccinated with p.g was x ( -fold increase in resistance) and with ~tg was x bacteria ( -laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld for control (liposomes with no lipid a) mice was ng lps; the ld for gg lipid a was rig lps ( -fold increase in resistance) and for xg was ng lps ( -fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld of franciseua in normal mice or mice inoculated with liposomes without lipid a was - bacteria. in contrast, mice vaccinated with liposomal lipid a ( ggl survived challenges as high as , bacteria, ( logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il- , and il- responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd . to evaluate if blocking lps-cd interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a hour period. to evaluate the effect of treatment with anti-cd mab, animals were given either nothing, an isotype control or anti-cd mab ( mg/kg) rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il- , i,- , il- and il- , and liver enzymes during a hour period revealed that treatment with anti-cd mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi ). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a kd n-terminal fragment (rbpi; ) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi has equivalent antibacterial activity to bpi against rough gnb but is up to x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi block the cd -dependent lpsinduced synthesis of the cytokines tnf, il- , el- and il- in vitro. rbpi has also been shown to inhibit the lps-induced synthesis of reactive metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi was not toxic to rats after daily consecutive i.v. doses of mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd ) of myeloid cells or as a soluble serum protein (scd ) lacking the gpi-anchor. binding of lps to mcd triggers cell activation while binding of lps-scd complexes to cells such as endothelial or epithelial cells that normally do not express mcd activates these cells. these pathways are shown in schematic form below. ~di mcd plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: mg/kg --- . mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il- , or il- , but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of - m and has a t / of • h in normal humans. methods: normal volunteers were randomized to receive either tnfr:fc vehicle (n= ), or low dose (ld) tnfr:fc ( mg/m iv, n= ), or high dose (hd) tnfr:fc ( mg/m iv, n= ) infused over rain prior to endotoxin (e) (escherichia coil : ; ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point ( - h) in subjects given tnfr:fc compared to vehicle ( h) (p=. ). at h, h (post fluid load ), and h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=. ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<. ) and later leukocytosis (p< . ). peak tnf bioactivity in subjects given vehicle was + pg/ml and < pg/ml in the ld or hd tnfr:fc groups (p<. ). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=. ) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<. ). decreased levels of il- (p=. t), granulocyte colony stimulating factor (p=. ), and il- receptor antagonist (p=. ) were found after ld or hd vs vehicle. il- levels were lower after ld vs vehicle or hd (p=. ). il- levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the ' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥ ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c inhibitor complexes ( ). in this in vitro plasma model we also found that methylpredoisolone alone in doses of mg/ml induced contact activation with the formation of kallikrein-c inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade ( ) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et ( %) and interleukin- ( % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c - nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c - nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c - nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c - nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type- in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type- (cr- ) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c /c convertase formation. we have recently purified the human recombinant cr- in a truncated form (brl , tp-io, scr- ) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: ) endotoxin-induced ards in paf primed animals; ) .- induced ards; ) thermal injury ( % body surface)-induced ards; ) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr- , - mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il- ), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr- in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that ( ) ptx suppresses lps-induced tnf release from activated macrophages; ( ) ptx prevents tnf-induced pmn activation; ( ) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; ( ) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse , d- m nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il- most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il- and il- . moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons ( . +. kg), were randomized to a control group receiving ° cfu/kg of intravenously administered e. co/i (n= ) and a treatment group (n = ), pretreated with . mg/kg of paf receptor antagonist ro - two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving ng/kg of intravenously administered endotoxin (lps) (n= ) and a treatment group (n= ) pretreated with mg of ro - eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for hours. results: baboons pretreated with the paf antagonist had improved oxygenation ( + mm hg v -+ in controls, p < . ) and creatinine clearance hours post e.coli ( . + . ml/min v . _+ . in controls, p < . ). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at hour post lps. this was in concert with a diminished peak cortisol ( +_ mmot/l v +- mmol/l in controls, p < . ), epinephrine secretion ( + nmol/l v + nmol/l in controls, p < . ). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il- and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il- ra, a naturally occurring il- antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha- a and e . future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa , and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn , have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il- and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd)i chain covalently linked to one of three different alpha chains (cd a, cd lb, cd lc) and exist on the cell surface as three distinct heterodimers. cd l a/cdi is expressed on all leukocytes, whereas cd l b/ cd and cd lc/cd are restricted to cells of myeloid origin. cd i/cd interacts with intracellular adhesion molecule- (icam- ), its ligand on endothelial cells. icam- is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = ) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria endotoxin) during the first days after inciosion in the study. the other randomized group (n = ) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of < wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one ( %) of patients who received immunoglobulln. by comparison, nine ( %) of control group patients died during this period (p <. ). within the first hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; ) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_ (meaning a mortality risk > %) were accepted into this protocol. patients were randomized to receive . g/kg of ivig or placebo on day (when they reached sepsis score> ), repeated on day + and + . at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: % vs, % respectively; p< , ). in conclusion, the results of our study in patients with severe surgical sepsis were the following: ) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', ) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg ( ) - ) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in patients with sepsis and septic shock (infection ~ ) - ), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score - ) and severity of sepsis (elebute sepsis score - ) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo ( .i % albumin) or polyglobin n" - ml ( . g)/kg on day and ml ( . g)/kg on day i. with an anticipatedpopulation size of patients the study should comply with the statlstical requirements (estimated mortality: %, with a % reduction in -day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november , more than patients had been included; patient enrollment will be finished in . previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il- , increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled patients at academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) . or . mg/kg/hr by continuous intravenous infusion for hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over days utilizing a linear dose-response model, assuming a log-normal distribution. results: patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< . endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d ) and tolerance of bn ( iv infusion of mg x /day over days) in severe sepsis has enrolled pts. the day mortality rate was % for the placebo group and % for the bn group (p = . ). the efficacy of bn was greater in pts with gram-negative sepsis: the -day mortality rate was % for the placebo group and % for the bn group (p = . ). bn also reduced mortality among pts with gram-negative septic shock (mortality was % for placebo vs % for bn ; p = . ). using statistical adjusments for pronostic factors, the relative risk of death of the bn group was . ( . - . , % confidence interval; p = . ). this risk corresponds to an adjusted reduction in mortality of % for pts receiving bn . no differences in mortality rates were found between the placebo and the bn groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn groups. bn is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~ lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for -day mortality (jamb. ; : , - ) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin- receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x = . , p [] . , log.normal) for all patients in the trial• survival benefit began st approximately % baseline risk of -day mortality. for the $ patients with a predicted risk > %, there was a % reduction (p= , $ log normal). when we examined the variation in patients above and below the % risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < % risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > % risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp- , in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp- on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population ( . %) of pop. low-risk pat. (ap< ; mortality: %) and the small groups of pop. pat. at risk lap= - ) and high risk lap_ ) with a significantly higher mortality ( % and %, mainly due to sepsis). subsequently, among consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n = ): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day : ml/kg, day : ml/kg). second study period (n= ): iggma (pentaglobin r, biotest, dreieich, frg, ml/kg on days to ). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n= , high-risk: n- ), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n= : p<o. ; iggma, n= : p: . ). in this group, ig led to higher (p< . ) response rates, defined as an ap score decrease -> within four days (igg: %, iggma: %; con.: %), and reduction in mortality (igg: %, iggma: %; con.: %), statistically significant (p< . ) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, aminoacids et (et ) in rat abdominal sepsis. methods. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et were determined with amersham tm endothelin rias , and h after sepsis induction. experimental groups: . cp control, . volume replacement (vr); , % saline ml/kg/h continous iv infusion started after h, . antibiotic; imipenem mg/kg iv after h, . e ®; mg/kg iv after h, . vr + imipenem + es® after h. results. high concentrations of both big et and et could be demonstrated after h and lasting for h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e ® significantly reduced et both , and h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et was the same as for e ® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e ® reduces circulating lps and tnf which is the probable mechanism of the suppressed et synthesis. the unaltered big et fraction after e ® treatment indicates conversion of big et to et as the site of action responsible for reduced et . conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et . e ® anti-lps antibody significantly reduces plasma et while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e- in congestive heart failure has been demonstrated aumann, ). therefore, we investigated the effect of bu-e- in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [ ] [ ] [ ] [ ] [ ] [ ] were anesthesized with fentanyl/dormicum, ventilated (n :o = : ) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study ( animals) to evaluate the effective concentration of bue- in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of ~g lps/kgkg/h ( :b , fa. difco). the hypodynamic state was defined as a decrease of cardiac output by % of steady state levels. a wedge pressure of - mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) groups with animals per group were studied. the groups were treated as follows: group i, lps and , % nac ; group ii, lps and bu-e- ( #g/kgkg/h); group iii, famotidine (h -blocker) pretreatment ( mg/kgkg), lps and bu-e- . results: the pilot study in healthy pigs revealed, that bu-e- had positive inotropic effects. these effects were inhibited by the h antagonist famotidin. bu-e- however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do ) were not significantly influenced by bu-e- application (group i versus group ii). bu-e- did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e- led to a further significant decrease of lvsw (p < , ). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h -agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci . % (control without endotoxin, n= ). group e : endotoxin tjg kg " bolus i.v. + naci . % (control with endotoxin, n= ). group c : endotoxin pg kg - bolus i.v. + cll u kg - bolus + u kg " h "~ i,v. (treatment group, n= ). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m ) and after (m ) and rain. (m ). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p< . ) between group e (lung , liver ) and c (lung , liver ) (group k : lung , liver ). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july to june , patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of . years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the st, rd, th and th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk receptor is constitutive. the bk receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk (bradycor [cp- ]),bk~ (cp- ) and bkz/bk (cp- ) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk agonist) in the lps-treated ( ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk agonists increased with time and at time min appeared maximally induced. constant iv infusions of cp- blocked bk but not bk~ and cp- bk~ but not bk responses. cp- ,cp- +cp- and antril+cp- blocked both bk and bk~ responses. antril alone had no effect on bk or bk~ responses. within - min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk ,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk receptors. in vitro studies demonstrated that beth bradycor and cp- (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp- is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september and july olt's in patients were performed at our department. the actuarial -year patient survival is %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from to hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss - ), mean temp. was . f, leukocytosis was k, pan was , fin was . , and peep was . at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t am and a thickness of nm. the ami-fungal efficacy is - times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than nm with a mutina ld of > mg/kg. it has been used in dosages up to mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a % response rate for invasive candida infections to % response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was % among placebo treated patients compared to % for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from % to % ill placebo and ambisome treated patients respectively (p< . ). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of mg/kg at day , , , , and after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of elisa units during months. cmv infection occurred in / ~eronegetlve and in / seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower ( / ] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f t ) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection ( v / ), but no difference in incidence of disease ( vs / ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from patients with different types of leucemia after autologous and allogenzc bmt had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in % of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity - days after intranasal inoculation of influenza virus a (h n - ) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest . consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years and . in the same period of time pri~ total hlp replacen~nts were performed under i entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than %. in the selected i cases the septic rate was % indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c ) and fourth (c ) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate ( "= - . , p < . ). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c , c , crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il- ra, il- , il- r, il- r, tnf-ct, tnf-~r (p ) and icam- . in contrast, pmn-elastase, il- and il- closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd +/cd + ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il- and il- , however, closely correlate to the clinical course and may prove valuable for follow-up. the cd +/cd + ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and ( ) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of mu/ml xanthine oxidase (xod), mm hypoxanthine (hx), mm fec and mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il- . cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& ) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, ) the trant~ of vim.©n~e traits between ~renr~a.,dsms and ) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il- receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h pital erasme, route de lennik , bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin ) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il- by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il and monocyte chemotactic activity when cultured. these activies were significantly increased ( -fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il- in patients with bacteriologic proven abdominal infection (intraabdominal abscess , diffuse peritonitis , pancreatic necrosis , pancreatic abscess ) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a to hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il- determination were drawn preoperatively, intraoperatively, and until h postoperatively in regular intervals (min /pat), results: preoperative apache ii was in median (rain , max ). patients fulfilled the criteria of sirs. of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p= , i, spearman coefficient). preoperative cardiovascular (systol. rr< mmhg) and respiratory (pao < mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p= , i, wilcoxon; pulmonal: p= , ) and il- (cardial: p= , ; pulmonal: p= . ) overall, lps, tnfa and il- values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i , mean pg/ml) than in those who did not fulfill these criteria (group b; n= , mean pg/ml; p= , i, wilcoxon). il- was significantly higher in group a (mean pg/ml) than in group b (mean pg/ml; p= , o i wilcoxon). conclusion: perioperative tnfa and il- were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel - , wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= ) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after hours. stastistieal calculations were performed on a personal computer with the spss programm vers. . (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla -activities [endotoxin range from . eu/i, sd= . to . eu/ , sd- . (p < ), ifn-¥ levels, range from . pg/ml, sd- . , to pg/ml, sd= (p < . ), circulating pla -activities range from . , sd= . to . u/ , sd= . (p < . ) and biopterin range from . nmol/l sd= . to . nmol/l, sd= . (p < . )]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = . , p < . ) and bioptefin synthesis (rv= . , p < . ). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = p < . .. p • , . . ) and to the pla -actwtues (rp = . , p < . ). the biopterin synthes~s correlates slightly with the pla -actn,ities (rp= : . ; p < . ). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of %. anaerobes were found in . %, anaerobes were isolated in . %. there were aerobic and anaerobic associations in . % and microflora was not found in . % of the cases. express method of anaerobes discovering let to receive information on - days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of patients with abdominal sepsis treated at the surgical intensive care unit during a -year period, ( %) had an age of years or more. were women and were men, a sex distribution not differing with patients younger than years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was (- ) days including in median days in the icu. figures in patients less than years of age were comparable ( (- ) days out of which in median days in the icu). apache ii and sss-scores did not significantly differ ( . vs and . vs . , respectively), between the groups. neither did the incidence of organ failure differ ( / vs / ). however, the incidence of multiple organ failure was significantly lower in elderly patients ( / vs / (p < . )). the mortality rate, however, did not differ between the groups ( / vs / ). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n-- in each group). at , , , , and hours (n= /timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of : i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was % both at and hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from hours and on and in the proximal and distal small intestine from hours and on. different types of alterations in capillary permeability seem to appear: ) a temporary short increase e.g. in the proximal small intestine and spleen; ) a temporary longer increase e.g. in the colon and kidneys; ) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p integrins (cd l a, b,c/cd ), and the fc'?r (cd , cd , and cd ), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score > ) or thermal injury (> ~ total body surface area, % full thickness), and healthy controls. the expression of cd b and c and to a lesser degree cdi a was significantly reduced on pmns. the expression of cd and cd but not cd was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, ~ ethesda ave, cincinnalt, oh, usa, - s b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet , dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e ) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il- . the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =. , mean b state = . , mean ~ state = . ). the accuraay of this estimate was prospectively analyzed in this group of m~itlple patients of whom % had sepsis and % had ssptlo ards. the survivors had a mean p death of . and the deaths had a mean p death of . . the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of patients who were enrolled into one of four treatment groups: ( ) . mg/kg of anti-tnf mab, ( ) . mg/kg, ( ) mg/kg or ( ) . mg/kg at study entry and the second dose hours later. the small sample size in each group (n= ) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin- may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x ) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of mg/kg tnf~ mab, . mg/kg tnf~ mab or placebo ( . % human albumin).patients received standard aggressive medical/surgical care during the day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca , was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca . as a single infusion or in combination with ha- a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage was an open label trial in which groups of patients each with the clinical diagnosis of sepsis received ascending doses of ca ( . , , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of ha- a ( mg) and placebo or one of doses of ca ( , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of placebo or one of doses of ca ( . , , mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il- , and il- levels.a total of patients were enrolled from clinical sites ( in stage , in stage and in stage ). primary analyses were performed on patients in stage and . there were patients who received ca exclusively and patients received placebo. administration of ca was well tolerated at doses up to mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in % ( / ) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca . the mean half-life was - hrs ( - hrs). a dose related decrease in tnf concentration was observed hr post infusion of ca . tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak f. it is a f(ab') fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak f or placebo were administered ( , ; , and i mg/kg) over a perid of hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il , il and tnf), and outcome. at this time only an interim analysis of patients is available i indicating that mak f in all dosage groups resulted in a decrease in il . this contrasted to a further in crease of il in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all patients in the study will be presented and discussed.$ s staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a atom-chain spacer. the detoxification capacity was ug[et/ml column material. the biocompatbility resulted in ~ platelet recovery. the column contained ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine . iu/h in the inflowline after bolus injection of . iu. hp was performed on pigs at a rate of ml/min by means of a roller-pump until the animals succumbed (h). animals served as controls (c). serum et levels rose from . pg/ml to , pg/ml after hours in the c and from . pg/ml only to pg/ml in the h group after hours whieh was highly significant. survival time could be extrended from to min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b( rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from to consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in °, and by septic-toxic event in %. all patients required mechanical ventilation (fio > , ) . ° showed hyperdynamic shock. % had renal and % hepatic failure. medium appache ii score amounted to , points. cvvh was performed in postdilution mode with a polyamide membrane (fh ) and high volume exchange ( l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in %, hemodynamic in % and renal in % of the cases. % of the patients survived and were discharged from hospital. of non-survivors ( %) died because of fatal mof within h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web ), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin ( p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n= ), or after pretreatment with web ( xl -gm, n= ), or diclofenac ( #g/ml, n- ). furthermore, the application of specific antitoxin (mg/ml, n= ) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, . mg/ml) (n= ) and the combination of immunogiobulins, web and diclofenac (n= ). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of . times higher than the control, starting after min and a delayed onset of edema formation resulting in a mean weight gain of g after min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about times that of controls in the toxin treated lungs. pretreatment with web or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st ( -n,n,n trimethylammonium-(r)- -isovaleroyloxy-butanoic acid z- -( -chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of h-paf (ki= . x - m) to rabbit platelets. the present study shows that pretreatment of c bl/ mice with st , administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli :b injected at mg/kg intraperitoneally). very interestingly, st administered at the same doses as above (i.e. . , . , and mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia ( . °c) induces heat shock protein ('asp) and increases smvival - fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp- by sodium arsenite improves survival in a rat sepsis model (abstract a am. rev. resp. dis. vol. , ) . the effects of heat are complex and in addition to formation of lisp- include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp- in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite ( mg/kg) and ethanol ( ~ of % ethanol) caused marked induction of hsp- in lung, gut, kidney, and liver, which was comparable to heat-induced hsp- . female nd mice weighing - gms were pretreated with arsenite or alcohol hours prior to challenge with escherichia coli endotoxin (-ld ) and survival was compared to control mice. results: survival at hrs. for arsenite treated and alcohol treated mice was % and % respectively and was statistically different from the % survival for control mice. (p< . ) (n= mice per group). however, at days post endotoxin, there were no differences in survival in the groups, i.e., ~ % survival for all groups. in contrast, the protective effect of hyperthermia remains present at days, i.e., ~ % survival vs % survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp- formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered x per day from the day be ~ fore the operation until the th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in / after pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from . % in the placebo-group to . % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from . % in the placebo-group to . % in the treatment-group (p ~ . ;fisher's exact test). in march final results with more than patients will be presented for the first time. (= po < mm hg, b s-creatinin > mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only of deaths occurred during the st week, all other deaths occurred late (after - weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. -- ) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only % of deaths today occur eady in the course of the disease, whereas this percentage varied between - % just years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for to years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in patients with acute pancreatitis ( males, females; median age: years, range: - years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type (slcam- ) were determined using immunoassays (henning, bender, t cell sciences). / had increased concentrations of stnf-r compared to the th percentile obtained in healthy controls (> . ng/ml), and / patients had increased neopterin (> . nmol/i), / presented with elevated slcam- (> u/i). all patients with increased neopterin also had increased stnf-r, patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = . , p < . ). weak associations between age and stnf-r (rs= . , p=o. ) or neopterin (rs= . , p = . ) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as ( , ) and characterized by increased serum or urine amylase levels (in about % of patients) in the fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the ffs, studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery ( , , ) . in a recent study ( ), evidence of pancreatic cellular injury was found in out of patients undergoing cardiac surgery, with out of these patients presenting abdominal signs or symptoms and developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), hours alter surgery and in the folfowing days alter surgery (days , , , and ). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day . we measured abnormal levels of trypsin and pancteatic iso-amylase in % of patients and observed simultaneous releases of these enzymes, the fi,'st one in the hours after surgery and the second more intense from day and pa~icularly on day after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about % of our patients : pancrealic abnormalities were revealed by scan in patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion ( ). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin- (il- ) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii- (elisa), transferrin and crp (nephelometry) were analysed daily along the first i days of hospitalisation by patients with acute pancreatitis admitted to our hospital from / to / . it was judged whether the patients have either interstitial (aip) (n= ) or necrotizing (anp) (n=lg) pancreatitis. patients with anp have died during the course of pancreatitis (mortality= . %). results: -severity o~ pancreatitis: signifcant differences (p<o. , mann-whitney test) could be calculated between aip and anp for endotoxin, il- , transferrin and crp during the i days of monitoring. -mortality: except of il- on days , , and after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. -organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il- and crp along the i days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il- and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from / - / , patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n= ) and n (n=l ) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in patients. the etiology of ap was gallstones in , alcohol abuse in , hyperlipidemia in and other or unknown causes in patients.five of patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il- concentration in blood reached up to pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of pg/ml was correct in patients ( %, sensitivity (se): %= specificity (sp): %, (z: , ). high levels of il- over pg/ml correlated well with the prognosis ifri in the first week (accuracy: %, se: %, sp: %, c~: , ). the blood levels of il- reached a maximum of pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of l,- blood levels indicated correctly the severity of ap in out of patients using a cut offtevel of pglml (accuracy: %, se: %, sp: %, ~: , ). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il- and il- . in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il- , and to a lesser extent of [l- , can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n= ), around the pancreatic duct (group b, n= ) or around both ducts (group c, n= ). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a mtcalbumin colloid with a diameter of nm, was measured before, , and days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day , the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day . the opossums in groups a & had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day in group a & b (p<o, ) and at day it~ group c (p< ,ol). at day , res function in group c was significantly worse than that in group a & b (p< , ). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day (p< . ), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b ) in lewis rats weighing - g. there were five groups: group a (n= ) were untreated controls; group b (n= ) were given isotonic saline intraperitoneally and observed for hours; group c (n= ) ml x e. coli intraperitoneally and observed for hours; group d (n= ) were given ml x e. coli and observed for hours; group e (n= ) ml x i e. coli and observed for hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser ). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p= , ) and spleen (p= , )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p< , ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel - , wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar( - ocalized jo-diffuse, -~eneralized) and -hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen- , the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, -com:lissural ileus). he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; ) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and ) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding /co hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate - wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. grafts were cryo'd in % dmso, quick-thawed & sutured onto x cm excised full-thickness dorsal wounds on athymic mice. "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained > % cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in - , x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through days of followup. histologic exams on days - showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than % total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we ] as ready availability of urdform lis~ue~. on dec~mt~r , , the fda assumed control of as tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, " ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki- (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since when galllco et el reported theh'use in two brothers with greater than % total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day ), the full thickness wounds were covered with epigard, a synthetic dressing until day . after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from patients at the beginning of surgical treatment (day ) and after , , and days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf- , double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.( ) pharmatec gmbh, frankfurt ( ) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group , cells migrating through skin: + % t lymphocytes (cd ), + % langerhans and dendritic cells (cdla, hla dr, s ), + % cd , + % cd , no b cells (cd , ), % cd r (memory cells), + % il r. approximately % cells possessed cdlla and antigens. cd lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group , cells in skin: langerhans cells were found only in epidermis, cd , and , cd r , rb, ila/ cells around venules, cd (macrophages) uniformly dispersed, no il r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group , one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il r cells, perivenular infiltrates of cd , r but less cd cells, high density of cdlla/ cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht bioassay). the hollow lipid microcylinders ( microns in length and i micron in diameter) show an initial burst ( - ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed , , and days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd and cd , mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code , naval research laboratory, washington, dc. - . expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet ) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to go of the origional size (t %) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from juvenile human bum patients, immediately after wound debridemerit mad on day after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*' ~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of psoriatics and healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx t-lymphocytes were then incubated alone or in coculture with x irradiated autologous lymphocytes in culture medium containing - mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce -surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t si-hsa over a rain period, the chemotac~c peptide imlp ( . . ng) and bradykinin were used to induce cell.dependent and cell- ndependent leakage respectively, the antibodies used were . e (cdis), nri (cdlla) and antibody (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien ( .~. ffi . p.l to . z b.bttl and . ,- . ~ to . z . d respectively. . e ( . - . mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at . mk/kg, the plasma leakage was completely inhibited, antibody nr produced similar results, treatment with antibody did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . patients without infectious coz~lications and group - patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection patients of the group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i- days after injury revealed a considerable decrease of lymphocytes, ed ",$d ~ and cd cells amo~it in the blood, cd /cd ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the - days in pateents of+the i group. but stable ~eple$ion of cd and cd cells amount, lower cd /cd ratio and indexes of leukocyte migration inhibition test in patients of the group were observed~ besides that, these persons showed higher cd cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in % of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. , , st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p integrlns (cd a,b,c/cd ) and the fw receptors (cd , and cd ) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~ integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z ) or thermal injury (> ~; total body surface area, > ~ full thickness) and incubated /n v/tro with gm or g-csf. the j integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd lb, cd , and cd while gm bur not c-csf induced an increase in the percentage expressing cdi a, cd lc, and cd , gm-csf and to a lesser extent g-csf induced an increase in the density ( , fold) of the ~ integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, bethesda ave, cincinnati, oh, usa, - . funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in % and in association with bacterial infection(l %), viral infection( */.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a. of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e -ebiary, a. torres et al, reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of olt in adult patients olt were performed for hbsag positive liver disease (cirrhosis n= , fulminant liver failure n= , retransplantation n= ) in pat. all pat. received . u hig in the anhepatic phase and . u/per day for the first week. a small group of pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are % in pat. who were transplanted for fulminant hepatitis, % in pat. with cirrhosis and long term prophylaxis, and % ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was % under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in % in pat, negative for hbv dna. in hbv dna positive pat. infection rate was %, the total rate of reinfection for all pat. with long term prophylaxis was %the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢ capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il- , ltb , c a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi , poe, lt's), complement components (c a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il, , il- or tnf), and immunosuppressivc agents (poe , il- ). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, - th ave za- , seattle, wa usa key: cord- -rz r sj authors: nan title: abstracts for the th annual meeting of the japan neuroscience society (neuroscience ) date: - - journal: neuroscience research doi: . /j.neures. . . sha: doc_id: cord_uid: rz r sj nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns . sl - - frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl - - neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif a/kif bbeta), nmda type(kif ) and ampa type(kif s) glutamate receptors and mrnas(kif s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least rna related proteins such as hn rnp-u, staufen, and fmrps. kif a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award - what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in - months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v ). after complete ablation of v , the monkeys recover performance of visually guided saccades toward the blind field in months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi , kakenhi sy - - - distinct roles of two -pass transmembrane cadherins in neurite growth control tadashi uemura , , yasuyuki shima , , keisuke sehara , manabu nakayama , shinya kawaguchi , mikio hoshino , yoichi nabeshima , tomoo hirano , graduate school of biostudies, kyoto university, kyoto, japan; school of science, japan; kazusa dna research center at chiba, japan; graduate school of science, japan; graduate school of medicine, japan; crest, jst, japan drosophila flamingo (fmi) and mammalian celsr - , which are pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian -pass transmembrane cadherins celsr and celsr are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr enhanced neurite growth whereas celsr suppressed it. our result suggested that celsr had a stronger activity as g-protein coupled receptor than celsr did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of -pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin- is preferentially localized in axons; and nectin- , in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin- and dendritic nectin- is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi sy - - - lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa , fumikazu suto nagoya university, nagoya, japan; institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema a is expressed in ca pyramidal cells and principally suppresses invasion of mossy fibers to ca . second, the repulsive signal of sema a is mediated by plexin-a expressing in mossy fibers. third, the repulsive activities of sema a are attenuated by plexin-a in the proximal dendritic segments of ca pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on ( ) working memory maintenance and manipulation, ( ) rule representation and task-switching, and ( ) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy - - - towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy - - - neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of − genes on chromosome q . , has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi ( ) sy - - - a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and k songbird cdna array. we used the arrays to reveal a set of genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. - - - stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi sy - - - role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using -photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., ) . furthermore, in response to single spine stimulation using -photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration ( ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy - - - basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼ hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy - - - coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f- fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad- found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca + sensing protein was activated. synchronies of sv fusion are maintained by a ca + sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy - - - sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim . thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka , takeshi baba , sumiko mochida , yoshimi takai department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca + -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab system, munc- , munc- , doc- , and tomosyn. we have previously reported that tomosyn, a syntaxin- -binding protein, works as a molecular clamp that controls free syntaxin- availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin- , resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin- , which increases the affinity of syntaxin- for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca + sensor for neurotransmitter release. however, the physiological contributions of its ca + -binding domains (c a and c b) are still unclear. we have studied the roles of aspartate (asp) residues in the c b ca + -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell , p. ) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca + binding to the c b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca + binding to the c b because mutation in the second asp inhibits ca + binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin- (nt- ) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt- and its low-affinity receptor p genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c t in the noncoding region, bdnf-linked polymorphic region, and val met), three polymorphisms of the nt- gene (g- a, microsatellite in intron , and gly- glu) and a missene polymorphism of the p gene (ser leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val met) in the bdnf gene resulting in a prodomain substitution at position from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns sy - - - processing of bdnf and brain disorders masami kojima , aist, osaka, japan; sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p neurotrophin receptor (p ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); (t/g) and (t/g) of the human bdnf gene, causing amino acid substitution (r m and r l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy - - - pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣ leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣ , the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv- vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il ss+fgf- , and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi ( ) sy - - - hgf gene therapy for the treatment of spinal cord injury masaya nakamura , akio iwanami , kazuya kitamura , , yoshiaki toyama , hideyuki okano department of ophthalmology surgery, keio university, tokyo, japan; department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th level in adult rat days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. or weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap +fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., ) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., ) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., . nature neuroscience. nakamura et al., . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first - months after stroke, suggesting the presence of a critical period. in the cat, illert et al. ( ) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c -c segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. ( ) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c -c propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. ( ) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca + responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l cam(l ) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l . therefore, these syndromes have been reclassified as l syndrome. we performed a nation-wide l gene analysis and identified li gene mutations in families with l syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l -mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l syndrome. research funds: kakenhi ( ), ( ) sy - - - rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein gene (mecp ) located at xq is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like gene (cdkl ) manifest as atypical rtt. the mutations of mecp are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in -ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase- are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp , suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca , and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣ or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣ and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context - days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr , nr a, b, c), glycine transporter , and eaac , by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase ␣ -subunit (pde c), a component of cone-type pde. since genetic mutations of pde c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde in visual functions. shinichi nakagawa , masatoshi takeichi , , fumi kubo , nakagawa initiative research unit, riken, wako, japan; riken cdb, kobe, japan; department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi ( ) sy - - - identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea- to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea- during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea- double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea- -positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea- have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx and crx play critical roles in retinal photoreceptor development. while otx is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi ( , , ) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf found in the mesoderm. we find that although fgf can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf and is actually inhibited by fgf . for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy - - - how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel , encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein e was identified as the gene responsible for this behavioral difference among the species. obp e forms a gene cluster with obp d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp d/e region obtained from species phylogenetically located between d. melanogaster and d. pseudoobscula. sy - - - an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi , , akinori nishi , , toshihiko shiroishi , , tsuyoshi koide , sokendai, kanagawa, japan; national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c bl/ (b ) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b and msm, and those strains have the same genetic background as b except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b - msm, which have msm chromosome , showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi ( . ) sy - - - cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome (capsq ) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv ) was impaired in kjr neurons in contrast to that of c bl/ j mice. by searching the candidate genes, eh domain-containing four (ehd ), a trp-binding scaffold protein encoding gene was found. ehd binds to c-terminal of trpv . three mutations were found in ehd of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv . this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy - - - comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than different strains of mutant mice with the battery. surprisingly, more than % of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi ( , , , ) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least genes and loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt f caused by prx, and scan caused by tdp . sy - - - gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm , who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm /gd synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, ) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa ) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi ( ) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo- is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo- mediates the inhibition of axonal growth as a component of the ngr /p /lingo- and ngr /troy/lingo- signaling complex. inhibition of endogenous lingo- by soluble lingo- or dominant negative lingo- can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo- treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo- treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo- mediates the inhibition of differentiation and myelination. loss of lingo- function using dominant negative lingo- , lingo- rnai, or soluble lingo- or lingo- knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo- led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo- in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc - a sy - - - neuronal avalanches reveal neuronal wirings of layer / cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi ( ) sy - - - spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi , jean-pascal pfister , kazuyuki aihara , , wulfram gerstner department of complexity science and engineering, university of tokyo, japan; school of computer and communication science & bmi, epfl, japan; aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows j and sci. res. on priority areas from mext of japan, and swiss natl. sci. found. - / sy - - - timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as - ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi , ikue otaki , saya suzuki , yasunobu yasoshima , kazuto kobayashi laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin- receptor ␣ subunit (hil- r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil- r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. - - - integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy - - - molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj- is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are ( ) leukemia in patients exposed with atomic bomb during the iind world war, and ( ) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, ( ) many genetic/familial diseases show clinical symptoms at the middle or old age. ( ) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. ( ) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp , which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼ drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc /+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi k/akt signaling, which results in decreased gsk activity, has also been shown to ameliorate ami. both pka and gsk phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk at young ages, declining igf levels during aging may increase gsk activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk causes neuronal dysfunction during normal aging. research funds: kakenhi sy - - - molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, - - tsukiji, chuo-ku, tokyo - , japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy - - - identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue , department of geriatric medicine, university of tokyo hospital, tokyo, japan; research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of - - sigma, a negative cell cycle regulator that causes g arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka , hossein esteky , kiani roozbeh , tadashi sugihara , gang wang riken brain science institute, wako, saitama, japan; institute for studies in theoretical physics and mathematics, tehran, iran; kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of inferotemporal cells to object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to • rotation. research funds: kakenhi alexander thiele , gene stoner , louise s. delicato , mark roberts university of newcastle upon tyne, uk; the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy - - - context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy - - - effects of task demands on color processing in area te of the monkey hidehiko komatsu , , kowa koida national institute for physiological sciences, okazaki, japan; sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development . monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception - . here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy - - - dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui , hirotaka sakamoto , katsunori sasahara , hanako shikimi , kazuyoshi ukena , mitsuhiro kawata laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi ( and to kt) sy - - - roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi ( , ) sy - - - sex steroid receptor function in sexual behavior shigeaki kato , , takashi sato , takahiro matsumoto , imcb, university of tokyo, tokyo, japan; erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., ) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., ) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., ) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., . pnas usa , . sato, t., et al., . pnas, usa , . shiina, h., et al., research funds: probrain sy - - - annexin : a mediator of cell-cell communication in the neuroendocrine system julia buckingham , helen christian , john morris imperial college london, uk; department of human anatomy and genetics, university of oxford, uk annexin (anxa ) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca . the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori , yuki takayanagi , masahide yoshida , yoshiyuki kasahara , masaki kawamata graduate school of agricultural science, tohoku university, sendai, japan; department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) ( ) sy - - - cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f ); f activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m ). local field potential activity in f and m is also tuned to grasp, and there is strong beta coherence between f and m , indicating reciprocal transmission of information. this is also seen in synaptic responses of m neurones to stimulation of f (and vice-versa). single pulse stimulation in f strongly modulates corticospinal outputs from m through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy - - - where tactile signals are ordered in time shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., . s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy - - - decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo , biology/cns, california institute of technology, pasadena, ca, usa; jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. -channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha ( ) ( ) ( ) ( ) ( ) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy - - - why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust ( ) sy - - - recognizing self actions through externalized eyes atsushi iriki , symbolic cognitive development, riken brain science institute, saitama, japan; cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut , existing mostly in cortical pyramidal cells, and vglut , found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut -expressing terminals (n = ), but were found in- % of spines innervated also by vglut -expressing terminals (n = ). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy - - - gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼ -fold decrease in the potassium-chloride co-transporter (kcc ) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer / pyramidal cells in visual cortex share common excitatory inputs from layer and from within layer / , when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer / cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi ( , ) sy - - - local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer of rat visual cortex and layer / of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer , axondescending cells receive excitatory input predominantly from layer / while neurogliaform cells receive stronger input from deeper layers. layer / neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer / , while the cr− martinotti cells also receive strong excitation from layer . irregular-spiking basket cells also receive strong excitatory input from layers / and , but they often have a gap at the top of layer , with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers / and , and only weak input from deeper layers. research funds: nih sy - - - physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers ( - ) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r ey , mcknight neuroscience of brain disorders award sy - - - axon guidance mediated by localized ca + signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca + signals. its turning direction depends on the occurrence of ca + -induced ca + release (cicr) through the ryanodine receptor type (ryr ). the activity of ryr is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr , that serves as a key regulator of axon guidance. attractive ca + signals facilitate intracellular membrane transport to the leading front and subsequent vamp -mediated exocytosis on the side with elevated ca + . in contrast, repulsive ca + signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp -mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy - - - molecular mechanisms for signaling through plexin-a hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema a acts as a guidance cue for axons through a receptor complex comprising neuropilin- as the ligand-binding subunit and plexin-a as the signal-transducing subunit. the ferm domain-containing gef, farp , associates directly with plexin-a . sema a induces the dissociation of farp from plexin-a , resulting in activation of farp 's rac gef activity, rnd recruitment to plexin-a and down regulation of r-ras. simultaneously, the ferm domain of farp sequesters pipki␥ from talin, thereby inhibiting its kinase activity. these activities are necessary for sema a-mediated repulsion of outgrowing axons. plexin-a also functions as a ligand binding receptor of a transmembrane semaphorin, sema d and contributes to cardiac morphogenesis. sema d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a forms a receptor complex with off-track in the ventricle or with vegf receptor type in the conotruncal segment, which are responsible for the effects of sema d on the respective regions. research funds: crest sy - - - axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins a (sema a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema a stimulated the transport of neuropilin- ::egfp, plexin-a ::egfp, and fyn::egfp, which are the components of sema a signaling, but not neuropilin- ::egfp. interestingly, sema a accelerated the anterograde transport of trka::egfp. these facts suggest that sema a selectively facilitates the transport of its own signaling components and that sema a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein- (crmp- ) is enriched in growing axon, and play a crucial role in axon specification. crmp- interacts with tubulin dimers to promote microtubule-assembly, and binds to sra- , an effector of rac to regulate wave-dependent reorganization of actin filaments. crmp- links kinesin- to tubulin dimmers and sra- , and participates in the kinesin- -dependent transport of tubulin dimmers and the sra- /wave complex to growing axons. we also found that the par- /par- complex and the ras/pi -kinase/akt/gsk- b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk- b and inactivates its kinase activity downstream of ras/pi -kinase, thereby increasing non-phosphorylated active crmp- which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy - - - regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki , , takashi abe , , , shigeyuki kan , , satoru miyauchi , national institute of information and communications technology, kobe, japan; japan society for the promotion of science, tokyo, japan; graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; kyushu institute of technology, kitakyushu, japan; crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o -and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p x receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p x receptors produce a reduction of the neuropathic pain behaviour ( . nature , - ) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p x causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury ( ( . nature , ( - . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi ( ) sy - - - pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk , which labeled with carbon- can be used for positron emission tomography (pet). using c-(r)-pk pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy - - - application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy - - - brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality ( . neuropsychobiology , ), aging ( . neuroimage , , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, ) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia ( . biol. psychiatry , ; . neuroimage , ) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every . s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy - - - clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy - - - measurement of language related brain activities during recovery from aphasia eiju watanabe , yumiko muroi , chizuru nakajima department of neurosurgery, jichi medical university, tochigi, japan; department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy - - - neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy - - - neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy - - - neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy - - - bridging neuroscience and public: neuroethics in cultural contexts osamu sakura , interfaculty initiative in information studies, university of tokyo, tokyo, japan; research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura , , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira , argye hillis showa university school of medicine, japan; crest, jst, saitama, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi ( ), crest sy - - - impairment of surface perception in patients with ventral occipital damages shinichi koyama , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , masato taira , argye hillis showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient was a -year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory ) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient was a -year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy - - - how do pictorial cues influence d information processing in the parietal association cortex? masato taira , arish, nihon university, tokyo, japan; department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for d perception. basically, it is thought that the parietal association cortex processes d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence d information processing in the parietal association cortex. research funds: kakenhi sy - - - case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy - - - neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v + for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi ( ) sy - - - neural correlates of visual attention argye hillis , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira johns hopkins university, usa; showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp gene, encoding methyl-cpg binding protein , cause rtt. mecp acts as a transcriptional silencer. abnormal expression of some genes due to mecp dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp -downstream genes. using mecp -null mice (mecp −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp directly regulates expression of insulin-like growth factor binding protein (igfbp ) gene in human and mouse brains. pathological features of mecp −/y have the similarity of igfbp transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp due to lack of mecp may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements- or l (line- ) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute % of the mammalian genome with l elements alone representing %. the fact that l can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy - - - notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima , jun kohyma , tetsuya taga , masakazu namihira grad. sch. biol. sci., naist, ikoma, japan; inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat . a cpg dinucleotide in the stat recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat binding element in the gfap gene promoter. sy - - - neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. ( ) which type of brain infarction will be a future target of this therapeutic approach? ( ) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? ( ) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy - - - gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that ( ) motor function may recover after minor injury to the primary motor cortex, ( ) this recovery is, at least in part, associated with reorganization of cortical motor representation, ( ) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and ( ) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy - - - neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji , toshiya sato , mitsunori yamada department of neurology, the university of tokyo, tokyo, japan; center for bioresource-based researches, japan; department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with cag repeats. the q mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy - - - control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi , sy - - - mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima , dept. of anat., keio univ. sch. of med., tokyo, japan; inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day . mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca + signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca + elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca + and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp , rho gtpase, mapk and pi k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy - - - membranes, water and diffusion denis j. le bihan shfj/cea, france among einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy - - - diffusion tensor fiber tractography using a tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded -t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on -t tractography compared to . t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using -t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy - - - anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to % of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor ( - mg/kg) into male wistar rats (from days to weeks of age) significantly caused hyperactivity at - weeks old. immunohistochemical analyses of the brain tissues at weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy - - - non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about - meters from a base station. another remarkable observation in our studies is the fact that a significant (p < . ) neuronal damage is seen in rat brains days after a hour exposure to gsm at sar values , and mw/kg. we have followed up this observation in a study where animals were sacrificed and days respectively after an exposure for hours to gsm mobile phone electromagnetic fields at sar values , , , . and (controls) mw/kg. significant neuronal damage is seen after days and albumin leakage after . our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after and days. sy - - - the mitochondrial toxin -nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs , france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin -nitropropionic acid ( np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of np toxicity and show that np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by np. in summary, the np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of -ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at days of age showed behavioral hyperactivity at - weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of -ohda-induced neurotoxicity, using pc cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of -ohda. we also characterized the oxidation of cellular proteins by -ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by -ohda, ros and p-quinone. sy - - - environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥ subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥ subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥ subunit-containing receptors in gh and hek cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥ subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi ( ) sy - - - involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca + rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k a at nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k a application was started soon after hfs or when k a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi ( ) sy - - - autocrine mglur activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur -gq/ transduction pathway as well as being initiated by direct mglur activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy - - - regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca + -activated protease calpain or by disturbance of association of gaba a receptor ␥ subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy - - - contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy - - - detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai , , susumu takahashi department of psychology, kyoto university, kyoto, japan; crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl- s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi ( ) sy - - - impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto - , japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy - - - tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad the amount of the tonic inhibition is reduced in ca hippocampal interneurons, while unchanged in pyramidal cells. sy - - - modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca region yoshihiko yamazaki , yasukazu hozumi , kenya kaneko , satoshi fujii , hiroshi kato dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca region. research funds: kakenhi ( ) sy - - - carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of m carbachol induces beta oscillations which occur intermittently with the interval of - s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is . ± . hz (mean ± s.e.m.). the oscillations are induced via muscarinic m , , receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations ( - hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that ( ) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, ( ) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca and ca neurons discharged during both up and down states, ( ) gamma and fast (ripple) oscillations were observed in hippocampal ca area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: ( ) most of the mprf neurons were active during rem sleep; ( ) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; ( ) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi ( ) sy - - - clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) : h and ld : h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per and per rhythms suggest that light-on resets per rhythms in both light conditions, while per rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld : . the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per and per showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy - - - genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified genes that fluctuated between day and night in the livers, lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose- -phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy - - - neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy - - - a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e) . to e . , the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy - - - live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi ( ) sy - - - visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol , , -trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy - - - rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦ (glur␦ ) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦ into glur␦ -null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦ lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi sy - - - gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi ( ) sy - - - physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi ( ) sy - - - deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid- th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy - - - firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no. ) sy - - - characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama , department of neurological surgery, nihon university school of medicine, tokyo, japan; division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate ( . ± . hz, n = ) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca + -atpase isoform that is located on the th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that ( ) d -type dopamine receptors are downregulated presynaptically in the striatum, and ( ) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy - - - metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr ( . nature , ) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat ( . endocrinology , ) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca + imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi sy - - - metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy - - - molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi , sy - - - thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp c is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t and reverse t , but the transport activity of t is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp a , the transport activities of t and t are similar, is also expressed. the uptake of t and t by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t and t . research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy - - - alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi , masaki takahashi , yasuhiro yoshikawa , tomoko tashiro department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to -hydroxy- , , , , pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to -hydroxy- , , , , , , -heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites ( . dev. brain res. , ) . koibuchi et al. ( ) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. ( ) detected some pcb and oh-pcb congeners in human csf, of which oh-cb is the highest concentration. to determine its effects on developing neurons, we examined oh-cb in rodent cerebellar culture cells. interestingly, oh-cb promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or -htp to examine the relevance of serotoninergic function. the treatment of -htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., ) . reference kawano et al., . j. neurosci. res. , - . research funds: kakenhi os a- - neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama , kazumi takahashi , yukihiko kayama cluster of science and technology, fukushima university, fukushima, japan; department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic ( ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣ receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os a- - correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi , , kazuyoshi hirano , , yukimasa muraishi , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan; secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l ) and grey matter volume among students aged - , who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l . os a- - grammar center activation in honorification judgment of japanese sentences kanako momo , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f op/f t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os a- - top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui , , , kimitaka kaga , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, tokyo, japan; dept. of otolaryngology, univ. of tokyo school of medicine, japan; crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m ) (neurosci. res. , s ). in a subsequent study, we confirmed that m was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m elicited by the original songs and that by the modified ones. however, while m without the deviation showed no significant effect of lateralization, m with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os a- - cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os a- - hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima , , naoki fukui , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, komaba, japan; crest, jst; dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os a- - individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext ( ), jsps research fellowship ( ) os a- - brain activity of happy facial recognition in mother-daughter relationship jun shinozaki , nobukatsu sawamoto , toshiya murai , takashi hanakawa , hidenao fukuyama hbrc, kyoto univ. grad. sch. of med. kyoto, japan; neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os a- - anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto , , atsushi maki , , kuniyoshi l. sakai , advanced research laboratory, hitachi, ltd., tokyo, japan; crest, japan science and technology agency, saitama, japan; dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f t/f o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f t and f o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (− , , ) , (− , , ) , and (− , , − ) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os a- - effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada , sunao iwaki , mitsuo tonoike aist, osaka, japan; chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = ), and with those from an inner semantic category (middle, d = ), while others were not changed (normal, d = ). peak amplitudes of waveforms of the root mean square values on the components of ms (f ( / ) = . , p = . ) and ms (f ( / ) = . , p = . ) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the and ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os p- - mechanisms of p y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono , xiaoming zhu , takashi tominaga , fumihito saitow , shiro konishi , waseda-olympus bioscience research institute, singapore, singapore; department of neurophysiology, tokushima bunri university, kagawa, japan; department of pharmacology, nippon medical school, tokyo, japan postsynaptic p y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and - min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were ± . % and ± . % of the controls, respectively. pretreatment with the protein kinase a inhibitor h- , but not the calmodulin kinase ii inhibitor kn- , completely abolished the atp-induced ipsc enhancement. os p- - activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca + permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca + influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein (arip ). activin-induced nmdar activation persists more than h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os p- - roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr phosphorylation following ltp induction in rat hippocampal ca region. like camkii activation following ltp (fukunaga et al., ) , the increased camki-thr phosphorylation remained elevated at least for min after ltp induction. the increased camki-thr phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto- significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing ( ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing ( ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi ( ) os p- - mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p ) rats that had received a single brdu injection at p were substituted with the corresponding region of entorhino-hippocampal slices from p rats. after five days in vitro, newborn gcs, detected by brdu and prox , migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os p- - cdk is required for neuroblast migration in the adult mouse brain yuki hirota , , , toshio ohshima , takuji iwasato , ashok b. kulkarni , hideyuki okano , , kazunobu sawamoto , , bridgestone lab. keio univ., tokyo, japan; dept. physiol., keio univ., tokyo, japan; dev. neurobiol. riken, tokyo, japan; behavioral gen. riken, tokyo, japan; nih, bethesda, usa; sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase (cdk ) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk mice to emx cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk has a cell autonomous function in neuroblast migration in the adult brain. os p- - colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi , wu sheng-xi , yuchio yanagawa , , kunihiko obata , nobuaki tamamaki kumamoto univ., kumamoto, japan; fourth military medical univ., xi'an, people's republic of china; gunma univ., maebashi, japan; sokendai, hayama, japan; riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng , plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad -gfp knock-in mouse neocortex at e -p by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s -␤ expression in the gabaergic neuron progenitors. os p- - role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi , , , masanori sakaguchi , toru yamashita , , , yuko fujita , yukiko gotoh , arturo alvarez-buylla , takeshi kawase , hideyuki okano , kazunobu sawamoto , neurosurgery, keio univ. sch. med., tokyo, japan; bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; physiol., keio univ. sch. med., tokyo, japan; inst. mol. cell biosciences, univ. tokyo, tokyo, japan; neurosurgery, ucsf, san francisco, usa; neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt b. to examine the molecular interactions between c-tsk and wnt b, we co-electroporated them into the optic vesicle at stage - chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) in developing cortical neurons. an expression vector of gfp-tagged hdac was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac was primarily localized in nuclear until week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac contributes to neuronal differentiation in cortical development. research funds: kakenhi ( ) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., ) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum . ns) was measured. in high-voltage electron microscopy (hitachi m, nips, , gap junctions (average size . m long) were present in contacts. in confocal laser-scanning imaging, connexin localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be /m and a single channel conductance is ps, the conductance of each junction would be ns. the presence of seven junctions between a pair will lead to estimate a total junction of ns. the measured conductance could allow to estimate a fraction of open channels as . %. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi ( ), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os p- - multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer stimuli and propagated horizontally in the layer / . in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os p- - presence of two phases in the sensitive period of orientation plasticity shigeru tanaka , toshiki tani , kazunori o'hashi , , jerome ribot brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after weeks of goggle rearing starting around the age of weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os p- - residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida , , , tadashi isa , , dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; sch. life sci., grad. univ. adv. stud., hayama, japan; crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield ( - % correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v lesion is unlike normal, near-threshold vision. research funds: kakenhi , kakenhi and crest, jst os p- - comparison of the angle representation in macaque visual areas v and v minami ito , , hidehiko komatsu , national institute for physiological sciences, okazaki, japan; the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v neurons has angle selectivity. here, we studied the angle selectivity of area v , which is the major source of inputs to area v . we conducted single-unit recordings from the superficial layer of area v , while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v neurons responded mainly to sharp angles ( • ), straight lines ( • ) or right corners ( • ), but not to intermediate angles ( • or • angle width). this contrasted with area v , where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v . os p- - firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os p- - integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v , we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os p- - probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner , , pei sun , keiji tanaka , david j. heeger , kang cheng department of psychology and center for neural science, new york university, usa; laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri ( . mm × . mm × mm voxels) in human v . we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t *-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih ( f ey - ) os p- - relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations ( - ms; isi = s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase ( - ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase ( - ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼ % of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os p- - the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe , kazuto terai , osamu hieda , shigeru kinoshita dept. of ophthal., kyoto second red cross hospital, kyoto, japan; dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from . to . . the logmar average was . . eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was . , improved from . . the residual five eyes showed less or no improvement. the change of logmar average was from . to . . conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os p- - the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada , hideomi tanaka , , satomi nakayama , miki iwasaki , , hitoshi okamoto , laboratory for developmental gene regulation, bsi, riken, japan; crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) and migrate tangentially to r near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr and frizzled a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os p- - disrupted-in-schizophrenia (disc ) regulates the transport of the nudel/lis complex to axons via direct interaction with kinesin- shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc . disc is reported to interact with nudel, which forms a complex with lis . although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc remain largely unknown. here we report that disc interacted with kinesin- , a motor protein of anterograde axonal transport. kinesin- interacted with the nudel/lis complex through disc , and these molecules accumulated at the distal part of axons. the knockdown of disc by rnai of disc induced the delocalization of nudel and lis from the axons and reduced axonal growth. the knockdown of kinesin- induced the delocalization of disc from the axons. taken together, these results indicate that disc links kinesin- to the nudel/lis complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os p- - role of a novel collapsin response mediator protein- interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein (slp ) can mediate the vesicle transport. slp is known to associate with rab . we found that slp associates with collapsin response mediator protein- (crmp- ), which is a key regulator of axon formation. slp could form the trimeric complex with rab b and crmp- , and also associate with kinesin- through crmp- . slp is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp functions as a mediator of recruitment of certain receptor depending on crmp- and kinesin- . os p- - absolute quantification of mdr a, mrp , mrp and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie , , yuki katsukura , , sumio ohtsuki , , xiao-kun cai , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr a, mrp , mrp , and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between and fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr a, mrp , bcrp were . , . and . fmol/g, respectively, while it of mrp was under detection limit of standard curve. this quantitative profile suggests that mrp and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr a. os p- - dominant expression of claudin- in highly purified brain capillary endothelial cells sumio ohtsuki , , hirofumi yamaguchi , saori sato , tmoko asashima , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam- ) and magnetic cell sorting. mrna expression of caludin- - was measured by real-time rt-pcr. claudin- showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin- and - were . % and . % of that of claudin- . claudin- mrna was concentrated in the purified bcecs, while claudin- and - mrna in the purified bcecs were lower than that in the whole brain. rat claudin- mrna was also concentrated in rat brain capillary fraction, but claudin- mrna did not. these results suggest that claudin- is a dominant tjs protein in bcecs, and expression of claudin- and - , which was reported as tj protein in bcecs, are not restricted in bcecs. os p- - effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad , a. pauzi m. yusof , p.d. mourad , m. bainbridge , s. ab ghani universiti sains malaysia; university of washington, seattle, usa; brody school of medicine, east carolina university, nc, usa h o is the main peroxides produced in mammalian cells that consume o . the main source of h o in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h o towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h o microsensor, we investigated the level of h o in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h o level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h o was injected into the brain. fatality occurs when the injected h o exceeds m. these finding showed that the altered paracellular permeability in the presence of h o is caused by a series of events that happen one after another. research funds: short term grants pkimia and pfar-masi os p- - somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t - ) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t - spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os p- - responses of renal sympathetic nerve activity and sodium excretion to days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions ( , , , meq./l nacl) over days. the sodium loading with meq./l nacl suppressed rsna significantly and then it gradually recovered while either meq./l nacl or meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os p- - cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku , haruko masumiya , yasumasa okada dept. physiol., hyogo col. med., nishinomiya, japan; dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c ventral root (c vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: ( ) a continuous column extending from the para-facial region to the pre-bötzinger complex, and ( ) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os p- - slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca + -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi , os p- - the effect of music to sex hormones of elderly person hajime fukui , kumiko toyoshima , kiyoto kuda , katsuhiko iguchi nara univ. of edu., nara, japan; grad. school of human sciences, osaka univ. japan; nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a -kda protein band in csf of % (n = ) of confirmed and % (n = ) of suspected tbm patients out of tbm patients. the -kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv c (ag a) and rv c (ag b) and one host derived protein as the components of the tbm specific -kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q , vitamin d and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os p- - involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh , boon chock , dennis l. murphy , chuang c. chiueh dept. applied pharmacol., univ. toayama, toyama, japan; lab. bioch., nhlbi, nih, md, usa; lab. clin. sci., nimh, nih, md, usa; cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk / and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy y cells. consistently, trx increased the expression of mnsod and bcl- supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl- proteins for protecting against mpp + -induced neurotoxicity. os p- - pgd induces neuronal apoptosis via d- , -pgj tatsurou yagami , noboru okamura , toshiyuki sakaeda facul. health care sci., himeji dokkyo univ., himeji, japan; kobe univ. grad. sch. med., japan prostaglandin d (pgd ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd induced neuronal apoptosis in rat cortical cultures. however, a pgd receptor blocker did not suppress neurotoxicity of pgd . little pgd receptor was detected, suggesting an involvement of pgd metabolites in the apoptosis. among pgd metabolites, -deoxy- , -prostaglandin j ( d- , -pgj ) caused neuronal apoptosis most potently and rapidly. although d- , -pgj is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that d- , -pgj induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ h] d- , -pgj (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd induced apoptosis via d- , -pgj in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the d- , -pgj -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c to t ). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi os p- - compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi ( ) os p- - timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae , xiaofeng lu , , yusuke uchida , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of targets following a gosignal given at a random timing between and ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: ( ) those that showed buildup activity during the delay period until the go-signal, and ( ) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, - h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (> ms) than larvae (> hpf, ms). m-cell activity monitored with confocal ca + imaging during the tail flip (> hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (< %) after hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca + release through ryanodine receptors, triggered by glutamate receptor-mediated ca + influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os p- - distribution of voltage-gated calcium channel ␣ ␦- mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka - , japan the ␣ ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ ␦- to ␣ ␦- . the distributions of ␣ ␦- , - , - mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ ␦- remains to be determined. we cloned ␣ ␦- cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ ␦- mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ ␦- mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ ␦- subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os p- - hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb ) depressed the excitation, while bicuculline depressed the inhibition. h or h histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os a- - activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c bl/ mice. by recording field epsp (fepsp) along m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at - div, then the amplitudes of fepsps in the ventral side began to decrease at - div, and dominated in the dorsal area at div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at - div but the axons terminals in the ventral area were eliminated until div. this synapse elimination from the ventral side was blocked by apv application from div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until div. the number of those ventral axons is reduced before div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during - div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os a- - transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os a- - segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa in r is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa function resulted in selective loss of eph expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa dependent cues are required to determine the territory for whisker representation in r and the assembly of a somatosensory circuit. os a- - the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p and those in the venrtolateral (vl) area were eliminated from p to p and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p , reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os a- - proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi ( ), kakenhi ( ) os a- - the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m , s , sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms ( , , hz, stimulations) was compared in dp patients. the pain relief was evaluated with visual analogue scale. high frequency ( , hz) rtms of m was the only effective stimulation for treating intractable pain in of patients ( %). the pain relief continued for h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m . os a- - involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p x r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p x , , / rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p x , , , , / , / r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p x rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p x r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os a- - the cantor-like patterns in rat hippocampal ca pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after days of age, before which they had been reared with song tutors from birth. when the noise stopped at - days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization ( days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, ) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, ) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada ( ) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca , may be encoded in ca in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca , using -compartment model of neuron, and we found the cantor coding of information of time series in the model ca . it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in patients with chronic ischemia due to diverse vascular occlusive diseases (n = ) and moyamoya disease (n = ). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os a- - hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata , , heng zhao , gary steinberg department of neurology, brain research institute, niigata university, niigata, japan; department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia ( • c) reduced infarct size by %. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < . ). p-␦pkc (t ) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s ) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, -vessle occlusion was induced. fluo- /am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu- , yokogawa, japan) equipped with × objective lens] was inserted into the brain. -vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo- increased up to %, and persistently increased up to % during -min reperfusion, indicating the long-lasting ca + increase in the ca region. in contrast, in the frontal cortex, -min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca + in situ. research funds: kakenhi # , # os a- - reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto , h. watanabe , y. kumon , t. ohnishi , chi ii , y. imai , j. tanaka dept. neurosurgery, ehime university, japan; dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b and cd b recognized with ox antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba + mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba + amoeboid-shaped mg started to accumulate days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os a- - insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki , , chihiro tamaki , sumio ohtsuki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein (lrp- ) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp- expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤( - ), which reached . -fold greater uptake than non-treated control after min treatment. increase of the hepatic uptake of a␤( - ) by insulin was concentration dependent (ec = pm), and was completely suppressed by rap ( m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp- to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os a- - mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used n astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of n cells was inhibited by an inhibitor of ip receptors, -aminoethoxydiphenyl borate ( -apb) or an endoplasmic reticulum ca + -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca + was inhibited by -apb and cpa. moreover, removal of external ca + accelerated the reversal of thrombin effects. these results suggest that refilling of ca + store by ca + entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age and a -year-clinical course. the brain showed severe atrophy ( g). microscopic examination disclosed that all telencephalic neocortices had nft of more than and sp of more than . all limbic cortices and nuclei had nft of more than and sp of more than . although there was no sp, various numbers of nft were observed in the following structures: claustrum , caudate , globus pallidus , hypothalamus , meynert's nucleus , thalamus , substantia nigra , central gray , locus ceruleus , purkinje cells , posterior root ganglion , adrenal medulla . this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os a- - transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv was expressed in soma, dendrites and synapses in the neurons. since trpv was firstly cloned as an osmotically activated channel, we hypothesized trpv might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv -deficient neurons at • c, and found rmp in wild type was more depolarized by approximately mv than rmp in trpv -deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv -deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv is involved in regulation of both neural activity and spine motility in hippocampus. os p- - a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at locations per ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by -photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os p- - d -like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a receptors (adora ) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora in the ng decreased by > % of that in the non-treated ng, being not accompanied by a change in mrna level for a a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase- activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf blockers such as brefeldin a, exo , and arf n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf and subsequent pld may regulate the k + -current response induced by da. os p- - p gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa , toshihiko kuriu , ayako m. watabe , toshiya manabe , shigeo okabe , tadashi yamamoto div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; dept. of cell biol., tokyo medical and dental univ., tokyo, japan; div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p gap, which interacts with the nr b subunit of nmdar. within neurons, p gap was highly concentrated in the postsynaptic density and co-localized with nr b and an actin-binding protein, cortactin. p gap promoted gtp hydrolysis of cdc and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p gap. when over-expressed in dissociated neuron, p gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os p- - non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os p- - spatial frequency tuning of disparity-selective neurons in macaque v hironori kumano , seiji tanabe , ichiro fujita grad. sch. of engineering science, osaka univ., osaka, japan; grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional ( -d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using -d filtered noise stereograms. the disparity tuning of most v neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os p- - predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v neurons. the results suggest that v neurons are involved in mediating stereoacuity. os p- - the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field -d ternary dynamic random noise, generally refreshed every - ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v /v neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v , and especially areas beyond. research funds: mext( ), jsps( ), coe os p- - analysis of center-surround organization of v neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area (v ) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of cells analyzed, % showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ± degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os p- - spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki , izumi ohzawa , grad. school of eng. sci., osaka univ., japan; grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with × dots was presented over an area to times larger than the rf of a complex cell. for each dot location, a nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of nd-order kernels at all locations. nd-order kernels had roughly comparable extent as the rf, and contained . subregions on average (n = ). among complex cells, whose rf envelopes were elongated, cells exhibited the horizontal elongation. research funds: mext( ), jsps( ), coe os p- - orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito , osamu sadakane , masahiro okamoto , hironobu osaki , hiromichi sato grad. sch. med., osaka univ., osaka, japan; grad. sch. front. biosci., osaka univ., osaka, japan; med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about % lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that % of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os p- - temporal dynamics of suppressive receptive field surround in cat v satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v ), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes ( ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (< ms) but not in middle ( - ms) and late responses ( - ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from . deg (early response) to deg (middle response) and . deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi ( ) os p- - spatial-frequency dependent surround suppression in cat v ayako ishikawa , satoshi shimegi , hiroyuki kida , hiromichi sato grad. sch. front. biosci., osaka univ., osaka, japan; grad. sch. med., osaka univ., japan; grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v . we used a stationary flash (duration, ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus ( ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = ) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os p- - contrast-dependency of spatial summation property in cat v and lgn masahiro okamoto , tomoyuki naito , osamu sadakane , hiromichi sato grad. sch. front. biosci., osaka univ., japan; grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v ) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by . and . times in v and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v , the strength of surround suppression did not affected by stimulus contrast on average. we conclude that ) rog model describes the surround suppression better than dog model both in v and lgn, ) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida , satoshi shimegi , ayako ishikawa , hiroshi sakamoto , hiromichi sato grad. sch. eng. sci., osaka univ., japan; grad. sch. med. sci., osaka univ., japan; grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v ), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into flanks ( • step), and stationary stimulated with an annulus, oppositely-faced flanks ( -fk) or a flank ( -fk) stimulus. the durations of stimulus presentation were ms for crf and ms for srf stimulation. localized srf stimulation with either -fk or -fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for -fk stimulation throughout the crf response. however, -fk stimulation of end position to crf had strong and long-lasting suppression on responses during - ms after onset. os p- - temporal-frequency dependency of receptive field size and surround suppression in lgn and v osamu sadakane , tomoyuki naito , hironobu osaki , masahiro okamoto , hiromichi sato grad. sch. med., osaka univ., japan; med. sch., osaka univ., japan; grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v ) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v neurons, suggesting that the subcortical tf tuning property contributes to that in v . ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. ; yang and maunsell ) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using . t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around %) was smaller than that of bold (> %), the temporal profile showed a constant precedence of the diffusion signal by . s. os p- - new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os p- - spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international - system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad ( . ), approaching unity (∼ . ) for oh, and in between for control ( . ). statistical analysis was performed and significant differences were observed for these groups (p < . ). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: ( ) resting, ( ) staring at a standing human object, ( ) thinking of standing, ( ) standing with eyes open, ( ) standing with eyes closed. regional cbf data analyzed by spm were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os p- - potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono , , eiji shigetomi , kiyoharu inoue , fusao kato dept. neurol., jikei univ., sch. med., tokyo, japan; lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os p- - effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan , -methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin ( -ht) release and cause degeneration of -htergic nerve terminals via -ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the -htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on -ht release and -htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant -htergic neurons around the raphe nuclei. treatment with a -htergic neurotoxin , -dihydroxytryptamine dramatically reduced the tissue contents of -ht and its metabolite, which was blocked by a selective -ht reuptake inhibitor. mdma and meth ( . - m) increased -ht release, and reduced the tissue contents of -ht and its metabolite at higher doses. the mesencephalic slice culture including the -htergic raphe nuclei may be useful to examine the mechanisms underlying -htergic neurotoxic effect of mdma in vitro. os p- - studies on drug dependence (rept. ): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os p- - prostaglandin d is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd by hpgds and that astrocytes expressed both dp and dp receptors and were activated by pgd . we found that gliosis and demyelination were well suppressed in hpgds-or dp -null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd is a key molecule of neuroinflammation involved in the demyelination. research funds: , os p- - on a sodium channel distribution enabling high frequency signal processing go ashida , , kousuke abe , kazuo funabiki grad. sch. medicine, kyoto univ., kyoto, japan; grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below khz, while that in the node does up to khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated nl cells from owls and achieved intracellular recordings in of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = ± mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, vision impaired ( blind and low vision) and sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os p- - autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada , runa araya , naoto kitamura , yuji mishinsa yamada unit, riken bsi, saitama, japan; nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba -positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os p- - fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita , , hideomi tanaka , , sachiko tsuruoka , hironori wada , , hitoshi okamoto , riken bsi, wako, japan; jst crest, kawaguchi, japan; riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose , dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi ( ) os p- - initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd -homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin (fas ) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas genetically expressed on a muscle, we found fas accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas is crucial for the postsynaptic localization of fas . in addition, postsynaptic localization of a scaffolding protein dlg, psd -homolog, and glutamate receptors was impaired in fas mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor a (mef a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef a that is sumoylated at lys promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef a at ser and thereby promoted a switch from sumoylation to acetylation at lys , leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns , ag ps a-a characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., ) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., . mol. brain res., , - . research funds: kakenhi ( ) ps a-a the distribution of snap- protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap- derived from exon splicing are expressed in brain. we generated two specific antibodies for snap- a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap- a and b during early postnatal period. snap- b was low at the birth and increased remarkably thereafter. by the contrast, snap- a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap- b but not snap- a was recovered in raft-enriched fractions of triton x- -treated lp membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap- b was widely distributed throughout brain, whereas, snap- a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap- protein are regulated differently in isoformspecific manners, and snap- a and snap- b play different functional roles in brain. ps a-a erc(elks/rab ip /cast) regulates syaptic short-term plasticity by recruiting bmunc - to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain ( . mol. brain res. , - ) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt- , are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt- , the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt- (-/-) mice, and a novel antagonist of glial glutamate transporters, ( s, s)- -[ -( -methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately %) of functional transporters, glast and/or glt- , is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat- gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat- mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat- vglut. a mutation in avr- glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr- functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps a-a biochemical characterisation of the vesicular glutamate transporter stephan schenck, shigeo takamori department of neurology and neurological science, st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut and vglut are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut and vglut has been found. to further characterise the specific properties of the transporters we make use of the vglut -ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut by yeast two-hybrid screening and found endophilin a . as expected for the interacting molecule to vglut , endophilin a was typically localized to vglut -positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in ∼ min, followed by spontaneously recovered after min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) was demonstrated in the lactate medium. results suggested that increased mct expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu , kazuhito tomizawa , shuang liang , iori ohmori , teiichi nishiki , kohji takei , hideki matsui dept. of physiol., okayama univ., okayama, japan; dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca + -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos- cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately ( th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm - and fm - , and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca + channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca + channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca + influx and epscs, suggesting that exocytotic machinery downstream of ca + influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca + or g-proteins. ps a-b spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca + channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap- subunit can directly bind with synprint site. this interaction was ca + -dependent, being weaker at concentrations higher than nm. in contrast, the interaction of synaptotagmin with synprint was optimal at m ca + , being weaker at lower or higher concentrations. the binding domain of synprint for ap- and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca + channel synprint site makes ca + -dependent interactions with ap- and synaptotagmin thereby contributing to vesicular endocytosis. ps a-b acl- , an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl- ˜ , and generated their deletion mutants. the mutants of acl- gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat- mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl- expression, suggesting that certain phospholipid species produced by acl- are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku , hussam jourdi , akiyoshi kakita , tadasato nagano , hitoshi takahashi , nobuyuki takei , hiroyuki nawa dept. mol. neurobiol., brain res. inst., niigata univ., japan; brain resource center, brain res. inst., niigata univ., japan; dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb ) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip and sap . moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima , eriko miura , keiko matsuda , tetsuro kondo , , masahiko watanabe , michisuke yuzaki dept. physiol., sch. med., keio univ., tokyo, japan; dept. anatomy, hokkaido univ., sch. med., sapporo, japan; mol. neurophysiol., aist, tsukuba, japan cbln is a member of the c q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln - , are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln , cbln and cbln were secreted as glycoproteins, whereas cbln was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd- and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd- . more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd- at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf /ra (ra ), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca +independent homophilic trans-cell adhesion activity. ra participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra -/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra interacted with par- being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par- was colocalized in the cell adherent region of p embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra +/+ testis, whereas it was undetectable in the spermatid of the ra −/− testis. ra and jam-c were localized around the head portion of spermatid and ra deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra and par- . research funds: izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd- clusters in cultured hippocampal interneurons. higher density of dendritic psd- clusters was observed in interneurons at div. at div, this difference was less prominent, mainly due to > -fold increase of psds in excitatory neurons. psd- -gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd- -gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps a-c two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in - div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, ; shigetomi & kato, ) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites ( s, -micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi ( ) ps a-c ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of d-and d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps a-c integrative spike dynamics of rat ca neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur connects with syntenin at the dendrites and mglur with pick at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur that was known to be ectopicly expressed in the dendrites of mglur -deficient on cone bipolar cells. we observed no pick but only syntenin at the mglur -deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur immunopositivity. these findings suggest that mglur connects with syntenin in place of mglur that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca + -permeable channels. trpv , expressed in sensory neurons, is activated by noxious heat above • c, whereas trpv , expressed in keratinocytes, is sensitive to moderate temperatures (> • c). here we examined the role of trpv and in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to • c did not induce the change in bt, desensitization of trpv with capsaicin resulted in the increase in bt. on the other hand, in trpv -knockout mouse, moderate thermal radiation (> • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv and respectively. research funds: kakenhi ( ) ps a-c generation and biochemical analysis of a glur␣ knockout mouse hirotsugu azechi , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan glur␣ (glur ) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣ , we generated a conditional glur␣ knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra f using c bl/ (b ) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra f was used as a target of glur␣ line. by crossing gra f and tlcn-cre that expressed cre in germ line cells, glur␣ null ko mice were produced, but most of them died within days after birth. to overcome the lethality, the glur␣ mutation was transferred onto b / or b /cd- genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣ in the distribution of functional ampa receptors in vivo. ps a-c gisp: a novel brain specific protein that binds to the gaba b subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b and gaba b from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b protein and also promotes gaba b surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b /gaba b complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b -binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut - ) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut and vglut seem to participate in this glutamate accumulation. in the present study, we found that vglut mrna is also expressed in the adult rat pinealocytes. vglut immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut -ir or vglut -ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito , , , kenji nakamura , toshikazu kakizaki , , satoe ebihara , masakazu uematsu , shigeo takamori , minesuke yokoyama , shiro konishi , masayoshi mishina , , jun-ichi miyazaki , kunihiko obata , yuchio yanagawa , gunma univ., maebashi, japan; sokendai, hayama, japan; sorst, kawaguchi, japan; mitsubishi kagaku inst. life sci., machida, japan; kumamoto univ., kumamoto, japan; toyohashi univ. tech., toyohashi, japan; tokyo med. den. univ., tokyo, japan; univ. tokyo, tokyo, japan; osaka univ., suita, japan; riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi ( ) ps a-c postnatal changes in the colocalization of vglut and vglut immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura , , akiya watakabe , hiroyuki hioki , fumino fujiyama , yasuyo tanaka , tetsuo yamamori , takeshi kaneko , dept. morphol brain sci., grad. sch. med., kyoto univ., japan; crest, jst, japan; div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut) and vglut accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut and vglut in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p . the cc was higher in area s than areas m and area v throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r and r . the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin ( -hydroxytryptamine, -ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several -ht receptor subtypes, -ht transporter ( -htt) and vesicular monoamine transporter (vmat ) in primary cultured mouse osteoblasts and mc t -e osteoblastic cells. sustained exposure to fluoxetine, a selective -ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc t -e . these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦ glutamate receptor (glur␦ ). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦ were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho j, was caused by a new type of intragenic deletion of the grid gene, which was indeed translated as glur␦ lacking -amino acids in the n-terminus. mutant glur␦ proteins were retained in the soma of purkinje cells and degraded. as a result, ho j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦ signaling and cerebellar functions. research funds: kakenhi ( ) ps a-d role of the dry motif in melanin-concentrating hormone receptor in signaling yumiko saito , yoshimi aizaki , mituse nakano , kei maruyama dept. pharamacol., saitama med. sch., saitama, japan; international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp , arg and tyr in the dry of rat melanin-concentrating hormone receptor (mch r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a r and p y r electrophysiologically using xenopus oocyte expression system. when a r and p y r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/ pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps a-d dual signaling via metabotropic glutamate receptor ␣ is regulated by a cytoskeletal protein . g michihiro tateyama , , yoshihiro kubo , department of biophysics and neurology, nips, aichi, japan; sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor ␣ (mglur ) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur are differentially regulated by different types of ligands, glutamate and gd + . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur . these proteins, such as homer and . g, are also known to change the membrane expression of and modulate the function of mglur . here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur . interestingly, the functional couplings of mglur to gq and gs pathways were altered by co-expression of . g, but not by homer. deletion of the c-terminal tail abolished the effect of . g, indicating that the interaction of . g with the c-terminal tail of mglur regulates the multi path signaling. ps a-d modulation of the eaac -mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto , , saori akiduki , age dimension research center, aist, ibaraki, japan; graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein - (gtrap - ), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier (eaac ). it contains two potential pkc phosphorylation motifs at positions - and - . however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac -mediated glutamate transport activity in c bu- cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine into alanine (s a) or at serine into alanine (s a). as compared with wt, s a had no inhibitory effect on glutamate transport activity under exposure to nm pma, and had increased glutamate transport activity under normal condition. by contrast, s a had the same glutamate transport activity as that of wt. thus, the eaac -mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine in addicsin. kaori akashi , manabu abe , toshikazu kakizaki , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, japan; sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤ - (glur - ) and glur␥ - (ka - ) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤ ko mice from c bl/ es cell line. the glur␤ ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥ and glur␥ proteins were significantly decreased in the crude fraction of ca region of glur␤ ko. furthermore, subcellular localizations of both subunits were also changed in glur␤ ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤ subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps a-d sema d/plexin-b activates gsk- ␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin d (sema d) receptor plexin-b induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b -mediated r-ras gap activity, leading to growth cone collapse. sema d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk- ␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk- ␤ antagonist suppressed the sema d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b -induced dephosphorylation of akt and gsk- ␤. plexin-a also induced dephosphorylation of akt and gsk- ␤ through its r-ras gap activity. thus, we conclude that plexin-b dephosphorylates akt and gsk- ␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur . hops was co-immunoprecipitated with grip (glutamate receptor interacting protein ) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur via grip binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc- . a neural amino acid transporter snat /ata also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc- immunoreactivity (asc- -ir) was detected in dendrites and somata of pyramidal neurons. the snat -ir was widely detected in neurons, whose intracellular localization was similar to that of asc- -ir. deferent from asc- -ir, snat -ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc- and snat to the neuronal uptake of l-serine. snat might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps a-d neuronal glutamate transporter eaat controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake , si-young song , shiro konishi , keiji imoto natl. inst. physiol. sci. (nips), okazaki, japan; mitsubishi kagaku inst life sci, tokyo, japan; tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat /glt- blocker threo- -methylglutamate, but not the glt- blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat was abundant. the results suggest that eaat plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat expression in postsynaptic pcs. chitoshi takayama , yoshiro inoue department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k- cl co-transporter (nkcc ) and k-cl co-transporter (kcc ). in this study, we focused on kcc , which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc with special reference to the neuronal development in the cerebellum. kcc was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc . the kcc -protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc -expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima , hisashi mori molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d -like and d -like according to their pharmacological properties. the d -like receptors consist of d and d receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop ( i loop) domain of d r fused with egfp effectively reduce camp production mediated by d and d receptors. interestingly, we also identified that the i loop domain of d r interfere with gs coupled beta adrenergic receptor signaling. these results suggest that the third intracellular loop of the d receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d -like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama , yoichi ogawa , hiroki yoshino , masayuki yamashita , toshifumi kishimto dept. psychiatr.; dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca pyramidal cells. da ( m) profoundly decreased gaba a receptor-mediated postsynaptic currents to % in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to % in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d -like receptor, since skf , a selective agonist for pi-linked d -like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d -like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina , jinko konno , sachine yoshida , hideki ohmomo , hitoshi kawano , fumihiro shutoh , haruo nogami , setsuji hisano graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut or - mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day (e ), vglut immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e , vglut -ir was seen as puncta close to l -ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l or tag -ir in the imz, whereas vglut -ir was first observed in the sp and upper imz where l -ir existed. these results show that vglut ir corticofugal fibers appose to elongating vglut -ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps a-d developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca regions michie kon , yoichi avil , hiroshi tsubokawa , dept. of information engineering, tohoku univ., sendai, japan; grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine ( . - mg/kg) produced a dose-dependent cpp. using a -day schedule of conditioning, it was found that intra-ca administration of nmda receptor antagonist, mk- ( and g/rat) significantly attenuated the morphine ( . mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda ( . , . and g/rat) significantly potentiated the morphine ( . mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk- receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura , kouichi nakamura , , hiroyuki hioki , masanori uemura , takeshi kaneko , dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d receptor are expressed in striatonigral neurons and d receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d , d , d , d , d ). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p - ) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since ( ) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, ( ) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, ( ) depolarizing agent released tpa from isolated neurosecretosomes, ( ) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u st, galnac - st, d st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda , maki ishii , isao nagata , yumiko shimazaki dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo- epitopes, glca( s)␤ - galnac( s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa , mineko kengaku , nobuaki maeda dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka , tohru marunouchi division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp or sulffp appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of -o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of -hydroxytryptoamine ( -ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day or rat cerebral cortex was cultured in the presence of -ht, na or da. after days, we analyzed dendrite formation using anti-map antibody. after - days, we analyzed synaptogenesis with anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( - nm), na ( - nm) or da ( - nm) increased dendritic length of pyramidal neurons. -ht ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by -ht a receptor, ␣ a receptor and d receptor, while inhibited by -ht a and ␤receptors. in addition, synaptogenesis was promoted by -ht a and -ht c receptors, whereas inhibited by -ht a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in t cell, singar associated with p and p , the subunits of pi kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps a-e lrfn , a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd- naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd- , a postsynaptic scaffold protein. we examined the physical interaction between lrfn and psd- by immunocrecipitation and pull-down assay, since lrfn contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn associated with psd- /nmda receptor complex in the brain extracts and lrfn directly bound to psd- via its pdz domain-binding motif. in this study, we suggest that lrfn may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt - ). among the prmt proteins, the prmt has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt in the rat brain. the prmt was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt in the brain, we tried to identify novel interacting proteins with the prmt in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps a-e proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi , , michihiro igarashi , div mol cell biol, grad. sch. med dent sci; trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi ; project-promoting grant from niigata univ ps a-e netrin- is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda , keisuke watanabe , kazuhiro ikenaka , katsuhiko ono , hiroyuki yaginuma dept. anat., fukushima med univ. sch. of med., fukushima, japan; div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin- is known to be a chemorepellent for a subset of axons, the role of netrin- for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin- in this repulsion. in the mouse embryo at e , netrin- is expressed in the floor plate and the dermamyotome, and the netrin- receptor unc c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin- repelled chick e drg axons. moreover, using function-blocking antibody against netrin- , we revealed the fact that netrin- plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin- to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p neonates, but the ventral neurons became unresponsive after p . the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe , hirohide takebayashi , , kazuhiro ikenaka , katsuhiko ono div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; dev stem cell biol. program, ucsf, usa netrin- is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin- also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin- −/− background, using olig -lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig +/lacz ;netrin- −/− mice, ventral migration of olig cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig -positive cells. these results suggest that netrin- exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g and netrin-g are vertebrate-specific membrane-anchored members of the unc- /netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g and netrin-g proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g and netrin-g differentially bind to lrrcontaining proteins, ngl- and a related molecule nag , in vitro. ngl- and nag in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g and netrin-g , respectively. furthermore, in netrin-g and netrin-g deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang , ryuichi sakai , teiichi furuichi lab. molecular neurogenesis, brain science institute of riken, saitama, japan; division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p -p , stimulation of parallel fibers produces excitation of purkinje cells lasting for - ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p -p . since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p and that they then locate in purkinje cells, in cell body on p and in primary dendrites on p -p . the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya- and mmahya- ) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya- protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya- is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya- for the early cerebellum development. toshitaka morishima , erina fukushi , kazuto kobayashi , naohiro hozumi , sachiko yoshida toyohashi university of technology, toyohashi, japan; honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto , hideyuki dekimoto , tomiyoshi setsu , masahiko watanabe , mikio hoshino , yo-ichi nabeshima , toshio terashima dept. of anat., kobe univ. grad. sch. of med., kobe, japan; dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida , tomoko nishiyama , hitoshi tatsumi , masahiro sokabe , department of physiology, nagoya university graduate school of medicine, nagoya, japan; icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately h and the elongation started at approximately h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca . once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca , which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) or e rat. dendritic outgrowth of pyramidal neurons was analyzed after days using anti-map antibody. synapse formation was analyzed after - weeks, using anti-psd- and anti-synaptophysin antibodies. in the presence of cgrp ( - nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of hydroxytryptoamine ( -ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day rat hippocampus was cultured in the presence of -ht or na. after days, we analyzed formation of dendrites using anti-map antibody. after days, we analyzed formation of synapses using anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( nm) or na ( nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites -ht ( - nm) and na ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, -ht a receptor and ␣ a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di- anepeq) and the imaging system (micam ). in dscam-ko mice, the optical signal which precedes c inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps a-f hippocampal cells cultured on d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after weeks in vitro, neurons formed a dense layer, > . mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, ( ) collagenasespecific inhibitor did not affect cell depth, ( ) limiting gliosis by antimitotics reduced the thickness of the tgl by %, ( ), neither glial nor neuronal cell body were found in the tgl by gfap/map detection, ( ) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca + by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama , kazuto kujira , tetsuya kawabe , takao ito , yoshihiro tsuruo department of cell biology and anatomy, wakayama medical university, wakayama, japan; department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) ( ) ps a-f metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama , hitoo nishino , takehiro yahata nagoya city university graduate school of medical sciences, nagoya, japan; nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit , anand goswami , nobuyuki nukina , nihar ranjan jana national brain research centre, india; laboratory for structural neuropathology, riken brain science institute, - hirosawa, wakoshi, saitama - , japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin- for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc . finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of , ests ( , non-redundant sequences). of them matched known genes of rodents in the genbank databases, but remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of , , or weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps a-f transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb- , an inhibitor of the type tgf-␤ receptor (t␤r ). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps ( h restraint; min forced-swimming; ether anesthesia) then they were put in untouchable situation for days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c bl/ j mice. pups were separated from mothers daily ( h) on postnatal days through . starting at weeks of age they were tested for ( ) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; ( ) responses to social stimuli in social investigation (sit) and social preference tests; and ( ) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of db (so), or were forced to swim (sw). these manipulations were performed for min once a day at weeks after birth (from postnatal days to ) or weeks after birth (from postnatal day to ). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of weeks. it was found that the spatial learning activity of both so and sw mice manipulated weeks after birth was impaired as compared to no mice. so mice manipulated weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo , yukiko yamada , mitsuo iinuma , yasuo tamura , fumihiko iwaku , kazuko watanabe , minoru onozuka dept. oral anat., asahi univ. sch. dent., japan; dept. ped. dent., asahi univ. sch. dent.; dept. physiol., gifu univ. sch. med., japan; dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara , yayoi kitamura , junichi tanaka , yukio hattori , yasushi hayashi department of human nutrition, notre dame seishin university, okayama, japan; department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c bl/ j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a -min interval, to measure the basal temperature (t ) and stress-enhanced temperature (t ), respectively. the difference ( t = t − t ) gives the sih. in control mice, t was approximately • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t . furthermore, spermidine and spermine also lowered t and t at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox- and cox- . it has been known that, under some stressful conditions, cox- is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for h and subjected them to the elevated plus maze task h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox- by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps a-g imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang , takesi sakurai , yasuitirou fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. pharmacol., univ. tsukuba, japan; dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of - weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between and weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between and weeks of age, and in the hippocampus at the age of weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi # ps a-g a systematic analysis of genetic factors associated with behavioral diversity between msm and c bl/ koide tsuyoshi , , aki takahashi , , toshihiko shiroishi , , akinori nishi mgrl, national institute of genetics, mishima, japan; sokendai, hayama, japan; mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c bl/ . in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b - cmsm, which carries chromosome of msm, showed extreme behavioral differences from c bl/ . the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga , yu kajiyama , shigenobu shibata , hiroshi kunugi department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e ) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e ) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps a-g synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi ( ) ps a-g involvement of nr b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary , takanobu nakazawa , yuji kiyama , toshiya manabe , tadashi yamamoto div. of oncology, ims, univ. of tokyo, tokyo, japan; div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr b is tyrosine-phosphorylated, with tyr- being its major phosphorylation site. to investigate the role of tyr- phosphorylation, we generated mice with a tyr phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr- and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr b is involved in the control of emotional responses by regulating crf content. ps a-g increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca + signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for weeks, locomotor activity and longinterval ( , and ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui , , yosky kataoka , , yasuhisa tamura , yasuyoshi watanabe , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at - times/min), inhibition periods (less than times/min) were often observed h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns- , a selective cox- inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps a-g impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima , naoya aoki , andras csillag biology, hokkaido univ., sapporo, japan; agriculture, nagoya univ., nagoya, japan; anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task , chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to s. in task , chicks chose between small reward located at cm and large reward at − cm, where cues signaled the distance of invisible food. task was similar to the task , except that cues signaled the food quantity. in task , total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi ( , ) ps a-g analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe , which had a missense mutation in the casy- gene. several casy- deletion mutants also showed learning defects. casy- has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x l/mint . however, after dissociation of x l, alcadein is susceptible to cleavage by protease(s). we found that casy- was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy- can also be proteolytically cleaved. ps a-g insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama , atsushi kuhara , akiko mohri , , kotaro kimura , , masatoshi okumura , masahiro tomioka , yuichi iino , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; mol. genet. res. lab., univ. of tokyo, japan; inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins- encoding insulin homologue caused defective associative learning, mutations in daf- and age- encoding the homologues of insulin receptor and pi -kinase, respectively, suppressed the defect of ins- , and the mutation in daf- encoding forkhead transcriptional factor caused the learning defect. this suggests that ins- antagonizes daf- insulin-like signaling for associative learning. interestingly, age- animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age- in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins- . we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps a-g analysis of ttx- : novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx- (nj ) and ttx- (nj ) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx- encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx- is similar to ric- , identified in c. elegans at first and conserved among several species (halevi et al., (halevi et al., , . ric- is thought to be required for the maturation of acetylcoline receptor (halevi et al., ) , so ttx- may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps a-g analysis of aho- mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio , akiko mohri , , eiji kodama , atsushi kuhara , mizuho koike , kotaro kimura , , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho- (nj ) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., ) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho- gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho- is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho- gene further. we have demonstrated aversive conditioning in lymnaea using mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b , located in the buccal ganglion. an associative memory, lasting h, was produced with pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura , dai hatakeyama , tetsuro horikoshi , etsuro ito , manabu sakakibara lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno , minoru saitoe , , tim tully tokyo metropolitan institute for neuroscience, tokyo, japan; department of biology, tokyo metropolitan university, japan; cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi ke zhang , jian z. guo , ai k. guo , institute of neuroscience, chinese academy of sciences, china; institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal /uas-shi ts system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (< min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari , , kazuo okanoya , , lab. for biolinguistics, riken-bsi, wako, japan; grad. sch. of sci. and tech., chiba university, chiba, japan; presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day , developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps a-h facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi ps a-h the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment , rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of . m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment , rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi ( ) ps a-h transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps a-h gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c bl/ mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over %, while those in the mscand ptx-injected mice decreased to %. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦ that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps a-h post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao , , takehito okamoto , fumihiro shutoh , lab for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan; dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of h daily training for week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi ( ) ps a-h modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri , , takehito okamoto , shin-ichi nishimura , fumihiro shutoh , , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; univ of the air, chiba, japan; dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., ) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps a-h influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho , , t kobayashi , , e hori , , k umeno , , t ono , h nishijo , system emotional science, univ. of toyama, toyama, japan; crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of place cells recorded, activity of was significantly modulated by the car speed and/or distance between the car and rat; , and cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a . - . mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas and , contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca + but is blocked by mg + in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg + binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l is a component of the ubiquitin system. uch-l is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl . gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca . in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps a-i learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage ). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band ( - hz) was strong at stage and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps a-i longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about . mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal ubiquitously expresses in brain. bmal functions by forming a heterodimer with either clock or npas , which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal function in a forebrain. using a dominant negative mutant of bmal (bmal r a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., ), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal r a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn ( , , or db; intermittent white noise). db bgn induced significant decreases in number of error and time to goal in maze compared with and db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn , , anucha pongpanparadorn , wasana pratchayasakul , anchalee pongchaidacha , nipon chattipakorn fac. dent. cmu, chiang mai, thailand; cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were ± , ± and ± in the groups administered , and mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was . ± . , . ± . and . ± . for , and mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a -h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a -h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses ( , and min). percentage inhibition of cache activity following a single tde injection at doses and mg/kg significantly decreased at and min after tde injection, but not at min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣ and ␣ -nachr and kinases involved in pi k-akt pathway, and increase the levels of phosphorylated akt and bcl- . these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi k-akt pathway via ␣ and ␣ -nachrs. ps a-i arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae , yoshiyuki ishikura , shigeaki fujikawa , yoshinobu kiso , manabu sakakibara lab. neurobiol. engr., tokai univ., numazu, japan; inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in -month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps a-i thimet oligopeptidase co-exists in gfap-and cd b-positive glia in rat pc/rsc treated with mk- takeshi kato , mohammad arif , michiyuki yamada , toshiyuki chikuma , md. mahiuddin ahmed lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; grad. sch. integr. sci., yokohama city univ., yokohama, japan; dept. hygien. chem., showa pharmaceut. univ., machida, japan; dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep . ) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk- activated gfapand cd b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) day after the treatment. mk- also increased ep . and prolyl oligopeptidase. immunohistochem data showed that ep . co-localized with gfap and cd b positive glial cells. since mk- causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep . in the pc/rsc. these data suggest that ep . in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps a-i effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) and in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) and , code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps and ps with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps a-i effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of -week-old adult mice. in this study, a␤ peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤ peptide. we found that treatment of nsc with oligomeric form of a␤ peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤ peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein- (hmgb ) is a non-histone chromosomal protein. here, hmgb was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb immunoreactivity was observed around dying neurons in the kainic acid-and a␤ - (a␤ )-injected rat hippocampi. hmgb was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb delayed the clearance of a␤ and accelerated neurodegeneration in a␤ -injected rats. these results suggest that hmgb released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps a-i x -like protein regulates metabolism of app in the mouse brain yoshitake sano , , tadashi nakaya , shigeyoshi itohara , toshiharu suzuki riken bsi, saitama, japan; hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x -like protein (x l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells ( . j. biol. chem. , . j. biol. chem. , ) . there is no evidence, however, that endogenous x l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x l in app metabolism in the brain, we generated x l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin- protein by d-electrophoresis and tof-mass delineate that mutant at and htt proteins similarly reduce transcriptional factor x and x . immunoprecipitation and pull-down assays support interaction between polyq and factor x and x . immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x and x into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x and x ameliorates poly-q pathology in htt-/at -expressing neurons and transgenic drosophila. these results suggest that factor x and x are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps a-j ba - was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, rats/group (g). group received e at . g/kg per day; groups and received ycj at ml, and ml/kg day, respectively, once everyday. group received ycj ml/kg plus e at . g/kg day twice a week, all for weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e in group was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid - antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer and layer of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group was lesser than ovx and control groups. amyloid deposition was correlated with e serum at r = − . . ps a-j correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki , shigeo kinomura , kazunori sato , shinya uchida , ryoi goto , kentaro inoue , ichiro tsuji , hiroyuki arai , ryuta kawashima , hiroshi fukuda department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; tohoku univ. grad. school of med., sendai, japan; niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h -kenko- ), (h -choju- , h -choju- ) ps a-j effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook , d.g. goodenowe , y. yamazaki , j. flax phenomenome discoveries inc., saskatoon, canada; precisionmed inc., san diego, ca, usa dementia affects about % of the population over the age of and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an -metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the -metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin- (chr ). in this study, we quantitatively investigated the rapidness of the light-gated current of chr expressed in pc cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about -fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori , shigeki ohta , kenji hamada , naofumi yamada-okabe , yonehiro kanemura , hideyuki okano , yutaka kawakami , masahiro toda , neuroimmnology research group, keio univ., tokyo, japan; chugai co. ltd., kanagawa, japan; inst. cli. res., onh, japan; physiology, keio univ., tokyo, japan; cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day were dissociated. after the cells were treated with nm estradiol- beta (e ) or ethanol for days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis ( d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than spots were detected from d dige. compared with ethanol treatment, e increased the expression of spots in the hypothalamic cells and spots in the cortical cells (p < . , difference > . ). proteomics analysis showed different effects of e for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp client protein family. -allylamino- -demethoxygeldanamycin ( -aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. -aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of -aag in a mouse model of sbma. administration of -aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of -aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp function by -aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately - % of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps a-k selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura , yoshiyuki ishii , kazuto kobayashi , yasufumi sato , keiichi itoi lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; dept. mol. genet., fukushima med. univ., japan; inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a , a and a ) and dopaminergic (a , a and a ) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg b has been shown to play an important role in the processing of lc , a mammalian homologue of yeast atg , but the tissue distribution of atg b remains unknown. to understand the role of atg b in rat tissue cells, we prepared an antibody to atg b and pc cells in which atg b expression was knocked down by rnai. in rnaitreated pc cells where atg b expression was % of that in the wild-type pc cells, the expression of cytosolic lc -i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc to lc -i. moreover, the expression of atg b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg b was intense in neurons, especially in the cerebellum. these results suggest that atg b plays a major role in the processing of lc , while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps a-k spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev n strain into the central nervous system was examined after its subcutaneous inoculation ( pfu) in the rat hind footpad. on day post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki , hiroshi kameda , hisashi nakamura , taro okunomiya , koji ohira , kouichi nakamura , , takahiro furuta , takeshi kaneko , dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn , rta , mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣ and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr increased the surface expression of glur in cos cells compared with grip/glur or ␦-catenin/glur transfection. this study points to ␦-catenin as a regulator of glur receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca area in wild type (wt) and gad knockout (ko) mice. this parameter was twice lower in gad ko mice (wt: . ± . pa/pf, n = ; gad ko: . ± . pa/pf, n = ; p = . ). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba ( m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad ko animals (wt: . ± . ms, n = ; gad ko: . ± . ms, n = ; p = . ). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p to p ) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur but not by mglur because it was antagonized by the mglur antagonist ly but not by the mglur antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps p-a involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita , michiko ohtomi cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d antagonist sch into pfc by using a microdialysis method impaired md-pfc ltp but the d antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by - %). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer / pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya - , japan we showed in previous study that somatic inhibitory synapses of neocortical layer pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate ( nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr in the regulatory mechanisms for ltp in the amygdala, we utilized ntr -knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr -ko mice than in wild-type mice. the bla-ltp in ntr -ko mice was attenuated by sulpiride, a d receptor antagonist. these results suggest that d -ntr interaction regulates the extent of ltp in the mouse la-bla synapses. ps p-a facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over -day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca + sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca + at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin- , and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps p-a inhibitory modulation of the hippocampal ca transmission and plasticity by glucagon-like peptide- jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode- (glp- ) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp- (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp- effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp- on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices ( m) were prepared from to days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca in the whole-cell mode using glass microelectrodes (resistance: - m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp- ( nm- m) inhibited spontaneous excitatory postsynaptic current. glp- ( nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp- may play an inhibitory role in the dg-ca transmission. ps p-b quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp- and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a , a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, hz for s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, hz for s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of m apv, m bicuculline, but not m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at − mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine ( - m) or capsaicin ( . - m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin ( m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv ), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji , akira komatu , akinao nose dept. phys., univ. tokyo, tokyo, japan; dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca + -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr * flies did not show mg + blockade, and dnr * flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf- , and notch. we examined how mg + blockade of dnr links to these transcription factors. research funds: kakenhi ps p-b response properties of wind-sensitive giant interneurons in the th-instar nymphs of the cricket tetsuya matsuura , masamichi kanou dept. of welfare eng., iwate univ., morioka, japan; dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) - , - , - and - in the th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the response magnitudes of gi - reached a plateau at a stimulus velocity of mm/s in most stimulus directions. the response magnitudes of gis - and - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi - , the ipsilateralfront and contralateral-rear in gi - , the ipsilateral-rear in gi - and the ipsilateral-front in gi - , designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca . serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca + , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca region. we found that ptp induced by the -hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca + channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca + channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma , kunihiko kaneko , , makoto asashima , grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca + concentration ([ca + ] i ) may change because of nonsynaptic communication. we loaded pc cells with a ca + indicator, indo- am, and the [ca + ] i of more than , cells was measured using flowcytometry. the [ca + ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca + ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca + -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss from human and mouse cns. in mouse cns, four isoforms (types - ) were expressed. subcellular localization analysis revealed that type (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc and nb a derived from neuronal cell express only type , whereas os and kt- derived from astrocyte express both type and type . interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os and kt- . we analyzed promoter region of m-spinesin gene, and identified that -flanking region from − to − bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme -phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca + signals, such as intra-and intercellular ca + waves; however, their physiological roles remain elusive. the overexpression of an ip -hydrolyzing enzyme, ip -phosphatase, suppressed the spontaneous ca + signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca + -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca + -signal-deficient astrocytes. these results indicate that spontaneous ca + signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai , noriko osumi , tohoku univ. sch. med., japan; crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax in postnatal mouse brain and found that pax was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax heterozygote mutant (sey/+) mice at weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, and h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., ) . we previously reported a unique expression of dim ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., ) . we show here that mab dim also recognizes adult neural stem cells and glial cells at postnatal period. dim , brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad / / (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk . lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in s and s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in -ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after h ( and %) and h ( and %) exposure to − and − m -ohda respectively. it was accompanied by morphological changes and induction of gfap, s- and ox . -ohda ( − m, h) was found to cause a significant impairment in h glutamic acid uptake ( %) and gsh levels ( %). further neurons (in coculture with -ohda pre exposed glial cells) on exposure to -ohda ( − m), showed loss of th expression and significant neuronal cell death ( %). the results of the present study suggest that -ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at day after injection, then started to repair at days and returned to the normal levels at weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at ∼ days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng ) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba + cells accumulated in stab wounds and ischemic lesions were found to express ng , of which molecular weight of its core protein was higher by kda than that of ng expressed in contralateral brain region. this was due to the lack of shedding of ng in the brain lesions. we found that iba + cells accumulated in stab wounds and ischemic core lesion, most of which were ng +/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd and von willebrand factor. ng + mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng + mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from < to m . most of the fibers were myelinated and counts produced a mean of , ( , - , ). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from . to . m (from fetal age to years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p - . costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca , ca pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps p-d impact of s b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani , akiko seto-ohshima , shigeyoshi itohara , hajime hirase neuronal circuit mechanisms research group, japan; lab. for behavioral genetics, bsi, riken, wako, japan s b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s b, we have recorded hippocampal spontaneous activities from urethane anesthetized s b ko and wt mice. typical eeg patterns including theta ( - hz) and sharp wave associated fast ripple ( - hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca radiatum, where ictal event was characterized by hyper-synchronous gamma band ( - hz) activity. while both populations developed ictal event within min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s b related pathways could potentially be activated. yoshiko takagishi , erina okabe , xiaoyang sun , sen-ichi oda , yoshiharu murata riem, nagoya univ, nagoya, japan; grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps p-d regulation of hippocampal neurocircuit activity by glutamate transporter glt- noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt- , the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap ) and the glt- blocker (dhk) with the signal subtraction, we could visualize the activity of glt- as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt- by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt- function in the ca region was elicited by much lower concentration of dhk than that in the ca region. ps p-d monoclonal antibody rip specifically recognizes , -cyclic nucleotide -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg- cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was , -cyclic nucleotide phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg- cells. moreover, the same localization was also observed in rat cnp transfected hek t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca + signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca + signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca + signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki , , hisatsugu koshimizu , megumi kashihara , tomoko hara , masami kojima , research institute for cell engineering; sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that -day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p ) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p expressed in the neural progenitor cells at embryonic days (e ) mice brain. to ascertain the function of p on these cells, we treated the cultured e cells with the selective chemical inhibitor for p (sb ) for days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit , takashi yamazaki , , takeshi oya , yoko ishii , masakiyo sasahara pathology ii, university of toyama, toyama, japan; oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on to days after injury, and activation of pdgfrs, mapks, akt and p were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from to days after injury. p-tyr was highly detected from to days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to days after injury and then gradually decreased. activation of akt and p continuously increased from to day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln is a cerebellum-specific protein structurally related to the c q and tumor necrosis factor families. recently, we have shown that cbln is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to -untranslated region (utr) of bdnf mrna to investigate the effect of the utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps p-e bmp expression in the adult rat brain bone morphogenetic protein- (bmp ) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp expression in the adult rat cns using immunohistochemistry. bmp is intensely expressed in most neurons and their dendrites. in addition, intense bmp expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp protein. these data indicate that bmp is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp plays pivotal roles also in the adult brain. ps p-e hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl- protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl- expression, decreasing the level of bax expression in c bl/ mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase . here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of -methyl- -phenyl- , , , tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda , yoko tohyama , shinichi kohsaka , tadashi kurihara , kazuyuki nakajima , soka university, hachioji, tokyo, japan; dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt- , and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤ among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba , chiaki nakagawa , masako tsuzuki , motoko matsuda , satoshi ichisaka , yoshio hata inst. dev. res., aichi human ser. ctr., kasugai, japan; div. neurobiol., tottori univ., sch. med., yonago, japan; div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats h after beginning an -h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin- (nt- ) in selected brain regions from -day-old male rats and compared to those from adults. the changes in levels of bdnf and nt- were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci ( kdyne, ih injury device) at t / and were assigned to three groups; a (placebo), b (mp treated; mg/kg i.v. immediately after injury), c (g-csf treated; g/kg i.v. for days after injury). animals were assessed with the bbb locomotor rating scale for w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi ( ) ps p-e glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons ( div) exposed to glutamate for min at m. glutamate exposure led to a decrease in mrna of nd and nd , which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least - h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk- . these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj- is enhanced by oxidative stresses. although exact functional significance of dj- has still unknown, it is thus proposed that dj- is protective against neural damage under oxidative stresses. in this study, we tested expression of dj- in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj- was markedly increased in the molecular layer of the dentate gyrus on days and post tmt injection. on day post tmt injection, enhanced expression of dj- was observed in the stratum lucidum of the ca . in glutathione-depleted mice, tmt was more effective in enhancing expression of dj- , compared with that in untreated mice. furthermore, double staining of dj- and gfap demonstrated that most of cells highly immunoreactive to dj- were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj- is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt ( . mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus days later, with visually apparent recovery of the granule cell layer days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone ( . - mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of . mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb ( g/ml) on the pc cells was elicited through apoptotic cell death. activation of caspase- / was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin ( nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase- / activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc cells. ps p-e kinesin superfamily protein (kif ) regulates activity-dependent neuronal survival by suppressing parp- enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein (kif ), a microtubule-based molecular motor. the c-terminal domain of kif is a module that suppresses the activity of poly (adp-ribose) polymerase- (parp- ), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif from parp- , resulting in upregulation of parp- activity, which supports neuron survival. after dissociation from parp- , kif enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. research funds: ps p-e expression of hsp , apg- , and apg- in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt ( . mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp , apg- , and apg- in the granule cell layer of the dentate gyrus, but not in ca -ca pyramidal cell layer, h to days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp highly expressed in survival neurons in the culture, apg- and apg- highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day . thirty gray of x-irradiations were performed on the cultured cells at or days in vitro (div). then the cells were fixed at h after the irradiation with dapi. at -div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at -div. this indicates that the radiosensitivity of -div immature neurons is higher than that of -div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦ glutamate receptor (glur␦ ), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦ is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca + , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin- and found a novel gene down-regulated in response to mutant ataxin- in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca molecular pathology. ps p-f permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca + -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca + -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways. interestingly the up-regulation of bcl- , a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat probe. scat is an indicator protein for caspase- activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase- cleavage sequence. using this probe, we could monitor the activation of caspase- at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt ( . mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex days later. in cortical neurons, tmt exposure for h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases and , but not caspase , were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride ( . mg/kg, i.p.) in -( w) and -week-old ( w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in w mice. by contrast, slight, diffuse neuronal damage was found in the ca and ca subfields and dentate gyrus of w mice. the hippocampal putrescine level was elevated markedly in w mice on tmt administration, whereas a minor putrescine increase was detected in w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps p-f establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai , toshinori hongo , kimisato naito , shigeto sasaki department of physiology, kyorin university, school of medicine, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for . - . s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of . s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in volunteers. experiment was performed by three tasks, control task is a forearm rotation, task is simulation of tools-manipulation, task is execution of tools-manipulation. we found different brain regions by this experiment. task to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at khz, to record simultaneously and independently the • horizontal saccades of each eye for subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi ( ) ps p-f dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼ hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from healthy subjects, high and low scorers (n = each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band ( - hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a -t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = . s) with online monitoring of meps. the intensity of tms pulses was varied from % to % at a % step (frequency at ∼ . hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above % stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = . , p = . ). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps p-f shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda , , yukihiro gomi , itsuki imai , nobuaki shimoda , , hiroyuki kato , international university of health and welfare, ohtawara, japan; crest, jst, kawaguchi, japan; nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels ( channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of s task and s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky , , rieko osu , hiroshi imamizu , mitsuo kawato , computational brain project, icorp, jst, japan; atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed hz isometric force pulses to seven targets separated by • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a d strain gauge. twenty repetitions of each condition were performed in s blocks (tr = s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only . % (chance: . %), errors were distributed normally about the target such that . % (chance: . %) of the data was classified correctly to within • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from • c, fish showed repetition of short time swimming in the range . - • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in - days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps p-f to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu , ken-ich morishige , jun nakanishi , , hiroyuki miyamoto , mitsuo kawato atr computational neuroscience labs, kyoto, japan; kyushu institute of technology, japan; jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p pups of the surviving cases increased greatly between p ( % of the control value) and p ( %), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p , dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p and p pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p to p . we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully ( • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative , , and days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps p-g role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa , tatsumi nagahama dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps p-g neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto , sayaka hihara , kazuo okanoya , atsushi iriki lab for symbolic cognitive development, bsi, riken, japan; lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about . • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps p-g prostaglandin e -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, - - toyama, shinjuku, - , japan unilateral microinjection of pge into the region around the rostroventral wall of the third ventricle (av v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of - pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide ( . mm, nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge injection site was examined. this treatment alone had no effect on the o consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge -induced responses were blocked min after the pretreatment with bicuculline, and recovered at ∼ min. pretreatment with vehicle saline had no effect on the pge -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge -induced thermogenesis. tetsufumi ito , hiroyuki hioki , kouichi nakamura , takeshi kaneko , yoshiaki nojyo dept. anat., univ. of fukui, fukui, japan; dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad -ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad -positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad . we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad -ir. after cutting t -t vrs and drs, almost all gad -ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama , ning ma , reiji semba , satoru naruse dept. of anat. ii, mie univ. school of med., tsu, japan; inst. for developmental research, kasugai, japan; dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin (aqp ) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp -immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp -ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp -ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp -ir fibers were found both in the myenteric and submucosal plexi. many aqp -ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp -ir fibers penetrated into the muscle layers. these results suggest that aqp -ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi ( ) ps p-g abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days - . the increase in the volume and frequency setting of the artificial ventilator (fio = %, fico = %) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad -gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = ) in medullary transverse slices. about % of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about % of gabaergic neurons recorded in the superficial rvm were co inhibitory, and all of the remaining neurons were co insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with and mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with mv forehead spla, in comparison to subgroups mv forehead spla. such law was common for both for sg and for cg. in two subgroups of and mv nasal zone spla in sg the greater rt gain after il was in mv nasal spla subgroup, and for cg in mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin ( ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via ht receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c bl/ n) were pretreated with pcpa ( . g/ ml/kg body weight) or saline intraperitoneally for consecutive days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o inhalation was changed to stepwise , and % co in o inhalation every min. hypercapniainduced increases in tidal volume and minute ventilation during % co inhalation were reduced by pcpa pretreatment; these results suggest that ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a . % gosha-jinki-gan diet group. after weeks, continuous cystometry with physiological saline or . % acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with . % acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps p-g differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to db white-noise for min. db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase (gad ), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were . on the small neurons, . on the medium-sized neurons, . on the large neurons, and . on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about % of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large ( . m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps p-h neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c nerve and facial nerve activities. addition of mm k + to the standard superfusate decreased burst rate of c activity. addition of or mm k + caused initial inhibition of c burst and subsequent high frequency c burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi ( ) ps p-h regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp ( m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at − mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin receptor (nk r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps p-h effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min- cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm- , shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda , usa) at the right middle finger from volunteers ( . ± . years). mild thermal stimuli ( ± • or ± • ) were administered every min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm- . we examined the effects of color environments on cognitive function in healthy subjects and patients after traumatic head injury using p components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p latency in the red environment was significantly shorter in controls than in patients. the p amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps p-h neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi , , mikinobu takeuchi , , takahiro omote , naoyuki yosimura , etsuro hori , , kazuo sasaki , taketoshi ono , , hisao nishijo , system emotional science, univ. toyama, toyama, japan; crest, japan science and technology agency, japan; bio-information engineering, univ. toyama, toyama, japan; molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems ( channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate ( ) human facial stimuli with different gaze directions and ( ) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi , sachiko koyama , ayumu matani , andrzej cichocki riken, wako, japan; hokkaido university, sapporo, japan; university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: ( ) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. ( ) the functions were averaged across subjects. ( ) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. ( ) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi ( ) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps p-h the reflection of category perception of sound in the auditory evoked n m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for different fundamental frequencies (f ) and different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n m source strength for nonlinear sounds was significantly larger than that for linear sounds. n m peak latency only for vocal sounds was not affected by f . these results suggest that perceptual categorization was reflected in n m source strength and location (linear or nonlinear), and in n m latency (vocal or nonvocal). sunao iwaki , hiroko kou , kouichi sutani , mitsuo tonoike national institute of advanced industrial science and technology, osaka, japan; chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p , related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p components, suggested the involvement of frontal and anterior cingulate cortex in the early p component (p a) and the contribution of the parietal and temporal regions to the later component (p b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l and l , respectively). we compared the brain activity of chinese learners of japanese as l with that of japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm . while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l and l . neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a . t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya , mie matsui , , tsuneyuki kobayashi , , hisao nishijo , , michio suzuki , , yasuhiro kawasaki , , masayoshi kurachi , , jun nakazawa , kyo noguchi , hikaru seto neuropsychiatry, univ. toyama, toyama, japan; crest, japan science and technology corporation, japan; psychology, univ. toyama, toyama, japan; system emotional science, univ. toyama, toyama, japan; neuropsychiatry, univ. toyama, toyama, japan; developmental psychology, univ. chiba, chiba, japan; radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura , atsushi sekiguchi , keisuke wakusawa , , yuko sassa , , hyeonjeong jeong , , kaoru horie , , shigeru sato , , ryuta kawashima , miyagi university of education, sendai, japan; niche, tohoku univ., japan; dep. pediatrics, tohoku univ. school of medicine, japan; ristex, jst, japan; gsics, tohoku univ., japan; lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain , , points or lose , , points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps p-h brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara , masato taira , , toshio iijima , ken-ichiro tsutsui div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; arish, nihon university, tokyo, japan; appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either yen or a gain or loss of , , or yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps p-i neural activation during experience-based reasoning chisato suzuki , , takashi tsukiura , hiroko mochizuki-kawai , yayoi shigemune , , toshio iijima neurosci. res. inst., aist, japan; div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps p-i the difference of brain activity elicited by different styles of art hiromi yamamura , yasuyuki kowatari , , shigeru yamane , miyuki yamamoto , comprehensive human sciences, university of tsukuba, tsukuba, japan; system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm . while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba , ba ). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j- ) ps p-i effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were - aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi , ps p-i the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo , michio nomura , jun kawaguchi ntt commun. sci. labs., ntt corp., atsugi, japan; dept. psychol., tokai women's univ., kakamigahara, japan; dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo , , , yulri nonaka , katsumi mizuno , kazuo okanoya , riken, bsi, biolinguistics, saitama, japan; chiba university, chiba, japan; jsps, japan; department of pediatrics, showa university, tokyo, japan; presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for neonates within days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three ( ) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around - ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism ( : - : ) and agematched typically developing children ( : - : ) were instructed to name pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = . ± . , loudness = . ± . ) than in the children with autism (duration = . ± . , loudness = . ± . ) (p < . ). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at . /s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser ( nm, mw) to the muscle surface ( . mm ) significantly delayed its attenuation (p < . ). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < . ). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps p-i nedl , a novel e ubiquitin ligase for dishevelled- , targets mutant superoxide dismutase- and interacts with p yuanyuan li , , , kou miyazaki , toshinori ozaki , akira nakagawara division of biochemistry, chiba cancer center research institute, chiba, japan; production technology development center, the furukawa electric co., ltd., ichihara, japan; hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e ubiquitin ligase gene termed nedl . previous study has shown that nedl is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled- , a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl for uniquitination and proteasome-mediated degradation. on the other hand, nedl bound and ubiquitinated mutant (but not wild-type) sod in a mutant sod type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl physically bound p , and induced apoptosis in a p -dpendent manner. taken together, our results suggest that nedl may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod and p . spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc interacting protein), u-box type e ubiquitin ligase, has been shown to interact with hsp or hsp and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca + current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox- inhibitor, rofecoxib in pentylenetetrazol (ptz, mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the th day of ptz treatment. chronic treatment with rofecoxib ( . and . mg/kg, p.o.) for days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced ( mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin ( and mg/kg, p.o.), naproxen ( and mg/kg, p.o.), nimesulide ( - mg/kg, p.o.) or rofecoxib ( - mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib ( mg/kg) or nimesulide ( mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox- inhibitors. cox- inhibitors also antagonized the effect of flumazenil ( mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps p-j cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps p-j a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus ( mg/ min). after min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that -oxoguanine ( -oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that -oxog accumulated greatly in mtdna of the ca microglia. wild-type and mth -null mice, the latter lacking an ability to hydrolyze -oxo-dgtp and -oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca neuron loss after kainate administration, however, levels of -oxog accumulated in mtdna and cellular rna in the ca microglia were significantly increased in mth null mice in comparison to wild-type mice. we thus demonstrated that mth efficiently suppresses the accumulation of -oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps p-j transcription factor nrf regulates brain response to kainate-induced excitotoxicity yukihiko dan , kosuke kajitani , noriko yutsudo , ken itoh , masayuki yamamoto , yusaku nakabeppu kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e related factor (nrf ) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf has been reported to increase neuronal death under increased oxidative stress, nrf seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca subregion of hippocampus. to unveil the functional significance of nrf in the brain, we compared seizure responses between wild-type and nrf -null mice after systemic kainate administration. we found that nrf -null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps p-j synaptic plasticity and -aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine ( -ap) at the concentration below . mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the -ap induced discharges in ca region was studied. ten m -ap induced the epileptic discharges with the frequency of . ± . hz (mean ± s.e.m.; n = ) and the amplitude of . ± . mv. when ap- , an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap- was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of -ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps a-a influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno , hugh p.c. robinson engineering science, osaka university, osaka, japan; university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣ a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the -day-old rat pup spinal subarachnoid space at the rate of l/ . min. the rats were sacrificed h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣ a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps a-a on the membrane potential profile of ca pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga , , yoko tominaga dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; lab. for dynamics of emergent intelligence, riken bsi, hirosawa - , wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps a-a overexpression of inwardly rectifying k + channel . in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) . have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca pyramidal neurons. then, egfp and kir . expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer / neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types and of ryanodine receptors (ryr and ryr ) in the cerebellum of a ca + channel ␣ a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr but not ryr in the rmn cerebellum: a less ryr mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr -mediated ca + release, contributing altered ca + homeostasis in the rmn purkinje cells. ps a-a dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than s. d receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi , , hiroyoshi miyakawa , masashi inoue , masato okada , tokyo institute of technology, japan; brain science institute, riken, japan; tokyo university of pharmacy and life science, japan; the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai , maya yamazaki , , mika tsujita , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan we previously reported that stargazin family (␥ , ␥ , ␥ , and ␥ ) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., ) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥ subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥ subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day , while the reduction was not so significant in adult brain. these results suggested that ␥ might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph . ) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop ). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop -transfected cells. mvsop induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn + sensitivity. neutralization of a positive charge in the s -like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh ). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca region. in the pyramidal layer, the amplitudes of the responses to the -hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the - -hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels (asic ) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic -expressing cells in rat central nervous system (e -p ) using immunohistochemical techniques. asic positive fiber first appeared in brain stem and spinal cord at e - stage. asic -expresseing cells appeared in white matter of brain stem and spinal cord at e stage. in early postnatal stages asic expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval ( st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long st isi by whole-cell recordings in rat slices. application of m apamin, an sk-type ca + -activated k + (kca) channel blocker, shortened the st isi and decreased the amplitude of the slow ahp. the shortening of the st isi was observed when membrane potentials were depolarized. moreover, application of m mibefradil, a t-type ca + channel blocker, shortened the st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca + influx through t-type ca + channels. research funds: kakenhi (c) ( ) jafar vatanparast , , mahyar janahmadi , houri sepehri , ali haeri-rohani , ali reza asgari neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px ( . m, within min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca + spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca + dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm ). repeated menthol application induced trpm desensitization. this desensitization was depended on extracellular ca + , indicating that involvement with ca + -dependent kinase. pkc activator (pma) desensitized trpm and go (pkc inhibitor) abolished pma-induced trpm desensitization. pma similarly desensitized wild type trpm and mutant trpm , in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm . as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm causes pkc to desensitize trpm itself. yosuke sawada , hiroshi hosokawa , kiyoshi matsumura , shigeo kobayashi dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa is activated by pungent compounds stimuli. if trpa responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa is activated by cooling or not is still controversial. here, we investigated that trpa -expressing hek cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was . ± . • c. in inside-out singlechannel recording, cooling activated trpa directly. single channel conductance was . ± . ps. single channel currents showed inword rectification. in conclusion, trpa is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn - ) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn - demonstrated that samples from the nerve and the heart showed an hcn band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn , hcn and hcn could not be found in those samples. when an hcn channel blocker, zd was applied on the stimulus portion of sciatic nerve and the nerve was elicited at . /s by a duration of ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < . ). it is suggested that hcn channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir ) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir . and kir . channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps a-b proliferation of rat c glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c ), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control ( mm) to or mm. an increase in [na + ] i was revealed by an image processor in c cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr- , which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr- level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma ( - m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma ( m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi ( ) il-sung jang , in-sun choi , eun-ju park , jin-wha cho , man-gee lee , byung-ju choi kyungpook national university, school of dentistry, south korea; kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (< m), but decreased those induced by gaba at higher concentrations (> m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav . -deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav . -deficient mice as negative controls to examine nav . distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav . expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav . distribution, in the present study, higher magnification analysis revealed that nav . is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav . antibodies used in these previous studies, none of which utilized nav . -deficient mice. based on our findings, we propose that nav . might be involved in propagating action potential to presynapses. keiji miura , , masato okada , , , shun-ichi amari department of physics, kyoto university, kyoto, japan; "intelligent cooperation and control", presto, jst, japan; department of complexity science and engineering, university of tokyo, chiba, japan; brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. and ) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [ - c] glucose intravenously ( g/kg) or h after the intraventricular injection of colchicine ( g/ l) and the amino acid fractions were extracted from the brains at , or min after the glucose injection. the amount of [ - c] glucose in the cerebra was increased, however, the c incorporation into glutamine, glutamate, gaba and aspartate from [ - c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes or hes gene, phes -and phes -egfp transgenic (tg) mice were generated (ohtsuka et al., ) . in each transgenic mouse, a promoter of hes or hes gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes or hes regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day (e ) to postnatal day (p ). in the phes -egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e to p . in the phes -egfp tg mouse inner ear, gfp-ir cells were observed from e . to p . gfp-ir cells in phes -gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., . mol. cell neurosci. ps a-c expression of zfh- in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine , kenji nakamura , motoya katsuki , tetsuo yamamori national institute for basic biology, okazaki, japan; mitsubishi kagaku institute of life science, machida, japan zfh- is a transcription factor containing three homeodomains and zn fingers and expressed in differentiating neurons. we have reported that the level of zfh- mrna is negatively regulated by antisense transcripts of the zfh- gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh- antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh- mrna starts to be expressed. recently, we have raised an antibody against mouse zfh- and examined the expression profile of the zfh- protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh- protein was not detected even when the zfh- mrna was already expressed. this observation together with other data suggested that the zfh- protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue , maya ota , katsuhiko mikoshiba , jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan; laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic - ) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic /zic compound mutant mice. mice carrying homozygous zic mutant allele together with zic null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic -or zic -single mutant. these observations indicate that zic , in cooperation with zic , have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp , a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp transcripts are first detected at embryonic day in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day - embryos, rp transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e , rp transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c hc-type zinc finger motifs. using p cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf- and nzf- respectively. nzf- null mice are born alive, but die within min after birth with cyanosis. on the other hand, nzf- null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf- and nzf- by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , ) . in the present study, to examine the role of runx in the development of drg in more detail, we examined the development of drg neurons in runx -deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx −/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days . and . . shin hisahara , , susumu chiba , hiroyuki matsumoto , yoshiyuki horio department of pharmacology, sapporo medical university, sapporo, japan; department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt is still unclear. recent studies indicated that sirt interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes promoter. we performed overexpression of sirt and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt and n-cor in the sirt with n-cor. sirt and n-cor suppressed hes transcription by notch-icd in the luciferase assay. hes transcription was suppressed in overexpression of sirt and n-cor, suggesting that interaction between sirt and n-cor represses hes transcription. consistent with this, chip assays revealed that not only n-cor but also sirt bind to the promoter of hes gene. taken together, these results indicate that sirt and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes promoter site and repressing hes transcription. yasushi maruyama , mitsuhiko kurusu , masataka okabe , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, japan; natl. inst. genetics, mishima, japan; inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., ) . furthermore, we announced previously that runx mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi , hirohide takebayashi , takayuki manabe , kazuhiro ikenaka , akio wanaka dept. anatomy, nara med. univ., kashihara, nara, japan; division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng proteoglycan and olig at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig lineage cells in the forebrain is largely unknown. here we examined fates of olig expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig -creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) . or . , when most of olig + cells are observed in the basal forebrain. the olig + cells at e . gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig + cells at e . generated more glial cells than neurons. these results indicate that olig lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi , , masato yano , , satoru hayashi , , hirotaka j. okano , , hideyuki okano , dept. physiol., keio univ. sch. med, tokyo, japan; sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p , p and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf and nf , binding partner of nf , respectively. in mouse embryonic brains, we found that nf / expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf / formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er and brn ) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi ( ) ps a-d protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for - weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng + cells in the neocortex are the opcs. we investigated about subtypes of ng + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp and wnt a, a significant proportion of these neural cells become math + . the induced math + cells mitotically active and express markers characteristic of granule cells precursors (pax , zic , and zipro ). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l + /calbindin-d k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf rather than by wnt a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p cip , p kip and p kip mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p kip mrna was shown. transfection of p kip into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e . rat midbrain and hb .f cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p kip elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g /s phase did not activate mapk and pi -k signaling. yuichi tanaka , yusuke tozuka , dai muramatsu , kin-ichi nakashima , tatsuhiro hisatsune departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng + and olig + cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj- + or map- + neuronal cells by adding retinoic acid or bdnf. more than % of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi , michiko imanishi , teiko kuroda , masao tachibana , masahiko takada department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv . b and kv . were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv . in the frog retina and by kv . b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv . b. thus, the differential expression of kv channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou , , kazunori yamamoto , masayuki hara , akinori homma , akimichi kaneko , masahiro yamada tokyo metropolitan univ., hino, tokyo, japan; astellas pharma. inc., osaka, japan; sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko ( ) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by -hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as . ± . ph unit by mm k + . acidification of pho was suppressed by . m bafilomycin a , a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps a-e analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar -channel field potential recording system in slice preparations obtained from to days old mice. stimulation at ssc induced negative field potential with short latency and short duration ( - ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing m bicuculline, the same stimulus induced a large negative field response with long duration ( - ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya , ken tsutsui , kimiko tsutsui dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap- binding, and the c-terminal sh domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe- , also expresses amph ir. arpe- thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe- cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura , satoshi ichisaka , chihiro hayashi , hirotoshi maki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v ) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi ( ) ps a-e rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut) and vglut , respectively. a complementary distribution of vglut and vglut was established by postnatal day (p) and both signal intensities increased further by p - (peak cp). the immunoreactivity for vglut decreased around layer iv after brief monocular deprivation ( dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area , with a small projection to layer vi of areas and a. cells in layer vi of area projected to dlgn. in addition, cells in layer v of area projected to dlgn, which is not observed in normal rats. in area a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of a. cells in layers v and vi of area a projected to lp. smaller number of cells in layer v of area also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps a-e activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida , takemasa satoh , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v ) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v to the lgn. we focused on the density of type metabotropic glutamate receptor ␣ (mglur ␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur ␣ in the lgn decreased after elimination of v , showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur ␣ signal in the lgn of young rats. yu morishima , hiroshi sakamoto , takahumi akasaki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., japan; div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, ( ) how rapidly the reverse od shift proceeds and ( ) whether the shift is induced only in young animals. we infused the cortex with muscimol in -week-old kittens and in adults. the reverse od shift was observed after days md, but not significant after days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps a-e experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai , nafiseh atapour , john renger , john roder , peter seeburg , takao hensch neuronal circuit dev., riken, bsi, wako, japan; mount sinai hospital, toronto, canada; max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur , ) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation ( d md) rapidly alters both the phosphorylation state and surface expression of glur in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as d md eventually led to full od shifts. in glur ko mice, even d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx promotor and dlx / enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca and revealed a nonpyramidal neuron projecting to ca and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita , takao oishi , motoharu hayashi div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than % of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area ( . %) than in other areas, such as the prefrontal ( . %), primary motor ( . %), somatosensory ( . %) and secondary visual areas ( . %), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than % of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe , guy n. elston , , tomofumi oga , hiroshi tamura , , ichiro fujita , osaka univ., japan; univ. queensland, australia; crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v ). pyramidal cells in prefrontal cortex, such as area , have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v (n = ), area te (n = ), and area (n = ) of a -week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area , followed by area te, and v . thus, considerable heterogeneity in pyramidal cell structure already exists -weeks after birth. hiroaki matsushita , mahito ohkuma , masami watanabe , ei-ichi miyachi department of physiology, fujita health university, aichi, japan; department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura- based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the % of axotomized rgcs. bath application of m carbachol, an ach receptor agonist, rose [ca + ] i in % of normal rgcs. although the % of rgcs responded to carbachol at days after axotomy, no responsive rgcs appeared during - days. ach responsiveness recovered in axonal regenerated rgcs ( %). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi ( to em, to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., ) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor ( hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames ( ms) or only one frame ( ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps a-e motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima , , tohru kiryu , kazuhiko ukai , takeshiko bando div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura , kenichiro miura , masakatsu taki , hiromitsu tabata , naoko inaba , kenji kawano , frederick a. miles grad. schl. med., kyoto univ., kyoto, japan; lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn ) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency f and f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps ( hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the f forwards and the f backwards. the contrast of the f was fixed at , , or %, while the contrast of the f was varied from one-fourth to four times the contrast of the f. when the contrast of the f ( f) was less than about half that of the f ( f), the f ( f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < . ). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito , yaoxing chen , yukiko tanada dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; china agricul. univ., beijing, china twenty white-leghorn chicks (p - ) were perfused with % paraformaldehyde through the heart under deep anesthesia of nembutal ( mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, ). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to in tec-rgcs, in tha-rgcs, and in hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers - ). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie , kenji yuda , eiichi okawa , katsuyoshi maruyama , hiroshi uozato , hiroko horie , satomi nakajima , kenkichi tanioka , yuji ohkawa , tomoki matsubara , wolfram tetzlaff advanced res. centr. biological sci., waseda univ., tokyo, japan; technomaster co. ltd., yokohama, japan; kikuna yuda eye clinic, yokohama, japan; healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; nhk sci. technical res. labo., tokyo, japan; icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between and cm toward and away in a high speed (top speed: mm/s) from the subjects. the device significantly improved visual performances in over % of the school-aged children with myopia and % of adults with presbyopia. hiroyuki miyamoto , toral s. surti , takao k hensch laboratory for neuronal circuit development, riken brain science institute, wako, japan; san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v ) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v . here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa , miquel bosch , shuzo sakata , yoshio sakurai , tetsuo yamamori division of brain biology, national institute for basic biology, okazaki, japan; mit, ma, usa; rutgers university, nj, usa; department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., ) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki , youichi miyawaki , tsunehiro takeda department of complexity science and engineering, university of tokyo, chiba, japan; mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly ( ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = - ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa ( and ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p , n and area under curve of a late positive potential expanding from to ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse , ayumu matani , , tomoe hayakawa , , norio fujimaki university of tokyo, kashiwa, japan; nict, kobe, japan; teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p amplitude becomes. this suggested that a factor of the variability of the p amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. ) hitoshi sasaki , takuya ishida , masayoshi todorokihara , junichiro miyachi , tahei kitamura , ryozo aoki dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama , nobuo usui , izuru nose , , masato taira , department of applied system neuroscience, nihon university school of medical science, tokyo; faculty of human science, bunkyo university, saitama, japan; arish, nihon university, tokyo, japan when an object is moving, perception of its d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps a-f local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a ) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., ) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., ) . moreover, aaf has dense synaptic connections within the area (budinger et al., ) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were -ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima , yasuo takeda , yasushi shimoda , kazutada watanabe department of bioengineering, nagaoka university of technology, nagaoka, japan; department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb- that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb- expression is prominent in the auditory system. nb- knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb- is indispensable for the function of auditory system. here we report the detailed analysis of the nb- localization using anti-nb- monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb- was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb- immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb- regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation ( khz octave band, db, h), and then subjected to electrophoretic mobility shift assay to determine activator protein- (ap- ) dna binding. ap- binding was increased - h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea h later. taken together, ap- may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c bl/ mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli ( - khz for ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by khz were different from those elicited by or khz. however, the areas activated by khz were also activated by the mixture of plus khz but not by that of plus khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps a-f functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by - %, whereas the inhibition of dc resulted in reduction only by - %. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh- . a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato , , kazuo okanoya laboratory for biolinguistics, riken bsi, wako, saitama; graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, ) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented - times at a fixed interval, through a speaker at - db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (< hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (> hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki , , , , , hokkaido university, sapporo, japan; hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan; kobe shoin women's university, kobe, japan; riken, wako, japan; sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children ( - years old, n = ) and adults ( - years old, n = ). there were six gap conditions with durations of , , , , and ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n and p complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps a-f an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua , tamaki miyamoto , atsushi terao , sachiko koyama , takashi omori , harumitsu murohashi , shinya kuriki jst, saitama, japan; hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba ), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation ( subjects, p < . corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi , yuko toyosawa , fumiya takeuchi , michinao matsui , shinya kuriki research institute for elecronic science, hokkaido university, sappro, japan; jst, saitama, japan; hokkaido university, school of medicine, japan; kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = ). four synthesized vowels were used as stimuli (stimulus duration ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used ( / ms). for the native vowels, an early sf ( - ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf ( - ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi ( ) ps a-f spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto , , junsei horikawa department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh ) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli ( - khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe , go ashida , , kazuo funabiki graduate school of informatics, kyoto university, kyoto, japan; hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi , shigenori takebayashi , masahiko bundo , masazumi fujii , toshihiko wakabayashi , jun yoshida , hiroyuki fujisawa , kazunori ando , kazumasa hayasaka department of radiology, national hospital for geriatric medicine, obu, japan; department of neurosurgery, national hospital for geriatric medicine, obu, japan; department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using . tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in out of patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = ) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, ) . surprisingly, subjects tended to misidentify the second toe as the third ( . %), and the third toe as the fourth ( . %), while the reverse misidentification rarely occurred (third as second, . %; fourth as third, . %). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps a-g effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi , , shunjiro moizumi , ayami okuzumi , humine saito , shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, jst, saitama, japan morrone et al. ( ) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade ( • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps a-g function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda , , miki taoka , department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas b and of four conscious macaque monkeys was studied by recording single-neuron activities. a total of electrode penetrations were made in areas b and . in penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining penetrations, however, % of adjacent pairs ( / ) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas b and might be responsible for forming composite rfs of area neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi ( , ) miki taoka , michio tanaka , hisayuki ojima , atsushi iriki secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them ( % contra-, % bi-and % ipsilateral hand movements), we could determine receptive fields for only neurons ( %). most of activities ( neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta , , kouichi nakamura , takeshi kaneko department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata , , shanlin chen , hiroki kitaura , masaharu kudoh , minoru shibata , katsuei shibuki department of neurophysiology, brain research institute, niigata university; department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane ( . g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation ( hz for s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi , nanae fukushima , tetsuhiro fukuyama , akira kakegawa , tetsuji moriizumi department of anatomy, shinshu university school of medicine, matsumoto, japan; department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima , takeshi kawano , hidekuni takao , , , kazuaki sawada , , , hidekazu kaneko , , makoto ishida , , department electrical & electronic engineering, toyohashi university of technology, aichi, japan; issrc, toyohashi university of technology, aichi, japan; aist, hsbe, ibaraki, japan; jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, m in diameter, m in length; array: -m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = . ). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi ( ), the st century coe program "intelligent human sensing" ps a-g a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko , hiroshi tamura , shinya s. suzuki , takahiro kawashima aist, hsbe, ibaraki, japan; laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over h. seiji matsuda , takehiro terashita , tetsuya shimokawa , kyoujy miyawaki , yuji miguchi , takuya doihara , jie chen , shuang-yan gao , chun-yu li , min wang , zhong wang , bing xue , naoto kobayashi , , kazuhiro shigemoto department anatomy, ehime university of medicine, ehime, japan; medical education c, ehime university of medicine, ehime, japan; department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than . m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat , whereas other genes known to be regulated by stat were not detected. stat is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat and stat phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto , susumu hyodo , makoto kawasaki , minori shibata , takeshi saito , hiroaki fujihara , takashi higuchi , yoshio takei , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; department of integrative physiology, university of fukui, japan adrenomedullin (am ) (identical to intermedin) belongs to the super family of am. centrally administered am and am activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am was stronger than those of am and the effects of am on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am would have unknown receptor except cgrp and am receptor. arata oh-nishi , makoto saji , taku uchida , sen-ichi furudate , nobuyuki suzuki division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, ) . here we report that the density and morphology of spines of pyramidal neurons in ca region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca , both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day - , the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day . the staining of somatostain mrna in individual neurons was diminished when examined on day or , albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps a-g short chain sugar acid, -buten- -olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki , tatsushi onaka , hirofumi hashimoto , hiroaki fujihara , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; department of physiology, jichi medical school, tochigi, japan -buten- -olide ( -b o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of -b o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at - min after intraperitoneal (i.p.) administration of -b o ( mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei min after i.p. administration of -b o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of -b o. these results suggest that peripherally administered high dose of -b o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps a-g estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto , , shinpei higo , , yasushi hojo , , kouhei nakajima , , hironori nakanishi , , hirotaka ishii , , suguru kawato , department of biophysics & life science, university of tokyo, tokyo, japan; crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps a-g gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from − to - mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than - mv and most of them were ∼− mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr , galr and galr , in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr selective agonist m induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr . hiroaki fujihara , tomoki fujio , david murphy , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking % saline for days in , , and months old tg rats. in and months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats ( and months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps a-h prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera , hiroaki fujihara , hirofumi hashimoto , makoto kawasaki , tatsushi onaka , takakazu oka , sadatoshi tsuji , yoichi ueta department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; department of physiology, school of medicine, university of occupational and environmental health, japan; department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin , afsaneh eliassi , nasser naghdi , ali ghanbari ghsemi; pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above ( . mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from min up to the end of experiments ( min). at the peak acid response, the vmh microinjection skf ( . , ) significantly reduced the amount of gas (p < . ). there was no any effect by microinjected sch ( . ) into the vmh. injecting skf into the vmh, min after sch , had no effect on gas in compare with control. but, the acid suppressant effect of skf was completely removed by peripheral injection of sch (p < . ). our results show that the vmh d dopamine receptors have regulatory mechanisms of gas by interaction with h receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p y purinoceptors (p y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p y r agonists increased [ca ++ ] i in son, with p y r agonist being the most effective. the possibility that p y r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p y r antagonist, mrs . atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs ( m) in mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs . furthermore, combined treatment with mrs and ppads ( m, a p x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p y r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps a-h analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii , , yasuhiro sonoki , , aizo furukawa , , yasushi hojo , tetsuya kimoto , , suguru kawato , department of biophysics and life science, university of tokyo, tokyo, japan; crest, jst, japan; kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day (pd ) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd to pd with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai , , gen murakami , shirou kominami , john h morrison , william g.m janssen , tetsuya kimoto , , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo - , japan; crest project, jst, japan; faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima , japan; kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc- , mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca , ca , and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud , song t. yao , jing qiu , julian fr paton , david murphy university of bristol, hw-line, uk; department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic ( d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase , has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt - . delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt - cells cultured in dmem supplemented with % fbs for days. bk current was increased by addition of ␤-estradiol (e ) in culture medium in a physiological concentration range. this action of e was blocked by ici- , , a potent estrogen receptor (er) antagonist. we further examined whether e acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e but ppt had no effect. e had no effect on the i k . these results indicate that e increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi , ps a-h myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi , haruko kumanogoh , shun nakamura , seiji miyata , shohei maekawa department of biosystems science, kobe-university, kobe, japan; division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, kda protein, was identified as myelin protein zero (p ) from mass analysis and this result was confirmed by western blotting that a specific antibody to kda band reacted to an authentic p prepared from rat sciatic nerve myelin. p is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan ␤-estradiol (e ) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e (hojo et al., ) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca , ca and dg with a custom made multi-electrode measuring system (med ). ltd was enhanced by e dose-dependently in ca , ca and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito , takushi x. watanabe , tomoko urabe , hirofumi hashimoto , hiroaki fujihara , yukio hirata , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; peptide institute, inc., osaka, japan; department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto , hao-gang xue , yuji ishikawa , yoshitaka oka , hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan; national institute of radiological sciences, chiba, japan; department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki , eiji munetsuna , asuka kamogawa , suguru kawato , shiro kominami graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p arom in the hippocampus. method: the cultured hippocampus slices from days male rat were treated with - nm of tbt for h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p arom was determined by quantification of [ h]estradiol from [ h]testosterone. result: forty-eight hours treatment of hippocampal slices with nm tbt induced increases in mrna contents of p arom, and with nm tbt induced that of ␤-hsd. estradiol content was increased by the treatment with nm tbt, but not affected by nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of , , and ng in nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent h. the ng dose of bicuculline methoiodide had minimum effect whereas and ng produced arousal. maximal wakefulness was observed at dose of ng with no further increase in wakefulness at higher dose of ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe , hiroyuki tsuda department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee ). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, ( antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via -opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d is a potent somnogenic substance, and isomerized from pgh through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl decreased rapid eye movement (rem) and non-rem sleep for h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl administrations. these findings indicate that pgd plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd involved in sleep-wake regulation. under baseline conditions, h r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (< s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., ) . ps a-h dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = nm). the scn neurons responded to the light in intensities above photons/cm /s and were activated maximally at around photons/cm /s, they were about . log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca + in scn neurons. since cytosolic ca + regulates diverse cellular systems, we have hypothesized that the cytosolic ca + rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca + rhythms, mouse bmal and its dominant negative sequence (mbmf r ) are transfected into the organotypic culture of scn with a yellow cameleon ca + sensor by the gene gun. the results demonstrated that over-expression of bmal or mbmf r significantly inhibited the circadian ca + rhythms and thus we concluded that the native bmal rhythm is essential for cellular output processes of the murine clock system. ps a-h the activation of ␣ adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca region of rat hippocampal slices. carbachol ( m) induced ␤ oscillation with the frequency and the amplitude of . ± . hz, and . ± . mv, respectively (mean ± s.e.m.; n = ). epinephrine ( m) significantly increased the frequency of . ± . hz (**p < . ), not change the amplitude. clonidine ( m), an ␣ receptor agonist, alone significantly increased the frequency at the concentration of m (*p < . ). yohimbine, an ␣ receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣ receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart . mm from each other in the hippocampal ca region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency - ms), oscillatory ( - hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties ( / slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca region. ryozo aoki , hiroshi wake , hitoshi sasaki , kiyokazu agata dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as /f, ranging from − to + hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida , hiroki sato , , naoki tanaka , atsushi maki , japan science and technology agency, crest, saitama, japan; advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from to hz) decreases and the sleep spindle power (from to hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from to s in (i), and from to s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim , kazuki nakajima , yutaka oomura , kazuo sasaki div. of bio-information eng., univ. of toyama, toyama, japan; dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca + exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng , wei zhang , akira nakamura , masashi yanagisawa , yasuichiro fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. autonom. physiol., chiba univ., japan; dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb- (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb- . icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co -sensitivity at least during waking periods. research funds: kakenhi , junko hara , taizo matsuki , katsutoshi goto , masashi yanagisawa , takeshi sakurai , department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; yanagisawa orphan receptor project, erato, jst, tokyo, japan; howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps a-i prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto , kazuhiko kubo , shuji aou dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. ) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. , s.a.) shuji aou , tetsuya fujimoto , yumi ichihara , kimiya narikiyo , toru ishidao , hajime hori , yukiko fueta dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan -bromopropane ( -bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to -bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to ppm of -bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. -bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that -bp is the potential candidate of endocrine disruptors which affect brain development. ( ) ps a-i changes in hippocampal excitability of rats prenatally exposed to -bromopropane yukiko fueta , toru ishidao , susumu ueno , yasuhiro yoshida , hajime hori department of environmental management, school of health sciences; department of pharmacology; department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to -bromopropane ( -bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to -bp ( ppm) during gestational day - ( h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca at the pnd - . conversely, s/r curves of fepsp as well as ps in the ca were inhibited at the age of - weeks. our results suggest that -bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca of the offspring during development. research funds: grant-in-aid for exploratory research ( ) ps a-i effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura , , kyoko itoh , takeshi yaoi , tohru sugimoto , shinji fushiki dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously g/kg of bpa daily from embryonic day (e ). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e . and e . , whereas those cells increased in the cortical plate at e . , as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant keiko ikemoto , teruko uwano , hisao nishijo , taketoshi ono , masayuki ito , ikuko nagatsu , katsuji nishi , shin-ichi niwa dept. neuropsychiat., fukushima med. univ. sch. med.; toyama med. pharm. univ.; faculty med., mie univ., mie, japan; fujita health univ. sch. med., toyoake, japan; dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day and after fertilization. pups were perfused at postnatal day . in the frontal cortex, the number of largesized (more than m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c /a catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps a-i developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi , , , kaori morohoshi , , rie yanagisawa , erina saita , gen watanabe , , masatoshi morita , kazuyoshi taya , , hirohisa takano , , toshiyuki himi , , hideki imai , dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; res. inst. pharmaceut. sci., musashino univ., tokyo, japan; nation. inst. for environ. stud., ibaraki, japan; grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; wildlife rescue veterinarian associ., tokyo, japan; facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; the united grad. sch. veterinary sci., gifu univ., gifu, japan; div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps a-i the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic ( -ht) systems in stressinduced anxiety, we measured brain da and -ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of -ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and -ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although -ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl - ) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl and nl in autistic disorders. nls have a relative identical structure (∼ %); nl and nl localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl seems express in the olfactory glia, but nl distribution is unknown. here we have generated antibody against human nl , and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl is important for neuroendocrine function. nl cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl were rough and thicker than those of nl or nl transformants. it suggested the unique activity of nl for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting - % of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below . here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura , daisuke horiuchi , tomoyuki miyashita , minoru saitoe , hitoshi okazawa department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein- (pqbp- ) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp- disease that pqbp- mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp- in xlmr pathology, we produced two kinds of flies, human pqbp- overexpression flies (hpqbp flies) and drosophila pqbp- knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp- in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun , sentaro takahashi , yoshihisa kubota , rui zhang , chun cui , yoshihiro fukui natl. inst. radiol. sci., chiba, japan; sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr subunits paired with two nr subunits. nr and nr (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr /nr a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr /nr b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca pyramidal neurons from mice (at weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca region after ischemia. in blood, corticosterone levels increased biphasically at min and h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca region. by days after ischemia, mr and gr were not significantly altered in the ca region. from days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca region, and at days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase- and midkine in the mca area. stroke was induced by a -min mca occlusion using an intraluminal filament. rats were exercised for min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase- expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps a-j reversed astrocytic glutamate transporter glt- crucial to the ca + paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca + paradox" is the phenomenon whereby the intracellular concentration of ca + paradoxically increases during reperfusion with normal ca + -containing media after brief exposure to a low ca + solution. the present study aims to characterize the ca + paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca + solution for min significantly injured only neurons after reperfusion with a normal ca + medium for h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt- . the present findings suggested that ca + paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt- during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c t, which results in pro ser. the second patient carried the missense mutation c g, which results in arg pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps a-j mci - , a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol kinase madinyet niyaz , tadahiro numakawa , yoshinori matsuki , emi kumamaru , yuki yagazaki , harumi kitazawa , hiroshi kunugi , motoshige kudo pathology department of tokyo medical university, tokyo, japan; department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci - , a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci - prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci - exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways and that the mci - -dependent survival effect was blocked by the inhibitors, u (an mapk pathway inhibitor) or ly (a pi k pathway inhibitor). these results suggested that mci - exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru , noriko yoshimura , tetsuro sakamoto , takaaki kitano , naoko nisimaru , isao yokoi dept. physiol., sch. med., oita univ., japan; med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by p-nuclear magnetic resonance ( • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with mm ep (ep- ), acsf replaced by acsf with mm ep after ischemia (ep- ), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep- than in control (p < . ). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) # from mext to t.k. hideaki tamai , kuniko shimazaki , norimasa seo department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc ), yintang (ex-hn ) and sanyinjiao (sp ), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through -bromo- ,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc , ex-hn and sp . the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago , keiko takahashi , shigeo nakamura , akemichi baba , toshio matsuda laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara , tatsuya mishima , takefumi kofuji , kimio akagawa department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc- /syntaxin a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc- /syntaxin a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc- /syntaxin a knock-out mouse will be discussed. research funds: kakenhi ( ) ps a-k effects of local administration of the gaba agonists into the hippocampus ca area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka , hiroko miyakubo , junichi tanaka , yasushi hayashi , yukio hattori , masahiko nomura department of curriculum, teaching and memory, naruto university of education, tokushima, japan; department of human nutrition, notre dame seishin university, okayama, japan; department of physiology, saitama medical school, saitama, japan in fischer male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite -hydroxytryptamine ( -hiaa) concentration in the ca area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad- . fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka , sho koyasu , akinori nishi , tomoyuki furuyashiki , toshiyuki matsuoka , shuh narumiya department of pharmacology, university of kyoto, kyoto, japan; department of physiology, university of kurume, kurume, japan prostaglandins e (pge ) exert their actions in various organs through specific receptor, ep to . the previous study suggests that ep modulates da system. to investigate the roles of ep in da system, we examined ep ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼ % in both wild-type and ep ko mice. however, increase of locomotor activity in ep ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d receptor (d r) signaling, we tested the density of d r and d r signaling with phosphorylation of darpp- . there were no differences in d r binding. d r signaling was significantly attenuated in the striatal slices from ep ko mice. the effect of d r agonist on locomotor activity was also attenuated in ep ko mice. these results indicate that pge has enhancing effects on locomotor activity via ep by potentiating the d r signaling. central serotonin ( -ht) function has been implicated in impulsivity. the present study examined rats with -ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of -ht and its metabolites ( -hiaa) in the brain. for both tasks, significant negative correlations between learning performance and -ht and -hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and -ht and -hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of -ht roles on selective indices of impulsivity. yuki sato , , , tatsushi onaka , norimasa seo , eiji kobayashi dept. anesthesiol., jichi med. univ., tochigi, japan; dept. physiol., jichi med. univ., tochigi, japan; div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p - ; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p ; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p . within the vestibular nucleus of p - rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p in the vestibular nucleus as fuzziness about neuronal cell bodies. from p onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p . during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p . with ul, the pn network on the lesioned side remained compact until days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca region of rat hippocampal slices, hz/ pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at hz. ikuko yao , hiroshi takagi , hiroshi ageta , tomoaki kahyo , ken hatanaka , kaoru inokuchi , mitsutoshi setou , mitsubishi kagaku institute of life sciences, tokyo, japan; university of tokyo, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps p-a presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., mv, s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca + currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca + channel knockout mice. we conclude that the p/q-type specific ca + channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi , koji ikegami , ken hatanaka , , , yoko fujiwara-tsukamoto , mineo matsumoto , ikuko yao , mitsutoshi setou , , mitsubishi kagaku institute of life sciences, japan; presto, japan; school of pharmaceutical sciences, the university of tokyo, japan; okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa (−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki , shingo kimura , reiko fujita , shuji watanabe , kazuhiko sasaki dept. of physiol., sch. of med., iwate medical univ., morioka, japan; dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c , a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦ glutamate receptor (␦ r) plays a crucial role in cerebellar functions. although ␦ r has a putative channel pore domain, and ␦ r displayed ca + -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦ r functions as a channel. here we introduced a ␦ r transgene, which had a mutation (gln arg) in the putative channel pore conserved in ca + -permeable glutamate receptors, into ␦ −/− mice. surprisingly, a mutant ␦ r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦ −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca + influx through ␦ r is not required for its function in the cerebellum in vivo, and that wild-type ␦ r may not function as a ca + -permeable ion channel. research funds: kakenhi ( ) and takeda science foundation ps p-a distribution of tarp - on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya , mika tsujita , maya yamazaki , etsuko kushiya , manabu abe , kaori akashi , masanobu kano , haruyuki kamiya , kenji sakimura , masahiko watanabe department of anatomy, hokkaido university school of medicine, sapporo, japan; department of cellular neurobiology, brain research institute, niigata, japan; department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the - is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized - on excitatory synaptic and extrasynaptic membrane. in - -ko mice, ampars were reduced in hippocampal homogenates ( % of control) and psd fraction ( %). immunogold labeling also exhibited reduction of extrasynpatic ( %) and synaptic ( %) ampars in ca pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites ( %) and spines ( %). ampar-mediated responses were reduced at ca synapses ( %). therefore, - is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa , yugo fukazawa , elek molnar , masahiko watanabe , ryuichi shigemoto , div. cerebral structure, nips, okazaki, japan; mrc, univ. of bristol, bristol, uk; hokkaido univ., sapporo, japan; sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed . to times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed . to times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda , , , hitoshi kita dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; japan society for promotion of science, tokyo, japan; dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp abolished the sipsp. an mglur antagonist, but not an mglur antagonist, partially blocked the sdepo. in addition, cgp enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur -mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps p-a essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto , yoshikazu isomura , , michiko imanishi , tomoki fukai , masahiko takada system neurosci., tokyo met. inst. neurosci., tokyo, japan; neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for > s after the induction. gaba amediated cl-influx lasted for . s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura , yuko sekino , , toshiya manabe , division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < ), using electrophysiological methods and optical imaging. in immature animals (p ), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p ) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p - ). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p - compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣ , ␣ , and ␤ / subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣ , ␣ , and ␤ / subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ -or ␣ -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ -adrenoceptor agonist phenylephrine, while reduced by the ␣ -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi ( ) ps p-b involvement of basal pkc and erk / activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa , takahiko chimura , azumi matsumoto , kazuhiko yamaguchi lab. for motor learning control, riken, bsi, japan; lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk / activities at pf-pc synapses. taisuke miyazaki , kohichi tanaka , masahiko watanabe department of anatomy, school of medicine, hokkaido university, sapporo, japan; department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki , , kouichi hashimoto , taisuke miyazaki , masahiko watanabe , masanobu kano dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto , , takashi kodama , , ryuichi shigemoto , , , yugo fukazawa , , yasuo mori division of neural signaling, nips, okazaki, japan; school of life science, graduate university for advanced studies (sokendai), okazaki, japan; division of cerebral structure, nips, okazaki, japan; sorst, japan science and technology agency, kawaguchi, japan; department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v . (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v . mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v . exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n ) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n ) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps p-b target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from . ± . to . ± . (n = ; mean ± s.d.) by reducing [ca + ] o from to . mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps p-b response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at / s, and were depressed completely by cnqx. when stimulus rate was increased to /s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at /s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at / s, but not at /s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at /sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono , keisuke shiba , ken nakazawa , ichiro shimoyama department of otolaryngology, chiba university, chiba, japan; department of otolaryngology, kimitsu central hospital, chiba, japan; department of integrative neurophysiology, chiba university, chiba, japan; section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps p-b in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada , , eiji shigetomi , , fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan; dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi ( , ) ps p-b laminar sources of synaptic input to layer neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer / . neurogliaform cells receive more excitatory inputs from layers and . the dominant inhibitory synaptic inputs onto both types of cells originate from layer / . these neurons also get inhibitory inputs from layers and - . the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer are highly integrative. takako nishi , tsukasa gotow , shiro nakagawa ins. nat. sci., senshu univ., kawasaki, japan; dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p- , es- , ip- and ip- which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p- /es- of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip- /ip- . the present study was conducted to examine the functional significance of the above ip- /ip- , having a peak sensitivity at nm light. the body wall of the animal transmitted enough nm to hyperpolarize ip- /ip- in the ganglion covered by the body wall. ip- /ip- were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip- /ip- were not coupled to a-p- /es- one another. finally, ip- /ip- , the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps p-c cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the -day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e . it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e -e ), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada , nyberg tobias , nahoko kasai , yuriko furukawa , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, ntt corporation, kanagawa, japan; sorst, jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase -green fluorescence protein (gad -gfp) knock-in mice at postnatal day - were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps p-c pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda , toshiyuki ishii , , yasuhide shigematsu , toshihiko hosoya , yukio shimoda dept. physiol., keio univ. sch. med., tokyo, japan; bsi, riken, wako, japan; mri, tokyo womens' medical univ., tokyo, japan; dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p x -purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p x -purinoceptors. furthermore, a p x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca + . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca + oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of mesatp was observed. the inhibition of mesatp response by p y receptor specific antagonist mrs indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca + oscillations. data will be presented on the p receptor mediated regulation of synchronized ca + oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d -like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf was shown to selectively stimulate plc-linked d -like dopamine receptors. darpp- is a phosphoprotein that regulates the efficacy of d receptor signaling. activation of d receptors that couple to golf/pka signaling leads to darpp- phosphorylation at thr . we investigated the effect of skf on darpp- phosphorylation at thr by using mouse neostriatal slices. treatment of neostriatal slices with skf increased thr phosphorylation. the effect of skf was enhanced by a plc inhibitor, u . in contrast, the effect of skf was partially blocked by a d receptor antagonist, sch , and the sch -insensitive increase in thr phosphorylation was completely abolished by an adenosine a a receptor antagonist, zm . thus, analysis of darpp- phosphorylation revealed that skf activates at least three signaling cascades in the neostriatum: ( ) plc, ( ) d receptor/golf/pka, ( ) adenosine a a receptors. ps p-c thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c bl mouse were dyed with a voltage-sensitive dye rh . in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal ( mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx ( mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no. ps p-c brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur at psd within min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca + transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca + rise, that is, ca + release from ip r and ca + influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca + signaling regulation. research funds: health sciences research grant of nano- hiroyuki konishi , kazuhiko namikawa , , keiji shikata , yuji kobatake , taro tachibana , hiroshi kiyama dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; dept. anat., asahikawa med. col., hokkaido, japan; dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t , is involved in the control of cell morphology via the regulation of rho proteins via rho gdi . however, it is unclear whether t regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c rat glioma cells, which express t endogenously. rho gdi was dissociated from t in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t -specific sequences reduced the effects of bdnf. these results suggest that t regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d , bcl . the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf- in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind in response to igf- both at protein & rna levels but no change was observed in c-myc, bcl levels. interestingly, mapk-erk, which is also known to activate cd in other systems, had no effect on the cd level in neuronal cells. on the other hand, pi kinase not only activated jak/stat pathway but also affected on cyclind level. thus it appears that jak/stat is activated by pi k, which in turn upregulates cyclind level. pi k also activated erk without changing cd level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi k. cd is a one of the major downstream targets of jak/stat. hiroko inoue , haruhisa kawasaki , ritsuko fujii , tohru yoshioka , kazunori sango , toshihiko kadoya , hidenori horie sch. of sci. and eng., waseda univ., tokyo, japan; advanced res. center for biol. sci., waseda univ., tokyo, japan; tokyo metropolitan inst. for neurosci., tokyo, japan; pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin- (gal- /ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal- /ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal- /ox enhanced mrna expression of nerve regeneration-related factors such as il- , il- ␤. however, those of gdnf, ngf, bdnf, nt- , nt- , igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal- /ox. since il - , il- , and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal- /ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca + concentration, ␣camkii is activated by the interaction with ca + /cam. prolonged increase in ca + level leads to further activation of ␣camkii through autophosphorylation at t . to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca + level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy y cells, the increase in ca + level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca + /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦ isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦ - antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦ isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps p-d the role of ca + /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata , , hiroyuki sakagami , keiji imoto , , kunihiko obata , yuchio yanagawa okazaki, japan; sokendai, okazaki, japan; tohoku univ., sendai, japan; riken, wako, japan; gunma univ., maebashi, japan ca + /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k with r to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , # ) ps p-d characterization of ca + -dependent membrane binding proteins of rat brain shohei maekawa , katsutoshi taguchi , haruko kumanogoh , shun nakamura division of biosystems science, grad. sch. of sci/tech, kobe-u kobe - , japan; division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo - , japan ca + -dependent membrane binding proteins were purified through a ca + -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca + -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps p-d generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa ∼ ) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa ∼ ). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal -dbd with mutants of crest; crest c ( ∼ ) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps p-d characterization of iq-arfgef, a novel exchange factor for arf , in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor (arf ) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf . in this study, we determined the entire sequence of a partially identified cdna (mkiaa ) encoding a novel sec domain. the deduced protein contained coiled coil, iq, sec , plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf more selectively than arf . thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf and interaction with various pdz proteins. eriko miura , masahiro fukaya , tokiharu sato , kazushi sugihara , masahide asano , katsuji yoshioka , masahiko watanabe dept. anat., hokkaido univ. sch. med., sapporo, japan; dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap (or jip ) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk ∼ mrnas. in embryos, jsap immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori , shigeo uchino , tomoko isosaka , , shinichi kohsaka , takeshi yagi , shigeki yuasa dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr b phosphorylation and the nmda-induced calcium responses increased after dopamine d -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp : dystrobrevin binding protein ) at p . . however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi , naoko oda , ryuzo shingai , st coe program, iwate university, morioka, japan; department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa , masaki nomura , , toshio aoyagi , tomoki fukai riken brain science institute, saitama, japan; graduate school of informatics, kyoto university, kyoto, japan; crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki , , tatsumi hirata division of brain function, national institute of genetics, sok-endai, mishima, japan; prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps p-d an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc- cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama , masahiro matsuura , nobuhiko yamamoto grad. sch. of frontier biosci., osaka univ., suita japan; riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer . to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a , sema a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a , sema a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi , kakenhi ps p-e expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami , katsuhiko ono dept. anat., juntendo univ. sch. of med., tokyo, japan; div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin- expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin- receptor neogenin. slit- was expressed in the forebrain, but its receptor robo- was not expressed in gnrh neurons. semaphorin (sema) a expression was detected in the olfactory-forebrain region. within the forebrain, sema a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema a receptor neuropilin- , suggesting that sema a may act as a repellent during the migration of gnrh neurons. kazuto fujishima , , qing-hua jin , junko kurisu , tomoo hirano , mineko kengaku riken, bsi, lab. for neural cell polarity, saitama, japan; department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., ) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer /vldlr induces dab phosphorylation, dab is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab . reelin treatment of the neurons derived from dab deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab signal pathways. hidenobu mizuno , , tomoo hirano , , yoshiaki tagawa , dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area / border of the other hemisphere, where they terminate in layers / and . we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the / border and started to invade the cortical plate around p . they generated exuberant axonal arborizations by p then underwent remodeling to form the adult pattern by p . neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir . in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers / . area-specific targeting to the / border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps p-e identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp like protein (nplp) that has a distant homology to pmp /claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata , shin-ichi sakakibara , hirotaka j okano , hideyuki okano , dept. physiol., keio univ., sch. med., japan; dept. histol. neurobiol., dokkyo med. univ., japan ; crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi and msi , which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi and msi deficient mice. msi −/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi −/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi −/− msi −/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu , satoshi yamagoe , kazuo suzuki , michiko ohtomi department of biomolecular science, graduate school of science, toho university, chiba, japan; department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect (leukocyte cell-derived chemotaxin ) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect and tau or map in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect in neurons. at the early stages in cultured neurons, lect was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda , , takahiro moriya , hideyuki terazono , suguru kudo , masahiro yamaguchi , kenji yasuda , izumi nagata , kazuyuki shinohara div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e . nestin-promoter gfp transgenic mice were placed into pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng cells were co-cultured with vascular smooth muscle cells (sm- ). sm- cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin- mrna was specifically up regulated in ng cells. but the western blotting analysis did not show the significant increase of neuropilin- . sema a, a candidate of ligands for neuropilin- , was found in ng cells rather than in sm- cells. the immunocytochemical analysis revealed that neuropilin- was increased in the surface of ng cells co-cultured with sm- cells or cultured with the conditioned medium of sm- cells. these results suggest that neuropilin- may be transferred from cytoplasmic pool to surface of ng cells by some soluble factors released by sm- cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa , j. nakanishi , h. hamada , y. yokomachi , k. yamaguchi , n. takeda inst. biomed. eng., waseda univ., tokyo, japan; riken, japan; kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca- was shown to be required for cdc to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca- . toca- is strongly expressed in developing brain. knockdown of toca- in pc cells significantly enhances neurite elongation. consistently, overexpression of toca- suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca- in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca- regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi ( ) ps p-e pragmin, a novel effector of rnd gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd is specifically expressed in brain. although rnd and rnd display an antagonistic action for rhoa, signaling pathways of rnd are not well known. here we have performed a yeast two-hybrid screen using rnd as bait and identified a novel rnd -effector protein, pragmin (pragma of rnd ). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd among the rho family gtpases in a gtp-dependent manner. rnd -bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd , and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd controls neurite outgrowth by regulating rhoa activity through pragmin. ps p-e regulation of growth cone motility by collapsin response mediator protein- (crmp- ) masumi iketani , yanai shigeki , fumio nakamura , yoshio goshima , , kohtaro takei , dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein- (crmp- ) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp- within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp- in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp- in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp- activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps p-e eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani , , masaru ihara , , hiraki sakuta , , hiroo takahashi , , ikuko watakabe , masaharu noda , division of molecular neurobiology, national institute for basic biology, okazaki, japan; crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps p-e neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa and lpa . here we examine how lpa , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma (pc ) cells. pc cells expressed low levels of endogenous lpa mrna. when pc cells were infected with retrovirus expressing lpa and treated with nerve growth factor under serum-free conditions for days, neurite formation was observed in % of total infected cells, which was similar to that in control virus-infected pc cells. however, the mean length of neurites was increased in pc cells expressing lpa , compared with that in control pc cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa was involved in neuritogenesis and branch formation. masao sakurai , tomoko aoki , kyoko ishikawa , shingo yoshikawa , john b. thomas , chihiro hama cdb, riken, hyogo, japan; salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt as a regulatory factor for glomerular patterning. in the wnt mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt expression in orns. previous studies in embryos have shown that wnt repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt mutant. this and other genetic data suggest that drl is an antagonist for wnt signaling. we propose that wnt signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps p-e a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps p-e differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino , masatomo kobayasi , taiki nakajima , lydia michaut , indrayani ghangrekar , kuniaki takahashi , ryu ueda , kaoru saigo , walter j. gehring , katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan; biozentrum, univ. of basel, switzerland; genet. strains res. ctr., natl. inst. genet.; dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified , genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki , yoshie suzuki , hiroyuki aburatani , akinao nose dept. phys., univ. tokyo, tokyo, japan; rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, and , which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼ genes with more than two-fold difference in expression level. for candidate genes out of ( %), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles or , implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet- . to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet- signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet- . loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps p-e analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi , takaaki hirotsu , eriko kage , hideaki takeuchi , hirofumi kunitomo , yuichi iino , takeo kubo dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; dept. biol., fac. sci., kyushu univ. grad. sch., japan; mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr- is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr- mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr- functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura , ken-ichi ogura , yoshio goshima , dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc- is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc- is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc- is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc- during the axonal guidance of the hsn neurons. silencing of netrin/unc- only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc- only at the vulval precursor cells in unc- null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc- expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn a-hsp :gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal /uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere . these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) masahiko taniguchi , yoshinori mikami , tomoyuki masuda , tomoyuki yoshida , naoto matsuda , masayoshi mishina , takao shimizu department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; department of anatomy, fukushima medical university, japan; dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin d (sema d). the orf of sema d is bp ( aa). zebrafish sema d protein showed a . % identity with mouse sema d protein. to clarify the expression pattern of sema d, in situ hybridization was performed. in the embryos, sema d is expressed in the lens and hindbrain. we also found that sema d has the repulsive activity. these results indicate that sema d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext ( ) mayumi yamada , shohei maekawa , seiji miyata department of applied biology, kyoto institute of technology, kyoto, japan; division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps p-f semaphorin d inhibits ␤ integrin activity through the r-rasgap activity of plexin-b izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin d (sema d) receptor, plexin-b functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema d/plexin-b in cell migration. activation of plexin-b by sema d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi -k activation, and ␤ integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤ integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤ integrin activation and cell migration, and that inactivation of r-ras by sema d/plexin-b -mediated r-ras gap activity control cell migration by modulating the activity of ␤ integrins. shun hamada , emi fukuda , , shota katori , , takeshi yagi , dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; grad. sch. frontier biosci., osaka univ., osaka japan, crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise > cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga is expressed by layer / neurons throughout the neocortex at p mice, but is absent in adults. between e . and late p , stages that span the generation and radial migration of layer / neurons, mdga is expressed in patterns consistent with its expression by migrating layer / neurons, suggesting a role for mdga in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e . to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day , but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps p-f analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono , yoshiyuki kuroda , fumiko matsui , yoshihito tokita , keiko nakanishi , michiru ida , masahito ikawa , masaru okabe , katsuhiko ono , atsuhiko oohira institute for developmental research, aichi human service ctr., kasugai, japan; research institute for microbial diseases, osaka university, suita, japan; national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi ( ) ps p-f phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata , tatsuhisa watanabe , ikuko suzuki , junichi kitagawa , akiko ogawa , kenro kanda , kazunao kuramoto dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats ( - months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura , koichi obata , masafumi sakagami , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk , mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days and after cfa injection. furthermore, time course of phosphorylated-erk level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk in drg neurons may contribute to the development of inflammatory pain. ps p-f activation of erk in drg neurons contributes to acute pain toshiyuki mizushima , , koichi obata , takashi mashimo , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p in dorsal root ganglion (drg) neurons. however, there has been no study examining erk (also known as big mapk or bmk ) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk in small-to-medium diameter sensory neurons with a peak at min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and spinal cord and participate in pain-related behaviors in the l spinal nerve ligation (snl) model. l snl induced an increase in the phosphorylation of erk not only in the injured l drg, but also in the spared l drg at day after surgery. furthermore, l snl induced a striking increase in erk phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai , minoru asada , naoki seno , hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan; pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣- , which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps p-f interleukin- ␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda , jun kadoi , msanori nasu , masayuki takahashi , shigeji matsumoto dep. physiol and res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il- ␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il- ␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il- ␤ induced depolarization in inflamed rats was larger than that in control rats. il- ␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il- ␤ paracrine mechanism following peripheral inflammation. junichi kitagawa , mamoru takeda , jun kadoi , yoshiyuki tsuboi , shigeji matsumoto , koichi iwata dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day after ion treatment and the threshold decrement was lasting more than day . single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day - after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for min. at , , , and h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at h, peaked at h and then decreased. intense gfap immunoreactivities were also detected at and h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for min at m/min. after the running, rats were applied the weight on the gastrocnemius muscles for min, and then spinal cord and soleus muscles were removed from the rats. productions of il- beta, il- and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at h post-running, number of fos-positive neurons was increased, peaked at h and then decreased to control level at h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo , yusuke sakai , aya masawaki , takashi shimoda , masayuki moritani , motohide takemura dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep receptor (ep r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca + ] i ) in a ptx-sensitive manner in cho cells expressing canine ep r and mouse bk b receptor (b r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk ( nm) was applied twice with a -min interval to the cells expressing b r, the second [ca + ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h- ( m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk ( pm) treatment for h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca + ] i was at least in part mediated by pka. ps p-f contribution of peripheral ht a or ht receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral ht a or ht /r in male rats with tmj inflammation. here we tested if these two ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c . fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for days compared to naive rats. we tested if local ht a or ht /r antagonist affects fos expression in both groups. the number of fos-li in the vc/c but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral ht a and ht /rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of -ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼ % of sg neurons examined, -ht induced an outward current. the outward current was mimicked and suppressed by a -ht a agonist and -ht a antagonist, respectively. in ∼ % of sg neurons, -ht evoked an inward current which was mimicked by a -ht agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that -ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of -ht a and -ht receptors, respectively. the reciprocal postsynaptic actions of -ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by -ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps p-g nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi ( ) and km ( ) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about % of neurons examined, melittin ( m) superfused for min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at mv in the presence of bicuculline. this action was visible about min after the beginning of its superfusion and subsided within min after washout. these melittin actions were reduced in extent by a pla inhibitor -bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi ( ) ps p-g presynaptic p y receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied -methylthio adp ( mesadp, m), a p y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about % of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by mesadp with no change in the amplitude. the mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs ( m), a selective p y receptor antagonist. these results indicate that the activation of presynaptic p y receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p y receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi ( ), the japanese health sciences foundation (kh ) ps p-g presynaptic enhancement by proteinaseactivated receptor- agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)- but not par- agonist (each m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par- mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par- agonist (sfllrn, m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at - mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par- antagonist (yfllrnp, m). these results indicate that the activation of par- s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par- activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi ( ) ps p-g effect of tramadol metabolite m on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m , which is one of its metabolites, at mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at − mv. this action was not seen in the presence of a -opioid receptor antagonist ctap ( m). m also reduced the peak amplitudes of epscs which were monosynaptically evoked at − mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk- or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk- injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk- . these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps p-g studies on pain control system (rept. ): changes in phosphorylation of nr b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for weeks. under these conditions, the immunoreactivities of phosphorylated-ser- nr b (p-ser- nr b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr- nr b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps p-g prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, hz, a, s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by -ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps p-g the nav . sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav . , a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav . . four weeks post-sci, nav . protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav . caused a significant reduction in nav . expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda , hiroyuki ichikawa , ryuji terayama , tomosada sugimoto department of oral maxillofacial rehabilitation, okayama university, okayama, japan; department of oral function and anatomy, okayama university, okayama, japan ib -sap is a neurotoxin designed for targeting primary nociceptors with ib binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib -sap ( . l of . % solution for each ganglion) directly into the th and th lumbar (l and ) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib -binding. after counterstain, the cell body size of neurons were measured. ib -sap reduced the total number of drg neurons in l and ganglia combined by % ( ± on untreated side versus ± on treated side). small neurons (< m ) were reduced by % whereas large ones (≥ m ) were not affected. ib -binding neurons were mostly small (≥ %) and were reduced by %. the number of small neurons, that were not stained for ib -binding, increased by % ( ± versus ± ). schuichi koizumi , kaoru nasu-tada , makoto tsuda , emiko kunifusa , , kazuhide inoue div. pharmacol., natl. inst. hlth. sci., tokyo, japan; dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p x receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p x receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p x receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p x receptor in microglia, leading to neuropathic pain. research funds: mf , kakenhi ( ) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi ps p-g lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at mm decreased the protein levels of myelin basic protein (mbp) at h. this action was significantly inhibited by botulinum neurotoxin/c (bont/c ). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp , mag, p in cultured fibers. the maximal decrease was observed all at as early as h after the addition of lpa. bont/c and y abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at and , , , , days after operation. pre-emptive co-administration of mk- and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk- decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama , , naomi akiyama , mikie hirata , takayuki nakagawa , shuji kaneko , masabumi minami dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for h per day between postnatal day and . visceral sensitivities were assessed in stomach and colon at weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research ps p-g change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered to days after exercise in rats ( w old). c-fos expression in the superficial dorsal horn increased in l spinal segment when the edl muscle was compressed days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats , ( - ) and ( - ) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in w old rats, but not significant. after lec, the threshold started to decrease day after lec in all three age groups. it returned to the pre-lec level days after lec in and w old rats only. recovery of w old rats delayed up to days after lec. increased c-fos expression in the superficial dorsal horn was observed in l as well as in l in w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama , masaharu akita kanagawa life-science res., japan; kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day to of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days - of age, they did not show the first estrus before days of age with mean age of . ± . day. in contrast, most of normal mouse igg-treated controls showed the first estrus by days of age with mean age of . ± . day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss- gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days of age (d ), and first estrus on around d . brain tissues were obtained on d , , , and . kiss- mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d to and was kept at a high level thereafter. gpr mrna expression in the medial preoptic area increased significantly from d to . metastin-immunoreactive cells were not found on d but were apparent in the arc-me on d onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito , sei-etsu fujiwara , kenjiro konno , takashi yamaguchi , tetsu nemoto , etsuko kasuya , ryosuke sakumoto anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; grad. sch. sci. & eng., yamagata univ., yonezawa, japan; sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps p-g hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi , teruko uwano , , anh tran , , eturou hori , , taketoshi ono , , hisao nishijo system emotional science, univ. of toyama, japan; integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of hf neurons, responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue , atsu aiba , kaoru inokuchi mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; grad. sch. med., kobe univ., kobe, japan vesl- s/homer- a and vesl- l/homer- c are splicing isoforms encoded by the vesl- gene. vesl proteins bind and regulate mglur / , ip receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl- s is upregulated by tetanic stimulation that elicits l-ltp. vesl- s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl- s gene-targeting mice (ko) and examined whether vesl- s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl- s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta , r. migishima , s. kida , k. inokuchi , mitsubishi kagaku inst. life sci (mitils), japan, tokyo univ. agricul., japan; crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps p-h sex differences in the effects of chronic estrogen treatment on fear conditioning in c bl/ j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c bl/ j mice were implanted with a silastic capsule containing , , or g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps p-h analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps p-h analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps p-h analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor (cb ) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb and lvgccs, we also found that cb is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda , takahiro furuta , kouichi nakamura , , takeshi kaneko , ps p-h difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama , tomo unzai , kouichi nakamura , , sakashi nomura , takeshi kaneko , department of morphological brain science, kyoto university, kyoto, japan; department of physical therapy, kyoto university, kyoto, japan; crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut) , thalamostriatal inputs, were less dense in patch region, whereas vglut -positive corticostriatal inputs were evenly distributed. quantitative analysis revealed % of vglut positive synapses in patch region were formed with spines, whereas % in matrix region were made with dendritic shafts. in contrast, the targets of vglut -positive inputs were mainly spines in both regions. moreover, vglut -positive axospinous synapses in patch region were larger than vglut -positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi ( , , , ( ), ) ps p-h single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai , fumino fujiyama , takeshi kaneko , department of morphological brain science, university of kyoto, kyoto, japan; crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi ( , , , ( ) , ) ps p-h lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui , kayo nishizawa , nobuyuki kai , kazuto kobayashi , dept. of psychology, fukushima univ., japan; dept. mol. genet., fukushima medi. univ., japan; crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of -ohda injections into the nucleus accumbens (acb) in c bl/ j mice. all mice were trained to press the lever for a food pellet using a fixed ratio (fr ) schedule. the mice were injected with ascorbate vehicle or -ohda into the acb, and then tested post-surgically using the fr schedule again. the -ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area , it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area , suggesting different functional roles played by each subdivision of area in spatial memory and behavior. eiichi jodo , yoshiaki suzuki , tadahiro katayama , ken-yo hoshino , yukihiko kayama dep. of physiol., fukushima med. univ., fukushima, japan; dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, %) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about . s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in % out of vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only % out of it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., ) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., ) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., ) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of sessions in phase , although they learned easily the four problems in phases and . our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto , noriyuki higo , munetaka shidara neuroscience research inst., aist, tsukuba, japan; grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d receptor study using antisense cdna showed that d receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d receptor antipeptide antiserum. d receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area of the perirhinal cortex than in area teav. the differential localization between area and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano , natsuko yoshida , kiyohisa natsume kyushu kyoritsu university, fukuoka, japan; kyushu institute of technology, fukuoka, japan; kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f and f was the highest and suggests that the theta rhythm at f and f may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after -month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi ( ) ps p-h neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe , sho yagishita , hideyuki kikyo , department of physiology, the university of tokyo school of medicine, tokyo, japan; department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a . t scanner system was used (te = ms; tr = s; acquisition time = . s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong , motoaki sugiura , yuko sassa , keisuke wakusawa , , kaoru horie , , shigeru sato , , ryuta kawashima , gsics, tohoku university, sendai, japan; niche, tohoku university, japan; miyagi university of education, sendai, japan; department of pediatrics, school of medicine, tohoku university, japan; the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz , christie l. sahley , kenneth j. muller physio. & biophys., univ. of miami, miami, fl, usa; biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps p-h sensory/motor modules regulating the development of peer social relationship mamiko koshiba , , shun nakamura jst, crest, japan; ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata , minoru hattori department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) -s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi -s procedure over days. the -s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the -s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi -s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps p-i the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka , , ritz oshio , , norihiro sadato , , manabu honda , nips, okazaki, japan; jsps, tokyo, japan; nagoya univ., nagoya, japan; ristex, jst, tokyo, japan; sorst, jst, tokyo, japan; ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps p-i sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi ( ) ps p-i selective manipulation of working memory through d and d receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d and d receptors. the computer simulation with this model shows that d receptor activation mainly affect working memory activity itself, while d receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d and d receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or mg/kg mam on the th day of gestation. the activity of male and female offspring was measured for each h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every min interval for h under min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca pyramidal layer and ectopic neuron mass). ps p-i long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur . hamid gholamipour , shirin babri , khameneh saied department of physiology, university of tabriz, tabriz, iran; university of tabriz, iran; university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca region of hippocampus on memory formation. fifty male rats were divided into five groups. ( ) control ( ) sham operation ( ) test ( ) diabetic/saline ( ) diabetic/insulin. groups and were made diabetic by treatment with stz ( mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < . ). compared to sham group diabetic/insulin group showed increased latency (p < . ) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca + /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of per , in india. we studied the park polymorphism in north indian population and parkin expression in early parkinson's disease (n = ) and sporadic parkinson's disease (n = ). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon- , and in early pd and exon- and in sporadic pd, heterozygous mutations in exon and in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul , kittisak sripanichkulchai , david finkelstein faculty of medicine, srinakharinwirot university, thailand; faculty of medicine, khon kaen university, thailand; university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c bl/ mice were pretreated with beta-estradiol (e ), injected with mptp on the day and brains were collected on day . e pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps p-i effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki , fumihiko yoshino , masaichi chang-il lee dept. of food sci. and nutrition, showa women's univ., tokyo, japan; clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi ( ) ps p-i recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi , kaori sawada , haruo okado , atsushi nambu , masahiko takada tokyo met. inst. neurosci., fuchu, tokyo, japan; natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype- raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type- ) of raav (raav ) carrying αs cdna (raav -␣s), and a cocktail of raav -␣s and raav carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav -␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged - s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of . - (i), . - (ii), . - (iii), and . - (iv). the ␤waves were rated with total periods (%) of - hz wavelets in -s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated - % in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about % in group iv. the pvp ␤-waves were - %, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana , , hirokazu iwamuro , , masahiko takada , atsushi nambu , div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; sokendai; dept. neurosurg., univ. tokyo, tokyo, japan; dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd expression on microglia and also enhanced the mt -mmp expression to shed off cd at the cell surface and degrade surrounding ecm to open the migratory way. a t mutant ␣-synuclein showed greater level of cd shedding and cell migration. extracellular treated ␣-synuclein also increased cd and mt -mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant ( grant ( - ps p-j serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai , hiromasa yamada , yoshinari aimi , ikuko nagatsu department of anatomy, shiga university of medical science, otsu, japan; fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba -and tb distribution in microglia were immunohistochemically investigated at h and days after twice mptp-treatments (one treatment comprised of intraperitoneal injections of mg/kg mptp at h interval) to c bl/ and balb/c at months (mo) interval. the recognizable change of iba -and tb -distibution in microglia of both mice strains was observed even mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps p-j activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada , ryohei hishida , yoko hirata , kenji ono , hiromi suzuki , shin-ichi muramatsu , imaharu nakano , kunihiro tsuchida , toshiharu nagatsu , , makoto sawada school of medicine, fujita health university, aichi, japan; division of neurology, jichi medical university, japan; department of biomolecular science, gifu university, japan; research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps p-j phosphorylation of erp in adult rat brain with neonatal -ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of -ohda at (p ) and days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p , tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the -ohda lesioned rats at p , and it was identified as disulfide-isomerase a precursor (erp ) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi ( or nm, h) was applied to cultures at days in vitro and succeeding changes of da neurons were investigated up to days. more than % of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps p-j specific expression of proapoptotic factor pag on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p -activated gene (pag ), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag in the internal capsule of l-dopatreated and constitutive expression in the smi- -immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps p-j an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang , , yuzuru imai , haruhisa inoue , , ayane kataoka , sachiko iita , nobuyuki nukina , ryosuke takahashi , neurology, university of kyoto, kyoto, japan; bsi, riken, saitama, japan since loss of parkin e activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., ) have found that application of an antisense oligodeoxynucleotide specific for nr have successfully knockdown the expression of nr gene expression in the striatum of -hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr was re-addressed using a small interfering rna (sirna) specific for nr . in pc cells, reductions of nr proteins after a single application of nr sirna were found by western blot experiments. and after one single application of nr sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr immunofluorescence were found in the striatum after the sirna treatments. lai et al., . neurochem. int. , - . research funds: faculty research grant, frg/ - /ii- , hong kong baptist university ps p-j homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy ( . - mol in l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on th day. animals with mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps p-j ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca takahiro seki , takayuki shimahara , naoko adachi , naoaki saito , norio sakai dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type (sca ), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca . however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca + homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki , yo sasaki department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat ) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at min, but next increased at h and returned to basal at h after cocaine challenge. actin showed a similar pattern but was maintained at high level at h. both psd- and glur were increased between h and h like actin. by contrast, some proteins such as drebrin were decreased after the peak at h. interestingly, the s proteasome subunit demonstrated a dramatic upregulation at h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps p-k effects of mdma and -meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, , methylenedioxymethamphetamine (mdma) and -methoxy-n,ndiisopropyltryptamine ( -meo-dipt), on sert and dat were examined. as previously reported, mdma ( . - m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, -meo-dipt ( . - nm) evoked an outward current response in sertexpressing cells similarly to that of selective -ht reuptake inhibitors. no current response was observed when -meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that -meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka , nobue kitanaka , tomohiro tatsuta , , yoshio morita , motohiko takemura department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine ( mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of . mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of and mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth ( . mg/kg, i.p.), pretreatment with subchronic clorgyline ( . - mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura , nobue kitanaka , tomohiro tatsuta , , yoshio morita , junichi kitanaka department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline ( . - mg/kg, i.p.) min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum min after drug challenge. these results suggested that the inhibitory effect of lobeline ( . - mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with mg/kg meth and locomotor activity measured daily for days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, nmol ufp- (a nop receptor antagonist) and mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp- strongly suppressed locomotor activity. thus, it was unclear if ufp- suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp- co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine ( - mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine ( . - mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine ( , . and mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine ( mg/kg, i.p.) caused more than % mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine ( mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps p-d regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: ( ) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis (ca ) were increased after fs. the rule of this kind of changing was of region-and time-specific. ( ) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi , , tomoaki shirao dept of neurobiol & behav.; ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only . ± . % of drebrin in the spine is stable, with a turnover time of . ± . min. blockade of ampar by m cnqx reduces the population of stable drebrin ( . ± . %), and has no effect on a turnover time. on the other hand, blockade of nmdar by m ap has no effect on the population of stable drebrin, whereas shortens a turnover time ( . ± . min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu , yoshito saitoh , kaoru inokuchi , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki , , daisuke okada , kaoru inokuchi , , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; yokohama natl. univ. environment information sci., kanagawa, japan; crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto , mitsutoshi setou , , kaoru inokuchi laboratory for molecular gerontology, mitils, japan; laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps a-a an activity that traps vesl- s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl- s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at m, but not nm. because nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps a-a acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosening, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca region in rat hippocampal slices. an application of . dheas for min to the slice augmented instantly the epsp, which was terminated within min. however, even h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosensing, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan to know whether ␤-estradiol (e ) can protect ca neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion ( vo) for min, and the effect of e against this ischemic injury was examined. the electrophysiological properties of ca -ca synapses were examined by a real-time optical recording method days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e ( mg/kg) h before vo was able to protect ca neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt ( mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn ( mg/kg) did not. above results suggest that e can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps a-a non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima , mari ogiue-ikeda , yuki oishi , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca , ca , and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps a-a morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki , gen murakami , , tetsuya kimoto , , suguru kawato , college of humanities and sciences, nihon university, tokyo, japan; department of biophysics and life sciences, university of tokyo, tokyo, japan; crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of nm dex induced rapid modulation of the density and morphology of dendritic spines in ca pyramidal neurons within h. the total spine density increased from . spines/m to . spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura , yuji ikegaya , sadao shiosaka division of structural cell biology, naist, nara, japan; laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: st century coe research ps a-a actin organizations within single dendritic spines in ca pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of . min and min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds ( . - . um/min) and lengths ( . - . um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori , katsushige sato , kohichi tanaka , hiroko hamazaki mol. neurosci., tmdu, tokyo, japan; physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at or days post-hatching (p or p ), and at p . this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p , but they lose this ability at p . in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps a-b effect of estrogen on hippocampus in male and female mice takanori sugawara , shinji hayashi , victoria luine graduated school of integrated sience, yokohama city university, yokohama, japan; department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received ␤-estradiol (e ) or oil-vehicle injections at adult before golgi impregnation. spine densities m of apical dendrites of the pyramidal neurons in the hippocampus ca region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto , , takafumi inoue , , akinori kuruma , katsuhiko mikoshiba , , lab. developmental neurobiology, brain science inst., riken, saitama, japan; div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca + reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p mapk and c-jun nh -terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u , a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb and sp , antagonists for p mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps a-b regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey , chou-chi li , yumiko motoyama , junko yamaoka , masao ito riken brain sci. inst., japan; national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur . glur phospho-ser promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur -vcp interaction. yuichi kitagawa , , shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp- and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps a-b long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from to days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over days old, when song learning is nearly complete, but ltd remained possible in birds over days old who had been isolated from song tutors, and thus retained the capacity for learning. ps a-b involvement of ca + -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that exposures to glu of cultured rat hippocampal slices at h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur and glur , showed that the number of glur -positive puncta increased transiently after the repeated glu exposures, whereas the number of glur -positive puncta increased gradually and persistently. jstx (a ca + -permeable ampar blocker) suppressed fepsp amplitude recorded at ca -ca synapses by - % in the period corresponding to the transient increase of glur -positive puncta. this transient increase should represent the delivery of ca + -permeable (glur -lacking/glur -including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca + -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka - , japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd times ( h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of -ht ( m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the -ht b receptor agonist cp mimicked the presynaptic inhibitory effect of -ht. -ht b receptor antagonist nas- reversed the -ht inhibitory effect, indicating that the -ht induced inhibitory effect occurs by mediating -ht b receptors. the presynaptic inhibitory effect of -ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, -ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this -ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that -ht, upon activating -ht b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku , masato ano , yuki hashimotodani , tadasato nagano , masanobu kano dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, -ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a , a a, a b and a ). adenosine a and a receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a and a receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a agonist (cpa) and a agonist ( -cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and -cl-ib-meca as reported previously. although ca currents were decreased by -cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of -cl-ib-meca. at administration of cpa and -cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a receptor may inhibit adenosine a receptor throughout a intracellular pathway in neurons. ps a-c influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa , j. yamada , k. inoue , y. yanagawa , a. fukuda , dept. physiol., hamamatsu univ. sch. med., japan; dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad -gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa , hiroyuki hioki , kouichi nakamura , , hisashi nakamura , takeshi kaneko , dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter (vglut )-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut -positive cells showed immunoreactivity for -hydroxytryptamine ( ht), a significant proportion ( . %) of vglut -postive cells was ht-negative. in this study, triple immunofluorescence staining was performed for vglut , ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad ) and tyrosine hydroxylase (th). our results showed that all of the vglut -positive/ ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut positive/ ht-negative neurons didn't show any immunoreactivities for gad nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut -positive neurons in these nuclei. research funds: kakenhi , , ps a-c cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig , mika ushimaru , yoshiyuki kubota , akiya watakabe , tetsuo yamamori , yuchio yanagawa , yasuo kawaguchi div. cerebral circuitry, nips, okazaki, japan; div. brain biology, nibb, okazaki, japan; dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin ( -ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, % of the cs were inhibited by -ht through -ht a r and % were excited through -ht a r. however, % of the fs cells were inhibited and % excited. these results show that cs cells are more inhibited by -ht than fs. the expression of -htr was confirmed by in situ hybridization. research funds: jsps pe and ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v , teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer but also in layer and/or . in each of the cases, about - gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad , somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine ( ) their other features, ( ) possible species variability, ( ) their functional significance. supported by riken bsi. withdrawn ps a-c regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge , , susanna b. park , greg j. stuart , yasuo kawaguchi national institute for physiological sciences, japan; div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure ( m) inhibited layer pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers / were generally less responsive to ach, but ach inhibited layer cells in visual cortex. prefrontal layer pyramidal neurons were more responsive to ach than were layer cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations ( mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in % of non-fs interneurons ( of cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki , , kenzi saito , , yuchio yanagawa , department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; sorst, jst, kawaguchi, japan; sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad -deficient mouse fetus. ps a-c gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal to day in egl. its release, especially from premigratory zone, was inhibited by ni + , but cd + couldn't. we suggest that gaba excitation induces granule cell migration. ps a-c gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi , hiroaki wake , junichi nabekura , osamu takahashi department of histology, kanagawa dental college, yokosuka, japan; national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata , kunihiko obata , jonathan davies , mark bellingham , peter g. noakes lab. for memory & learning, riken-bsi, wako, japan; obata res. unit, riken-bsi, wako, japan; sch. biomed. sci., univ. queensland, queensland, , australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., ) . we investigated the involvement of gaba alone in the mn death using gad -deficient mice, in which cerebral gaba is reduced to less than % of the wild-type. mn numbers at embryonic day (e) were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e embryos and subjected to electrical recording from the c and c ventral roots and also gaba measurement. in gad -deficient embryos, increase in number of brachial mns ( %) and decrease in both spontaneous discharges in the c , c roots and gaba content (less than %) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣ ␤ ␥ and ␣ ␤ ␥ combinations ( % reduction) but had no effect at ␣ ␤ ␥ or ␣ ␤ ␥ . in slice recordings dmcm blocked ipscs by % in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥ subunits whereas in the central amygdala some inputs contain ␥ while others contain ␥ subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps a-d hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane , takashi iwai , yasuo nagai , junichiro oka , hideo kimura dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h s. we examined the effect of h s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na s), a donor of h s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na s. these observations suggest that h s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by ( l) lidocaein ( %) min before training. animals were allowed to self administer morphine ( mg/kg per inf.) ten consecutive daily -h session. during all morphine self administration session lever pressing was measured. our results show that: ( ) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. ( ) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca + wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of ␤ estradiol from glucocorticoid activity yuki oishi , suguru kawato department of physics, graduate school of science, university of tokyo, tokyo, japan; graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, ␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. ␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that ␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps a-d the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex ( . mg/ g b.w., sc) or sesame oil (vehicle control) everyday for - days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf- mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf- , estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf- and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps a-d mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of -hydroxydopamine. ps a-d the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba , kimi watanabe , munekazu komada institute for developmental research, aichi human service center, aichi, japan; graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida , , asif k mustafa , kenji hashimoto , kumiko fujii , , kazuhisa maeda , , hiroshi ujike , richard l. huganir , solomon h. snyder , akira sawa dept. of systems biology, obi, suita, japan; depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; chiba univ. forensic mental health, chiba, japan; dept. of psychiat, shiga univ. med. sci., shiga, japan; div. of neuropsychiat, tottori univ., yonago, japan; dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick knockout mice were significantly lower than those of their wildtype littermates at age of p , but not in adults, suggesting regulation of pick on sr at developing stage. in case-control association study, we observed an association of the pick gene with sz, which is more prominent in disorganized sz. our findings suggest that pick contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps a-d epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe , , werner kilb , ileana l. hanganu , taizhe qian , daiichiro nakahara , atsuo fukuda , heiko j. luhmann dept. of physiol., hamamatsu, japan; inst. of physiol., mainz, germany; dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca region of an intact preparation. mm taurine blocked epileptiform activity induced by mg + free acsf and m -ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps a-d responses of pge in undifferentiated and differentiated ng - cells kayoko matsushima , takashi imanishi , akinori kawaguchi , tetsuyuki wada , shigeru yoshida , seiji ichida school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of sci. & eng., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan our previous findings showed that -ht-and bk-induced [ca + ] i increases were enlarged in differentiated ng - cells. for the next stage, we investigated the effect of pge , an inflammatory mediator for -ht and bk, on the cells. ng - cells were loaded with fura- /am, and the change in [ca + ] i was monitored by an image processor. the results showed: ( ) pge -induced response was decreased when ng - cells were differentiated by bt camp, ( ) − m ah and sc irreversibly inhibited pge -induced response by about % and %, respectively, while − m ah and sulprostone had no effect, and ( ) pge -induced response was abolished under ca +free conditions in about % of both ng - cells. these results indicate that the response to pge , via ep and ep receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from -to -day-old rats. bath application of serotonin ( -ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during -ht application. under the current-clamp recording, -ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that -ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps a-d searching for endogenous ligands of trace amine receptors in mammals ( ) akira komatsu , airi yamaguchi , noriko makikusa , osamu koizumi dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by % dms, post-fixed by % formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps a-d effects of mg + on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa , , nahoko kasai , akiyoshi shimada , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, kanagawa, japan; sorst/jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg + plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca + concentration are largely affected by mg + removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg + removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg + concentration and their actions on neural activity using a mg + indicator, kmg- -am together with fluo -am. we observed different effects of the mg + removal on gabaergic and non-gabaergic neurons by using gad -gfp knock-in mice. research funds: jst/sorst ps a-e transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p -p . zn + cortical neurons were also labeled, intrinsic and extrinsic, from p . unlike in the adult, these were in layer , instead of layers , , and . at p , neurons occurred in layers , , and and, in some areas, layer . at p , zn + neurons first appeared in layer ; and at p , there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. ps a-e systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano , , ryohei kanzaki , kei ito , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi , , taizo nakazato , shigeru kitazawa neurol, juntendo univ., tokyo, japan; physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, ) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns ). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse ( to mv for da; to mv for ht) was applied at hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps a-e time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui , , yuko sekino , , tomoaki sirao dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; div. of neural network, inst. med. sci. univ. of tokyo, tokyo; crest, jst, kawaguchi, japan; jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at days in vitro (div), and analyzed the dynamic localization of gfp-da at div. glutamate stimulation started gfp-da translocating within s and completed in min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps a-e distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa , jun shiota , hiroyuki tsutsumishita , hiroyuki sakagami , masaaki tsuda , akiko tabuchi dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr- , c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi ( ) to a.t. shoko shimizu , shinsuke matsuzaki , tsuyoshi hattori , ko miyoshi , masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia (disc ) was identified as a novel gene disrupted by a ( ; ) (q . ;q . ) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc at centrosome and residues - of disc (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc to target to the centrosome. furthermore, we have shown that inhibition of the disc -kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc is important to the disc -kendrin interaction and that a truncated form of disc lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca + or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca + and cl − imaging. we found that the relatively low density a glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca + oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps a-e involvement of ca + influx in the unpolarized non-vesicular release of fgf- hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf- lacking a conventional signal peptide sequence. we found that fgf- is co-released with s a , a ca + binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf- is co-released with s a from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf- and s a required ca + . the overexpression of s a - mutant lacking an ability of interaction with fgf- inhibited the their release, suggesting that s a is a cargo molecule. the release of fgf- upon either stimulation was abolished by voltage-dependent n-type ca + channel blocker. these findings suggest that ca + influx may be involved in the unpolarized non-vesicular release of fgf- . in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that -ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca + ] i ) in pc cells. however, it is unclear how [ca + ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca + ] i on actin dynamics in pc cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a . treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to -ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp is endogenously expressed in pc cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp in actin dynamics in growth cones and found that knockdown of clp expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp deletion mutants that both of pdz and lim domains are necessary for the proper function of clp . ps a-e screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura , tsuyoshi kobayashi , yuchio yanagawa , kunihiko obata , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; grad. sch. of medicine, gunma univ., maebashi, japan; bsi, riken, wako, japan; sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the clones tested, two genes, neuronatin and seizure related gene (sez- ) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu , , tomoko matsumoto , , sakae mikami , takashi nagasawa , fujio murakami , grad. sch. of frontier biosci., osaka univ., japan; sorst, jst, japan; inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf- and its receptor cxcr in the precerebellar migration. we show that cxcr is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr and sdf- knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps a-e involvement of an immunoglobulin superfamily molecule, neph /mkirre in the migration of precerebellar neurons kazuhiko nishida , , kazuhide nakayama , saori yoshimura , , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph , neph /mkirre, and neph , only neph /mkirre was strongly expressed in pcns. expression of neph /mkirre was detected from e . when pcns migrate tangentially. the expression level became weaker at p , when pcns stop the radial migration, raising the possibility that neph /mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph /mkirre in the migration of pcns. hiroki umeshima , , toshio ohshima , tomoo hirano , mineko kengaku lab. for neural cell polarity, riken bsi, wako, japan; department of biophysics, kyoto university, kyoto, japan; lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk (cdk -dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin -gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk -dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito , , takahiko kawasaki , , tatsumi hirata , division of brain function, national institute of genetics, mishima, shizuoka, japan; department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc functions with unc- to regulate axonal transport in drosophila hiroaki mochizuki , hirofumi toda , , emiko suzuki , joseph gindhart , toshifumi tomoda , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; gene net. lab., natl. inst. genet. mishima; beckman res. inst., city of hope, ca, usa; dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc , which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc is essential for neural development in drosophila. loss of function of drosophila unc results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc genetically interacts with unc- , an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc mutants, unc- was separated from synaptotagmin vesicles. these results suggest that unc coordinates kinesin-cargo interaction via unc- to regulate dynamic axonal transport. ps a-e change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi , daisuke takahashi life science inst. sophia university, tokyo, japan; waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi ( ) and kakenhi on priority areas ( ) ps a-e generation and analysis of region-specific rac -deficient mice hidetoshi kassai , masahiro fukaya , eriko miura , mizuho sakahara , masahiko watanabe , , atsu aiba div. cell biol., kobe univ. grad. sch. med., kobe, japan; div. physiol. sci., hokkaido univ. grad. sch. med., japan rac is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac in the cns is poorly understood because of the embryonic lethality of rac knockout mice. in this study, we generated and analyzed region-specific rac -deficient mice (emx -rac ko mice) by the cre-loxp system, in which a promoter for emx homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx -rac ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx -rac ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map b containing nterminal amino acids promoted neuronal death. to reveal the mechanism of map b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map b by two-hybrid system. alpha-tubulin was found to interact with map b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map b n-terminal has not yet been reported. beta-tubulin was also found to interact with map b n-terminal. when ␤ tubulin was divided in fragment at between amino acid and , there was no interaction between ␤ tubulin fragments and map b n-terminal. interaction needs the continuous region over aa and . there were much proportion of round formed cos cells in n-terminal containing map b transfected cells than in n-terminal lacking map b transfected cells. there might be some interference in interaction between map b and tubulin in cells express map b containing n-terminal. otone endo , , masaaki mizuno , yasukazu kajita , jun yoshida department of neurosurgery, ja kainan hospital, aichi, japan; department of neurosurgery, nagoya university, nagoya, japan; department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta , masae yaguchi , yumi matsuzaki , yoshiaki toyama , yutaka kawakami , hideyuki okano , masahiro toda , neuroimmunology research group, keio univ., tokyo, japan; physiology, keio univ., tokyo, japan; orthopaedic surgery, keio univ., tokyo, japan; institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd c + dcs, cd ␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd ␣ + dcs compared to cd c + dcs. these results suggest that cd ␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps a-e von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo , hiroshi kanno , takaakira yokoyama , shuichi nakano , naoki sugimoto , nahoko kobayashi , tetsuhiko yoshida , isao yamamoto dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; fiber and dept. of chemistry, konan university, kobe, japan; toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly %, and peptide with the elongin c binding site (residues - ) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps a-e the effect of pueraria mirifica on erk / and s- following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e ) on the expression of erk / and s- following sciatic nerve crush and transection in rats. protein levels of perk / and s- in distal segments of nerve at day were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e , caused a significant decrease in the expression of perk / levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk / phosphorylation. following treatments, levels of s- were significantly decreased both in crushed and transected nerves with respect to control group at p < . . this estrogenic effect was blocked by ici , . because of their structural similarity to e , pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day . this study suggested that pm as well as g could enhance nerve regeneration like e by interfering with the injury-induced erk signaling pathway. yasuhiro kato , takafumi suzuki , kunihiko mabuchi department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf- and stat , which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p - wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y , a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin ( ht) positive cells increased in number progressively from day to weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and ht cells surrounded the axons and glia. thus, ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda , yukio someya , ryo kadota , chikato mannoji , tomohiro miyashita , atsushi murata , masashi yamazaki department of orthopaedic surgery, togane hospital, department of ortopaedic surgery, graduate school of medicine, chiba university, japan; division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt ( . mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), -bromo- -deoxyuridine- -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps a-f early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski , hanna kozlowska , marcin jurga , aleksandra habich , elzbieta wanacka , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed h, h, h and days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue , , and days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext , one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican- and syndecan- were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe , hiroe sagawa , masahiro ichikawa , yoshihito tokita dept. perinatol., int. dev. res., kasugai, japan; dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y , can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for d. after fixation, the neurites were stained with anti-tuj antibody to obtain number and length of tuj neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day , wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of -days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of -days-old embryonic mice by culturing in dmem/f medium for days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of - div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for - div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for days. dha increased the number of tuj (+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi ( ) ps a-f interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps a-f effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the d structure of surrounding stimuli defined by binocular disparity. ps a-f rotational motion aftereffect in positive direction for -dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for -dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known dimensional mae. this finding suggests that the visual system has a mechanism that detect -dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j takanori uka, ryo sasaki department of physiology , juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch ( deg) of a center/surround display presented degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always percent. for each of subjects, we calculated direction discrimination thresholds (at % correct) at each surround size. consistent with crowding, thresholds increased when surround size was . and degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was and degrees, relative to degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps a-f generation of receptive fields in higher visual areas based on v columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v and v , respond to stimuli with complex shapes. how do these neurons integrate signals from v ? in particular, does the well-known columnar organization of v play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about % of model neurons exhibit responses similar to those of v and v neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a -d surface slant is perceived (blakemore, ) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa ) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext( ), jsps( ), coe ps a-f consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi , kota sasaki , izumi ohzawa , grad. school of frontier biosci., osaka univ., japan; grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and -d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by -d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext( ), jsps( ), coe ps a-f firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate , , hiroshi tamura , , ichiro fujita , graduate school of frontier biosciences, osaka university, osaka, japan; jsps, japan; crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi - ps a-f spatial-frequency dependency of receptive field size and surround suppression in lgn and v hironobu osaki , tomoyuki naito , osamu sadakane , masahiro okamoto , hiromichi sato , med. sch., osaka univ.; grad. sch. med., osaka univ.; grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v ), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v (n = ) and lateral geniculate nucleus (lgn) (n = ) in anesthetized cats. first, we found that v neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura , akichika mikami , kazuo itoh department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v a, we injected a bi-directional tracer biotinylated dextran amine into the v a. our results indicated that the v a has connections with the occipital, parietal and temporal cortices. the v a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v a has commissural connections with the v , the v a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v . these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps a-g activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera ( fps), and were also monitored with a d or d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity - ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps a-g timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki , , arman abrahamyan , catherine stevens , andreas a. ioannides , brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately ms (n ) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto , shoutaro akaho , kenji fujikumi , yasuko sugase-miyamoto , masato okada aist, ibaraki, japan; ism, tokyo, japan; university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of neurons for visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps a-g relationship between color and shape selectivity in area teo of the monkey masaharu yasuda , , hidehiko komatsu , national institute for physiological science, okazaki, japan; sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps a-g neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a -choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was . , a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across - simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant ( ) ps a-g behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki , atsuko komori , tomonori manabe , toshihiko hosoya , hiroshi sagara , emiko suzuki , hitoshi okamoto , ichiro masai masai initiative research unit, riken, wako, japan; riken bsi, wako, japan; ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde c activity is important for the sensitivity of vision. ps a-g localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: - years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from to years of age, and showed a decline and lack of ir in older retinas ( - years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from to years of age, and absent at years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps a-g temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa , norio fujimaki , toshihide imaruoka nict, kobe, japan; kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak ( , ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity ( , ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano , , derek arnold , alan johnston , tsunehiro takeda univ. tokyo, chiba, japan; ntt cs lab., kanagawa, japan; univ. sydney, sydney, australia; ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, ) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around hz and matched with the alpha frequency of meg. as hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps a-g the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya , , kei ito , , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps a-g dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi , , toshiki tani , shigeru tanaka , graduate school of life science & systems engineering, kyushu institute of technology, japan; laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of weeks. we also found that when the goggles were removed after or weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, ) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than . hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps a-g retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., . neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada , yasuhiro enami , hiroshi jouhou , takehiko saito , kaj djupsund tokyo metropol. univ., hino, tokyo; astellas pharma. inc., osaka, japan; suny upstate med. univ., center for vision and ophthal., ny, usa; univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph only . units from the baseline of . , we found a remarkable reduction of the conduction velocity by - %, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps a-g analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi ( ) ps a-g stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of % ( of ) of neuron pairs (inter-neuronal distance > m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in % ( / ) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii , , toshihiko hosoya bsi, riken, japan; dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v ). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v . this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v neurons. masayoshi tsuruoka , masako maeda , bunsho hayashi , ikuko nagasawa , tomio inoue dept. physiol. showa univ. sch. dent. tokyo, japan; dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t -l dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention ( mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil ( %, ml). the increased emg activity significantly reduced following bilateral l -s ventral rhizotomies. a baseline emg did not significantly alter when the l -s ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps a-g hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba ) was significantly greater (p < . , uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < . , uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm agonist (l-menthol: . - mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = ) or boo rat (n = ). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm on the urothelium cell of the urinary bladder. ps a-g caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito , a.d. craig dept. physiol, shimane univ. sch. med., izumo, japan; atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol ) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns ps a-g ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps a-h fasting induced long-chain fatty acid receptor gpr expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor (gpr ) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of h fasting on gpr expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. h fasting induced gpr mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr -like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr at least fasting period. ps a-h ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that hydroxybutylate ( hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls ( v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of v respond to the change in hb. change in the intracellular calcium concentration ([ca + ] i ) in vitro was measured in dispersed ependymocytes taken from the v in rats. the present results showed that the [ca + ] i increased in response to hb, but the increase was blocked by ␣-cyano- -hydroxycinnamic acid, which is a monocarboxylate transporter (mct ) inhibitor. immunohistochemistry showed that mct -immunoreactivities were located on the v ependymocytes. these results indicate that the ependymocytes may sense hb through a mct -dependent mechanism. research funds: kakenhi ps a-h comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin ( . mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with -hydroxydopamine ( -ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at weeks after -ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at mm; anteroposterior, + mm; lateral and − and − mm; dorsoventral from bregma at weeks after -ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better -ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: ( ) retrieving the food with a rake (a motortool), ( ) retrieving the hidden food with a mirror-attached rake, ( ) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, ( ) moving a mirror hung along the rail by hand to find the food, ( ) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada , , shigehiro miyachi , michiko imanishi , masato taira , masahiko takada div. applied system neurosci., nihon univ. sch. med., tokyo, japan; dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area ( m), dorsal area ( d), and ventral area ( v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from m, d, and v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that m, v, and d receive multisynaptic projections from the entorhinal cortex, area , and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps a-h new neural activities of reward anticipation and task errors h. ogawa , h. ifuku , t. nakamura , s. hirata kumamoto kinoh hosp, kumamoto, japan; fac educ, kumamoto univ., kumamoto, japan; nat kikuchi hosp, kumamoto, japan; dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, & - ) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca s after led offset at correct trials. relations between cues and responses were reversed. of reward-related neurons found, . % showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = ) only at correct trial, i-type (n = ) at around possible reward onset only at incorrect trials, and c-i type (n = ) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai , takayuki hosokawa , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps a-h neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue ), and centrally allocated one of the objects presented in cue (cue ). the frequency of which object was presented as cue , i.e., task context, was manipulated across blocks. we focused on cue period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps a-h anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki , barry j. richmond , munetaka shidara , grad. sch. of tsukuba univ., ibaraki, japan; neurosci. ri., aist, tsukuba, japan; lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in conditions: ( ) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], ( ) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition / neurons responded in all trials. in the certain condition / neurons responded in the rewarded trials only. of these showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas ), aist masamichi sakagami , , kosuke sawa , xiaochuan pan , bsrc, tamagawa university, tokyo, japan; senshu university, kanagawa, japan; presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a -b -c and a -b -c . after monkeys could acquire the sequences, new pairs of stimuli (i.e., d and d , e and e , etc) were introduced to associated with b or b (d -b , d -b , etc). the asymmetric reward rule was instructed by pairing c (c or c ) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a and a , which were associated with c and c in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi ( ), hsfp, presto, jst ps a-h reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan , , kosuke sawa , , masamichi sakagami , bsrc, research institute, tamagawa university, japan; presto, jst, japan; department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a -b -c and a -b -c . the asymmetric reward rule was instructed by pairing c (c or c ) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of neurons from the lateral pfc, % showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about % of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps a-h neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima , hajime mushiake , jun tanji department of physiology, tohoku university school of medicine, sendai, japan; brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the -sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps a-h neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. ) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = ) preferentially when the instruction was associated with small reward. the instructions activated groups of putamen neurons preferring small reward-(n = ), large reward-action (n = ) and both types of action (n = ). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by - and - ms, respectively. putamen neuron activation lead that of cm neurons by - ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi ( ) ps a-h encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t ), second (t ) and third (t ) choices with reward probabilities of about , and %, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r , %). most da neurons ( / ) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t to t and to t trials, then decreased in r trial. in another task with two repetition trials (r and r , %), magnitude of start-cue responses decreased gradually from t to r and to r trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi ( ) ps a-h striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada , , hitoshi inokawa , minoru kimura dept. of physiol. kyoto prefect univ. med., kyoto, japan; jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded striatal projection neurons from a monkey. after depressing a start button, the monkey chose of target buttons with correct rates at st, nd, rd and repetition trials of , , and %, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice ( / ) rather than to upcoming choice of target ( / ). during the target choice, another subset of neurons showed firings selective to choosing particular target ( / ) rather than to expected reward probability ( / ). after the target choices, another group of neurons fired related to expected reward ( / ) rather than to chosen action ( / ). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi ( ), jsps fellows ps a-h encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose of target buttons at average correct rates of (first), (second), (third) and % (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans ( / ) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi ( ) ps a-i representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda , k. samejima , k. doya , m. kimura dept. physiol., kyoto pref. univ. med.; brain sci. res. center, tamagawa univ.; irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of , or %. a linear regression of neuronal discharge rates showed: neurons encoded reward values of either action during delay period before go signal, with most ( %) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = ), while other neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi ( ) ps a-i delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c ), first delay (d ), second cue (c ), second delay (d ), and response periods. activity during the d and d periods was analyzed in this study. firing rates during the d period linearly depended on c durations. on the other hand, d period activity depended on trial types (ls and sl), but not on the variety of c durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi ( ) ps a-i neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku , ryoi tamura , teruko uwano , taketoshi ono dept. integrative neurosci., univ. toyama, toyama, japan; dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi ( ) ps a-i reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori , mikio inagaki , wu lisa , taijiro doi , eishi hirasaki , hiroo kumakura , ichiro fujita osaka univ., japan; massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from and takeda science foundation ps a-i response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, ( ) performance of visually guided sex discrimination task and ( ) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) ( ) (sa) and coe program in kit from the mext ps a-i activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa , satoshi katai , masanori saruwatari , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro ) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto , tetsuya iidaka department of psychology, nagoya university, nagoya, japan; department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: - hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the - ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the - ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included images ( objects × views). novel objects were generated by deforming a prototype in four directions. for deg-interval object sets, views were obtained by rotating each object with the interval of deg, deg-interval set and deg-interval set were with deg and deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across deg separated views was significantly higher in the trials with deg-interval sets than those with deg-interval sets. similarly, the difference was also found in the performances across deg separated views between those with deg-interval sets and deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps a-i brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps a-i obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps a-i sirna-induced nr knockdown causes hypofunction of nmda-r and cognitive deficit m. saji , , t. utida , a. ohnishi , k. noda , m. ogata , h. akita , n. suzuki , physiol, health sci. sch. kitasato univ., sagamihara, japan; brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr using hvj-envelope vectors. we confirmed that marked down-regulation ( %) of nr expression occurred only in the hippocampus among various brain regions - days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr knockdown, the nr down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr knockdown, the nr down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka , , hiroshi ageta , ikuko yao , kaoru inokuchi , yutaka kirino , mitsutoshi setou , graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; mitsubishi kagaku institute for life sciences, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps a-i decreased interneurons in the pax mutant mouse limbic system hasumi haba , tadashi nomura , yoshinobu hara , , noriko osumi , div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at weeks, but not at weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp ) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine ( . mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: , ps a-i gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba , , satoko hattori , hiroaki hori , tetsuo nakabayashi , hiroshi kunugi , ryota hashimoto department of mental disorder research, national institute of neuroscience; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of patients with schizophrenia and age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: , ps a-i genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto , hiroko noguchi , hiroaki hori , tetsuo nakabayashi , satoko hattori , sachie chiba , seiichi harada , osamu saito , hiroshi kunugi department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; musashi hospital, ncnp, kodaira, japan; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in healthy volunteers and patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: , ps a-i detection of f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura , nicola bright , toshio yanagida , masatoshi takeda , paul grasby department of physiology, osaka university, japan; department of psychiatry, osaka university, japan; cyclotron unit, imperial college, hammersmith hospital, uk we used f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and f-dopa scans were obtained from a database of scans published in mcgowan et al. , archives general psychiatry. the schizophrenic patients all met dsm-iv criteria on medication and healthy volunteers were compared. we attempted to improve the quality of the f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > . ). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p component of the visual eventrelated potential in patients with schizophrenia and healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p amplitude between the faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area between the faces in controls, while in patients, there was no significant activity difference between the faces. stimulation with crying face induced higher activities in the and right areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps a-j inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura , shoji tanaka , koki yamashita , motoichiro kato , haruo kashima department of neuropsychiatry, school of medicine, keio university; department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps a-j pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc , a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as -ht are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in of the sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps a-j alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta , ryo yamamoto , shigeki sugiura , kaoru inokuchi , nobuo kato dept. integrat. brain sci., grad. sch. med., kyoto univ.; nara med. univ.; mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer a/vesl- s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer a protein by diffusion from patch pipettes. homer a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer a may be a biological mediator of ecs effects. masanori kasai , nozomi miyagi , norio kawashiro , daisuke torizuka dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant ( . mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the nd, th, th, st, th and th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc ( . ± . g/dl, n = ) on the the day was significantly lower than that in intact rats ( . ± . g/dl, n = ). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps a-j molecular cloning of a novel candidate for ethanol-responsive genes, yy ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy ap (yy -associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy -binding and transactivation domains previously demonstrated in yy ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy genes was demonstrated in the same neurons. analogous to yy ap as a co-activator of transcription factor yy , it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps a-j excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr b-selective nmda antagonists ifenprodil ( and m) and ro - ( . and m) markedly inhibited degeneration of orexin neurons induced by h application of nmda ( m) or qa ( . mm). we also show that stimulation of gaba a receptors by muscimol ( and m) or isoguvacine ( and m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba ( m) plus a gaba uptake blocker nipecotic acid ( mm) was abolished by a gaba a antagonist picrotoxin ( m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh ). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps a-j a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi , osamu fukayama , takashi sato , takafumi suzuki , kunihiko mabuchi , dept. biomed. eng., univ. tokyo, tokyo, japan; dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps a-j on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki , yasuhiko jimbo , kenji yasuda department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are khz in ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita , tetsu okumura , kazuo okanoya , jun tani lab. for behavior & dynamic cognition, riken-bsi, japan; lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps a-k local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura , yuichi yamashita , kazuo okanoya , jun tani behav & dynamic cognition, riken-bsi, saitama, japan; biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata , shinya ishii , munetaka shidara dept. of e&e engineering, oita univ., oita, japan; grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [ ] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki , shinya yamamoto , sunao uchida , shigeru kitazawa , faculty of hum sci., waseda univ., tokorozawa, japan; neurosci. res. inst, aist, tsukuba, japan; faculty of sport sci., waseda univ., tokorozawa, japan; dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ± ms, s.d. = ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi ( ) ps a-k single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama , fumiko sato , ken nakazawa , kenichi ono chiba university, japan; field of home economics, faculty of education, chiba univ., japan; department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm- , shi-madzu) over the frontal area ( channels) for volunteers ( - y). thirty tasks were presented visually every s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was − . +/− . between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi , , kosuke hamaguchi , masato okada , , department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; jst, japan; riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi ( ) and ( ) ps a-k auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa , , sachiko koyama , , takashi omori , takashi morotomi research institue for electronic science, hokkaido university, sapporo, japan; japan science and technology agency, saitama, japan; graduate school of information science and technology, hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = ). the erps were elicited by tones ( hz) with different durations ( , , , , , ms) and gaps ( , , , , , ms) embedded in a continuous pure tone ( hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for and ms were high both for the tones ( ms, %; ms, %) and the gaps ( ms, %; ms, %). ps a-k determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps a-k theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to % till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, , -diphenyl- , , -hexatriene, and -[ (trimethylammonio)phenyl]- -phenyl- , , -hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp expression correlates with wnts/bmps in the cortical hem, sp with fgfs in the cop, and sp with shh in the ventral midline and mge. by using in utero electroporation, we show that sp regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp and fgf exhibit reciprocal induction, indicating that sp is a positive feedback regulator of fgf . surprisingly, though, ectopic expression of both sp and its dominant active form shift cortical areas in the opposite manner to fgf , suggesting that sp activates additional targets that overcome fgf function. our results indicate that fgf is an additional target of sp , showing effect on patterning similar to sp . these findings indicate that sp balance the proper cortical arealization through fgf and fgf . research funds: nihr ns ps p-c fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira , kotaro hattori , takeshi yagi , shigeki yuasa dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi , kazuko keino-masu , tatsuyuki ohto , satoshi kunita , satoru takahashi , masayuki masu dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp -deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp /sulfatase- and sulffp /sulfatase- , which have hs -o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp and sulffp . here we report the isolation and the characterization of the third homologue, sulffp . sulffp has about % and % overall amino acid homology with sulffp and sulffp , respectively. at h postfertilization, sulffp is expressed in the ventral region of spinal cord, whereas sulffp is expressed only in the floor plate and sulffp is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp was shown to mediate the repulsive effect of sema a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp , , , and are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps p-c developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa , n. yamamoto , m. yoshimoto , t. yasuda , k. maruyama , t. kage , h. takeda , h. ito nat. inst. rad. sci., chiba, japan; nippon med. sch., japan; tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax -or dlx -positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er homologue (zfer ), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is % identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta , , hiroo takahashi , , takafumi shintani , , kazuma etani , masaharu noda , div. of mol. neurobiol., nibb, okazaki, japan; crest, jst, japan in the developing chick retina, the expression of bmp is relieved by that of bmp at around e with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina , are expressed in a double-gradient fashion from e on like ventroptin and bmp . when bmp expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp knockdown and ephrina -misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp to bmp thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en , wnt b and pcp /l gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and di- asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and di- asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or di- asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru , , takahisa taguchi aist, ikeda, japan; presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a -dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps p-d laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps p-d synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki , toshio aoyagi department of physics, kyoto university, kyoto, japan; graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf- mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into weekold wistar strain rats in several doses followed by successive injections of brdu for the next days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps p-d the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato , james e. goldman division of pharmacology, national institute of health sciences, tokyo, japan; department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p rat svz, sagital sections of forebrain were made at p and cultured for days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣ integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps p-d localization of neurotrophin receptors trka in pc cells: d reconstruction analysis of membrane proteins tomoki nishida , hiroshi jinnai , tatuo arii , akio takaoka , ryoichi yoshimura , yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan; department of polymer science and engineering, kyoto institute of technology, kyoto, japan; national institute for physiological sciences, myodaiji, okazaki, japan; osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin- in pc cells, analyzed by high-voltage electron microscopy, and reconstructed d structure of their subcellular distribution by imod. our results indicated that localization of caveolin- , known as an integral membrane protein of caveolae, was never found in the invagination structure in pc cells, but trka and caveolin- immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd , a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd in the wnt-mediated neuronal development. there are three subtypes of mouse ccd gene products, ccd a, ccd b and ccd c, which are generated by different promotor usage. mouse ccd a as well as zebrafish ccd a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi ( , ) ps p-d analysis of a role of r-spondin on proliferation of the cortical neuroepithelium yumiko hatanaka , masahiro yamaguchi , fujio murakami , masayuki masu grad. school of comprehensive human sci., univ. of tsukuba, japan; grad. school of med., univ. of tokyo; grad. school of frontier biosci., osaka univ r-spondin (rspo ) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. ) . rspo is expressed in the developing medial cerebral wall and transgenic mice expressing rspo in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. ) . since wnt a has a role for expansion of caudomedial cortical progenitor cells (lee et al. ) , these findings lead us to the idea that rspo may synergistically promote proliferation of cortical neuroepithlial cells together with wnt a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day . mouse. an application of wnt a on these cells increased level of the reporter expression, and an addition of rspo further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt a and rspo directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il- ␤, il- , lif, gdnf) or lowered o ( . %), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells ( . -fold) as compared to normal o . cytokine cocktail significantly increased th (+) cells ( . -fold) compared to nontreated control. treatment of lif and il- ␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps p-d structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui , terukazu nogi , mitsuharu hattori , kenji iwasaki , , junichi takagi research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer and vldlr), resulting in tyrosine phosphorylation of dab . in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic d reconstruction of four-domain reelin fragment (i.e. r - ) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r - ) was capable of binding to the receptor (apoer ), and was also able to induce tyrosine phospholylation of dab in primary neuronal culture. ps p-d effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax and pax compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps p-d neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res : , ) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi- k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps p-d fabp is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa , miho matsumata , , yuji owada , shigeki yuasa , noriko osumi , natl. inst. of neurosci., ncnp, tokyo, japan; tohoku univ. sch. of med., sendai, japan; crest, jst pax transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp , a downstream of pax , regulating maintenance of embryonic neural stem/progenitor cells (arai et al., ) . fabp was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). % of fabp -expressing cells co-expressed gfap (a marker for early progenitors), and % of them co-expressed psa-ncam (a marker for late progenitors). fabp expression was also overlapped with pax , and expression of fabp was down-regulated in the dg of pax deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp knockout mice. taking all together, it is concluded that fabp is required for maintenance of neural stem/progenitor cells in dg. ps p-d involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day - . sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani , , shiki okamoto , masato imada , kouichi itoh , atsushi irie , hitoshi sakuraba , hideo kubo department of molecular biologu, ohu univ., koriyama, japan; dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; dept. anatomy, nihon univ. shl. med., tokyo, japan; dept. mol. pharma., univ. tokushima bunri, sanuki, japan; dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam- shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam- is possible. in the present, we show that the isolated randam- high+ cells enrich nscs. the randam- high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam- low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam- is a usefule marker for the isolation of nscs by facs. yasuharu takamori , yasuhisa tamura , , yosky kataoka , , yilong cui , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b and b are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b (++), b (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b (+++), b (+), and mature neurons possess lamin a/c (++), b (+), b (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka , yuichi tanaka , tatsuhiro hisatsune department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps p-e new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba , , hideo namiki , tatsunori seki dept. of anat, juntendo univ. sch. of med., tokyo, japan; integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p . three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., ) . the dividing pattern is currently under investigation. akiya watakabe , noritaka ichinohe , sonoko ohsawa , tsutomu hashikawa , kathleen s. rockland , tetsuo yamamori div. of brain biol, nibb, okazaki, japan; lab. for cortical organization and systematics, bsi, riken, wako, japan, lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer neurons in several areas of monkey neocortex. we previously reported that nurr , ctgf and sema e mrnas are specifically expressed in subsets of layer neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer . by double ish, layer neurons in monkeys are roughly divisible into cck(+) and sema e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi ps p-e rbp-j regulates the cortical laminar formation kenji tanigaki , kazue muraki , norio yamamoto , tasuku honjo shiga medical center, research institute, shiga, japan; department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps p-e search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi , tomoko ikawa , yuya kasukawa , hironori ueda , , kazuki kurimoto , michinori saitou , fumio matsuzaki , lab. for asymmetric cell division, cdb, riken, kobe, japan; functional genomics subunit, cdb, riken, kobe, japan; lab. for systems biology, cdb, riken, kobe, japan; lab. for mammalian germ cell biology, cdb, riken, kobe, japan; crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi ( ) ryosuke tatsuno , tomoaki sai , , masahiro otsu , kuniko akama , takashi nakayama , tosifusa toda grad. sch. of sci. and tech., chiba univ., chiba, japan; lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; dept. orthop. surg., jikei univ. sch. med., tokyo. japan; dept. biochem., yokohama city univ. sch. med., yokohama, japan; proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using -d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps p-e identification of se , a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se in developing and matured cns. teiichi furuichi , akira sto , , yukiko sekine , noriyuki morita , tetsushi sadakata , satoshi shoji , jin-hong huang , toshio kojima laboratory for molecular neurogenesis, riken brain science institute, japan; comparative systems biology team, riken genome sciences center, yokohama - , japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki , makoto mizuno , kazuhisa sakai , yoshimoto kiyohara , kazuhiko yamaguchi , tsutomu hashikawa , hiroyuki nawa institute of biomedical engineering, waseda university, tokyo, japan; department of molecular neurobiology, brain research institute, niigata university, niigata, japan; laboratory for memory and learning, bsi, riken, saitama, japan; laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg knock out mice (nrg type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps p-e psd- protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama , , satoshi wakisaka , shiho honma , akira ito , , kei azuma , , mikihiko kogo department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd- implicates in formation and maturation of excitatory synapses. psd- regulates the localization of the nmda receptor by means of binding with nr . rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd- protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd- expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf . in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps p-e ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps p-e comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi , kenji mizutani , hiroto ogawa , kohji hotta , kotaro oka ctr. for biosci. and info, keio univ., yokohama, japan; neuro, karolinska inst, stockholm, sweden; bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion ( % or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps p-e three dimensional ( d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in monkeys. of a total of p-cells that were examined during both frontal and vergence-pursuit, % discharged for both, % only for vergence, and % only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had d pursuit signals. majority ( %) of these p-cells discharged before onset of vergence eye movements with the typical lead time of ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi ( ) ps p-e information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey , rr aya takemura , yumi murata , , kenji kawano , neurosci. res. insti, aist, tsukuba, japan; dept. integ brain sci., grad. sch. med., kyoto univ., japan; grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid ( mg/ml, - l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps p-e the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara , kenji kunita , kaoru maeda , takeo kiyota department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for ms with -ms intervals. the oscillation trial ( . -hz frequency and . -cm amplitude) ( - s) was repeated times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the th trial. a significant correlation was shown between th- st trial differences in mean speed and vep amplitude (r = . ). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps p-e primary motor cortex contributes to generating manual following response toshitaka kimura , naoki saijo , hiroaki gomi , ntt cs labs, kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m ), that ( ) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and ( ) intracortical inhibition within m assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m . such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in radial directions applied during reaching. the data show that the direction of the initial phase ( - ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase ( - ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps p-e spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps p-f influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka , takashi obu , kazuyuki kanosue asmew, waseda university, saitama, japan; graduate school of human sciences, waseda university, saitama, japan; faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = ) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at . , . , and . hz. over % success rate was observed in all tasks except oppo ( - %). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps p-f simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu , tetsuro muraoka , kazuyuki kanosue , , faculty of human sciences, waseda university, saitama, japan; faculty of sport sciences, waseda university, saitama, japan; asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at , . , and . hz. they were asked to perform tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps p-f activities of erector spinae muscles during jaw clenching in man kayoko yasunaga , , tadachika yabushita , kazuo toda , kunimichi soma orthodontic science, tokyo med. & dent. univ., tokyo, japan; div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada , sachiko motoyama , futoshi mori , shigemi mori department of veterinary physiology, yamaguchi university, yamaguchi, japan; national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for - weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps p-f neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima , futoshi mori , akira murata , masahiko inase dept. of physiol., kinki univ. schl. of med., osakasayama, japan; dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi ( ) ps p-f activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara , , nobuhiko hatanaka , , masahiko takada , atsushi nambu , school of life science, the graduate university for advanced studies, japan; division of system neurophysiology, national institute for physiological sciences, japan; tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu , sei-ichi sasaki , yuichiro cho , kenji sato anatomy and physiological science, tokyo medical and dental university, tokyo, japan; department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s and s spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl -s spinal segments which contain afferent fibers of eas muscles. after weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for consecutive days. we compared adaptation in day and that in day . the gain decrease for the first saccades in day ( . ± . ) was larger than that in day ( . ± . ) (p = . , n = , paired-t test). the rate of adaptation in day ( . ± . × − /sac) was higher than that in day ( . ± . × − /sac) (p = . ). the overall gain change ( saccades) in day ( . ± . ) was larger than that in day ( . ± . ) (p = . ). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi ( ) ps p-f asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v . the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = ) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in % of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida , xiaofeng lu , , shogo ohmae , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi ps p-f frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps p-f projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki , , kei ito , , dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; center for bioinform., imcb, univ. of tokyo, japan; bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening , strains, we identified about candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps p-f immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura , yoshitaka ohtubo , takashi kumazawa , kiyonori yoshii graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi ( ) and the st coe program (center # ) granted by mext of japan ps p-f inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa , atsushi kuhara , hitoshi inada , eiji kodama , takafumi mizuno , ikue mori , lab. of mol. neurobiol., nagoya univ., japan; institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx- gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx- mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx- mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps p-f postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut and - ) during rat ob development from postnatal day (p ) to p by in situ hybridization and immunohistochemistry. at p vglut immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut mrna signals were detectable in the mitral cell layer from p to p . in contrast, vglut ir was prominent in the glomerulus at all days examined, and only at p and p in mitral cells. despite mitral vglut ir disappeared at p , the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps p-f v r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito , , masumi ichikawa , yuji mori , kimiko hagino-yamagishi tokyo metrop. inst. med. sci., tokyo, japan; the univ. of tokyo, tokyo, japan, tokyo metrop. inst. neurosci., tokyo, japan in rodent, v r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v r gene, which is expressed in the olfactory epithelium (oe). to examine when the v rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified v r sequences. these v rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps p-f analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t r /t r heterodimer plays a role as a sweet taste receptor. but, mice lacking t r showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t r -independent sweetener binding site also exist in mice. in this study, to predict binding sites on t r /t r and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [( ) sucrose, glcose, fructose, ( ) saccharin, aspartame, acesulfame-k, glycine, ( ) d-phenylalanine, ( ) d-tryptophan, ( ) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t r and t r , respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu , sachiko saito , yuko murata , ding ming , tatsu kobayakawa , robert f. margolskee , yuzo ninomiya sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; saito sachiko taste and smell research institution, ibaraki, japan; national res. institute of fisheries sci., kanagawa, japan; dept. of physiol. & biophys., mount sinai sch. med., new york, usa; national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t r receptors which coupled with ggustducin. ps p-f newborn infant body odor attenuates their mother's postpartum moods shota nishitani , mayumi kokuryo , tsunetake miyamura , kazuyuki shinohara div. neurobiol. & behav., nagasaki university, japan; obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps p-f human prefrontal activity in taste encoding: an fnirs study masako okamoto , mari matsunami , haruka dan , tomoko kohata , kaoru kohyama , ippeita dan national food research institute, tsukuba, japan; nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = ) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes ( subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes ( subfamilies) were expressed in the dorso-medial zone (zone ) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone . these results indicate that there is a clear boundary between zone and non-zone areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi ( ) ps p-g taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to ( ) best-taste profiles and ( ) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi ( ) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼ members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi ( ) ps p-g identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka , kazuhisa sakai , tsutomu hashikawa , kensaku mori , masahiro yamaguchi dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps p-g nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi ( ), kakenhi ( ) ps p-g integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than % of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi ( ) ps p-g odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi ( gs ) ps p-g odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa , takaaki sato , akira shimizu , ken-ichiro tsutsui , toshio iijima div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range ( hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps p-g chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when m lysine was spotted on ammonium chloride background, . - . m and . m background did not influence lysine chemotaxis, while . m background augmented and . - . m background suppressed the chemotaxis. in contrast, when . m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps p-g glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu , , , takeshi ishihara , eisuke nishida , yuichi iino dept. biol., fac. sci., kyushu univ., japan; mol. genet. res. lab., univ. of tokyo, japan; grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr- , a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr- appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr- . in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr- . these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr- localization in aiy neurons. kohei ueno , yoshiaki kidokoro dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼ %), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal /uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps p-g spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki , graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps p-g medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka , yoshinori sahara , shinichiro horie , masumi ichikawa , shun nakamura st. marianna univ. schl. med., kawasaki, japan; ntl. inst. neurosci., ncnp, tokyo, japan; tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps p-g synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi ( ) ps p-g rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii , , ken yoshikawa , yuki takatori , koichiro kawashima dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca + signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca + signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m , m , m and m machr subtypes, and ␣ , ␣ , ␣ , ␣ , ␣ , ␣ and ␤ nachr subunits. transfection of anti-m , anti-m and anti-␣ small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca + signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m or anti-m sirna. ca + signals evoked by nicotine were reduced by anti-␣ sirna. these findings indicate that m , m machr and ␣ nachr subtypes play major roles in ca + signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi ( ) ps p-g is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto , sachiho kubo , seiji matsuda , naoki maruyama dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, ) research funds: kakenhi ( ) ps p-g dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day . electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day . analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c from kansai medical university ps p-h unusually folded sod species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno , yumiko simazaki , fuminori saitoh , ryosuke takahashi , toshiyuki araki national institute of neuroscience (ncnp), tokyo, japan; dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod protein is thought to be responsible for the selective loss of motoneurons in sod -related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod species are not yet clarified. since we have detected accumulation of unusual sod species in motoneuronal axons from g a sod -tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod species. immunoprecipitation analyses revealed specific interaction of unusual sod species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod -tg compared with wt sod -tg mice. these data strongly suggest that the toxic property of unusual sod proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c ( ) ps p-h relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately % of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day . the mice started to show ncl symptoms on p , and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from % to % of that in the wild-type mice. research funds: kakenhi ( ) ps p-h neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura , katsumi higaki , junichiro matsuda , yoshiyuki suzuki , eiji nanba division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; national institute of biomedical innovation, osaka, japan; clinical research center, international university of health and welfare, tochigi, japan g m -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm accumulation of lysosome and pm and the membrane fluidity was also reduced. gm -bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m -gangliosidosis. katsuya inoue , , katsuaki endo , takamitsu fujikawa , seijyun fukuda , tatsuo nakamura department of physical therapy, university of aino, osaka, japan; institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after min occulusion. ps p-h development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando , yuya kawanaka , minoru saito , hiroaki mochizuki , ken honjo , hirofumi toda , , toshifumi tomoda , akira sawa , katsuo furukubo-tokunaga grad. school of life & envir. sci., univ. tsukuba, japan; molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; beckman res. inst., city of hope. california, usa; dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia- (disc ) gene, originally identified at the breakpoint of a chromosome ( ; ) (q . ; q . ) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc functions in vivo, we expressed disc in drosophila and examined developmental and behavioral phenotypes. overexpression of disc resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps p-h effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake , ayumi kamo , kenji minato , hitoshi haruna , hiritsugu fukuda , yuji murata , takayoshi hosono department of bomedical engineering, osaka electro-communication university, japan; graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of of -day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing % oxygen at • c for min. after the insult, of the rats were put in a box at • c for h (hypothermia, h-group). the other rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps p-h tau hyperphosphorylation in ts cje, a partial trisomy mouse model for down syndrome ebrahim abdul , a. shimohata , w. yu , m. yamaguchi , m. murayama , d. chui , t. akagi , t. takeuchi , k. amano , h.s. karthik , t. hashikawa , h. sago , c.j. epstein , a. takashima , k. yamakawa research scientist; lab. for neural arch.; lab. for alzheimers disease; div. of fetal med. ncchd; ucsf, usa although down syndrome (ds) or trisomy is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts cje, a ds mouse model partially trisomic for chromosome , shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome q , has about genes. importantly, sod and app, which may contribute to the ds phenotype, are excluded from the ts cje trisomic segment. here we report that ts cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk ␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts cje segment other than app and sod can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: ps p-h increased oxidative stress and mitochondrial dysfunction in ts cje, a down syndrome mouse model atsushi shimohata , ebrahim a. s. , m. yamaguchi , w. yu , h. sago , c.j. epstein , k. yamakawa lab. for neurogenetics, riken-bsi, japan; div. of fetal med. ncchd, japan; dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome (hsa ) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome extending from sod to znf , containing genes syntenic with hsa . since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts cje brains. collectively, our results implicate dosage imbalanced genes other than sod and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts cje ds mental retardation-like phenotype. ps p-h polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan , , kouko tatsumi , takayuki manabe , takahira yamauchi , , eri makinodan , juro shimoda , toshifumi kishimoto , akio wanaka department of psychiatry, nara medical university, kashihara, japan; department of nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded ( q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. genes, encompassing around % of the drosophila genome, were screened, resulting in the identification of candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps p-h analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. ( ) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the , ,-cyclic nucleotide -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps p-h dysregulation of sodium channel ␤ subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤ (␤ ) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤ as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤ started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤ expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤ gene in pc cells. forskolin, an activator of the camp/pka pathway, did not affect b promoter activity, indicating that ␤ is not camp-responsive gene. these findings strongly suggest that sodium channel ␤ subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps p-h repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki , yuji takahashi , jun goto , mutsuo oyake , toshiya sato , shoji tsuji department of neurology, the university of tokyo, tokyo, japan; department of neurology, brain research institute, niigata university, niigata, japan; center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of (q ), (q ), (q ), or (q ). in the accelerating rotarod ( w), the latencies of q , q , q and q were %, %, % and %, respectively. in the open field, moving distances of q , q , and q were decreased to %, %, and %, respectively, while that of q was increased to %. home cage activity was decreased depending on the repeat length. the q mice, however, showed increased ratios of the activity during the light time to that during the total day at weeks ( %) and weeks ( %), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps p-h the mice lacking schnurri- show multiple behavioral abnormalities related to psychiatric disorders keizo takao , nobuyuki yamasaki , keiko toyama , tsuyoshi takagi , shunsuke ishii , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; riken, tsukuba, japan schnurri- (shn- ) is a zinc finger transcription factor, a mouse homologue of human hiv-ep , that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn- is known to play important roles in the mammalian immune systems. however, the role of shn- in the central nervous system (cns) is still unknown. to investigate the functional significance of shn- in mammalian brain, we analyzed the shn- knockout (ko) mice using a comprehensive behavioral test battery. shn- ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn- mice. our results demonstrate a critical role of shn- in cns and suggest that shn- ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca + /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi ( , , , ) , jst bird ps p-h effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c bl/ j, c bl/ n, c bl/ c, svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than mice (, including wild type and mutant mice from strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behaivoral analysis of ryanodine receptor type knockout mouse suzuko ohsako , koichi tanda , , nobuyuki yamasaki , keiko toyama , hiroshi takeshima , tsuyoshi miyakawa kyoto university graduate school of medicine, kyoto, japan; dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca + signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr deficiency, we subjected ryr knockoout mice to a battery of behavioral tests. ryr knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr in regulating general locomotor activity and social behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama , koichi tanda , , nobuyuki yamasaki , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi ( , , , ) , jst bird ps p-h primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi , keiko shimizu grad. sch. human and environmental std, kyoto univ., kyoto, japan; primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected -ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. -ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in -ohda injected monkeys. these results suggest that monkeys injected -ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi ( ) ps p-i training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata , , , noriyuki higo , , takao oishi , , , akiko yamashita , keiji matsuda , motoharu hayashi neurosci. res. inst, aist, tsukuba, japan; grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; crest, jst, kawaguchi, japan; dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext ( ) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about , g animals (dba/ j × enu-treated c bl/ j) for home-cage activity, open-field activity, and passive avoidance response, and obtained lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in of mutant lines. hyperactivity was predominant phenotype, and of mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps p-i ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., ) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg -knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., ) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc , and found that in our ncl model mice, positive signals for ubiquitin and lc were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)( ) and creative scientific research ( gs ) ps p-i activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama , eiichi jodo , yoshiaki suzuki , ken-yo hoshino , yukihiko kayama dept. of physiology, fukushima medical university, fukushima, japan; dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch , mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps p-i increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara , hiroyuki sakagami , hisatake kondoh , kohji fukunaga department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps p-i severity of audiogenic seizures is influenced by multiple factors in vlgr -mutated mice hideshi yagi , , makoto sato , division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr -mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c bl/ backcrossed vlgr -mutated mice and /svs backcrossed vlgr -mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr -mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps p-i reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota , ken kadota , department of bioenvironmental medicine, university of chiba, chiba, japan; chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of mg/kg of map i.p. for days (d d ) and then withdrawn from the drug for days (wd wd ). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg ) and mfc. the decrease in the density of pac was first observed in cg and then in mfc. the reduction began on d and advanced to higher levels on d and subsequently wd . these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg and mfc, particularly in layers ii and iii. ps p-i up-regulation of ␤ -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ -adrenergic receptor (␤ -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ -ar-immunoreactive cells were labeled with ki- , a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ -ar-immunoreactive cells also exhibited fgf- -ir. these findings suggest that ␤ -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf- . ps p-i effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita , norihiro muraoka , hiroshi hasuo , takashi akasu , minoru shigemori dept. of physiology, kurume univ. sch. of med., kurume, japan; dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh ). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity ( - v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was - v. the amplitude of slow peak in fpi group was about % larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps p-i comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca + imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca + imaging. the slices ( m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of ml/min. green autofluorescence (> nm) of the slices illuminated by blue light ( - nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca + imaging was also performed in the slices. slices were incubated in acsf medium containing m of fluo / am for h at • c. the ca + image was recorded with an excitation wavelength of - nmand an emission wave length of > nm. the autofluorescence and ca + responses wereobserved in slices perfused with l-glutamate ( mm). takuya hayashi , hiroshi sato , shinichi abe , takashi hanakawa , hiroshi watabe , hidenao fukuyama , babak aldekani , hidehiro iida department of investigative radiology, national cardiovascular center research institute, osaka, japan; human brain research center, kyoto university, kyoto, japan; nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area corresponded to that revealed by the tracer (mncl ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h -kokoro- ps p-i optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata , , yasutake mori , , tsuya taneda , , hiroaki okuda , , masaya tohyama , department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps p-i a model selection of glm applied to fmri data using aic jobu watanabe , fumikazu miwakeichi , andreas galka , , ryuta kawashima , tohru ozaki , sunao uchida , institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; department of medical system engineering, faculty of engineering, chiba university, chiba, japan; institute for statistical mathematics, tokyo, japan; institute of experimental and applied physics, university of kiel, keil, germany; new industry creation hatchery center, tohoku university, sendai, japan; faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for - min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps p-j flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata - , japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps p-j effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune , , takashi tsukiura , hiroko mochizuki-kawai , chisato suzuki , , toshio iijima neurosci. res. inst., aist, tsukuba, japan; div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps p-j different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno , kenji kansaku , yu aramaki , mitsuo kawato atr cns, kyoto, japan; institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = ) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, or yen is provided with a probability of . , but only with . if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < . ) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps p-j brain networks for communicative speech production: feeling inference and speech content production yuko sassa , , motoaki sugiura , hyeonjeong jeong , , keisuke wakusawa , , kaoru horie , shigeru sato , ryuta kawashima niche, tohoku university, sendai, japan; ristex, jst, tokyo, japan; miyagi university of education, sendai, japan; gsics, tohoku university, sendai, japan; department of pediatrics, tohoku university, sendai, japan; the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the st coe program ps p-j the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi , yasuhisa fujibayashi , yoshiharu yonekura , tatsuya asai department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps p-j imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai , kuniyoshi shinya , tetsuo araki , masahiro kusakabe , yasuhisa fujibayashi , yoshiharu yonekura department of nuclear power and energy safety engineering, university of fukui, fukui, japan; department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura , , winfried denk , michael hausser department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; university college london, london, uk; max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps p-k analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka , yasuhiro tanaka , takeshi kaneko , dept. of morphological brain science, kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad -gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after h incubation in nmdg-based solution than in nacl-based solution. cutting at • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at • c. the viability after h incubation was better kept at • c than at or • c. we thus recommend that slices be cut at • c in na-free solution, and incubated at • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi ( , , ) ps p-k contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh , yoshichika yoshioka faculty of engineering, iwate university, morioka, japan; iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal ( . ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals ( . , . , and . ppm). a phantom solution mimicking the normal condition (gsh:gssg = : ) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = : : : ), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was . mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area ) and the right superior and middle frontal gyri (brodmann area ) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps p-k cortical commissural connection in macaque and human callosum using diffusion mri rishu piao , takuya hayashi , hiroshi sato , shinichi abe , , takashi hanakawa , hidenao fukuyama , hidehiro iida national cardiovascular center, osaka, japan; human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps p-k optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong , niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the -d neural cortical activity using the -d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps a-c sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp ) nobuyuki karasawa , kazuko watanabe , keiki yamada seijoh universitry, tokai, japan, dept. physio., sch. med., gifu univ., gifu, japan, dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, , -diamino- -hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp ) mice. time course of immunoreactive intensity for serotonergic ( -ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that -ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and -ht neurons. katsuya yamada , , , yoshihiro matsumura , takashi miki , makoto wakui ␣-smooth muscle actin + arterioles were fewer in kir . (−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir . (−/−) barrel cortex than in wild-type one for short ( s, hz) but not long ( s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps a-h learning to use sensory-tools by japanese monkeys yumiko yamazaki , hiromi namba support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps p-h mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h receptor (h r) in cognition in physiological and pathological conditions by using h r mutant (h −/−) mice. in normal condition, several behavioral studies indicated h −/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h −/− mice. these results indicate h receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h −/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h −/− mice. mutation of h receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h r impairs cognition in normal conditions, whereas h r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi ( ; ) ps a-h -ht a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura , , hirohito-m. kondo , makio kashino , department of psychology, tokai women's university, kakamigahara, japan; ntt communication science laboratories, ntt corporation, atsugi, japan; shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of -ht neurotransmission. we examined whether this -ht a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the -ht a receptor gene polymorphism in impulsive behavior (nomura et al., ) , together with the present findings, this observation indicates the involvement of -ht a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps a-h role of cortical thin arterioles in neurovascular coupling; analyses of kir . -containing atpsensitive potassium channel-deficient mice ps p-g rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal , chinmay k. mukhopadhyay , birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india; special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv- and the direct infection of hsv- are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv- jrfl-infected macrophages or with hsv- . the generation of primary neurospheres did not suppressed by hiv- -infection or by hsv- infection at no more that moi . in the secondary neurospheres, cd + neural stem cells were intact in these infections, although beta- -tubulin + cells were decreased in hiv- infection and intact in hsv- -infection. in the differentiation assay, neun + nfp + neurons in hiv- -infection and gfap + s + astrocytes in hsv- infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv- infection and in hsv- infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv- and hsv- infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi grant-in-aid for young scientists(b) ps p-g effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga , , taeko yamauchi , toshihiro konagaya , hideki ohira department of psychology, nagoya university, japan; department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka , shen jin-song , saburo saito , shigeki yuasa dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori , tomoko yamamoto , kiyoshi matsumura , hiroshi hosokawa , shigeo kobayashi dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e (pge ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge but also induce an enzyme cyclooxygenase- (cox- ) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox- , and whether cox- is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox- was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox- specific inhibitor, ns- . these results indicate that aa itself or its metabolites induces cox- that accelerates the formation of pge from aa, and, hence, enhances fever. mitsunari abe , tatsuya mima , shinichi urayama , toshihiko aso , nobukatsu sawamoto , hidenao fukuyama human brain research center, kyoto university graduate school of medicine, japan; nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at . hz ( min; % of the rest motor threshold) applied over the left primary motor area (m ). we scanned sets of dwi (before, and min, min and min after rtms) using -t mr scanner, and calculated apparent diffusion coefficient (adc). in out of subjects, the adc decreased (mean . × − /mm s − ) in the left m just after the rtms, which recovered at min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v and v a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v a located more dorsal has a larger receptive field size then v . both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v and v a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v and v a.ps p-i measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro -channel magnetometer consisting of a × matrix of superconducting quantum interference device (squid) with its integrated base of . × . mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with - ms peak by the white led flashing stimulation and the aef wave with - ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of bits was used, along with a "blue dye" (rh ) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were . - . % of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto , yoko hoshi , chie sato , susumu terakawa tokyo institute of psychiatry, tokyo, japan; photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals ( and nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around -ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps p-j multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh , a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a -s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba ), superior occipital gyrus around the parieto-occipital sulcus (ba ) and superior parietal lobule (ba ). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps p-j influence of task difficulty during meter inspection: an fmri study naoki miura , , makoto takahashi , jobu watanabe , , shinya uchida , , shigeru sato , kaoru horie , masaharu kitamura , toshio wakabayashi , katsuki nakamura , , ryuta kawashima crest, jst, kawaguchi, japan; niche, tohoku univ. sendai, japan; graduate school of engineering, tohoku univ. sendai, japan; bme institute, waseda univ., tokyo, japan; idac, tohoku univ., sendai, japan; lbc research center, tohoku univ., sendai, japan; department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps p-j neural connectivity among brain areas related to language function shinya uchida , , , naoki miura , , jobu watanabe , shigeo kinomura , kazunori sato , yasuyuki taki , kentaro inoue , ryoi goto , ai fukushima , kaoru horie , shigeru sato , katsuki nakamura , hiroshi fukuda , ryuta kawashima department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; niche, tohoku university, sendai, japan; national institute of neuroscience, ncnp, kodaira, japan; japan science and technology agency, kawaguchi, japan; bme institute, asmew, waseda university, tokyo, japan; graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than % of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa , , motoaki sugiura , yuko sassa , , hyeonjeong jeong , , kaoru horie , shigeru sato , hiroyuki yokoyama , kazuie inuma , ryuta kawashima niche, tohoku univ., sendai, japan; department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; miyagi univ. of education, sendai, japan; ristex, jst, tohoku univ., sendai, japan; gsics, tohoku univ., sendai, japan; lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda , yasumasa okamoto , kazuhiro shishida , akiko kinoshita , shigeru toki , kazutaka ueda , hidehisa yamashita , shigeto yamawaki department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada , , , kenji yoshimi , , noriyuki higo , yong-ri ren , hideki mochizuki , yoshikuni mizuno , shigeru kitazawa , , dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; crest, jst, tokyo, japan; neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba- positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma − . mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections.